RF plasma texturing process is utilized to create surface nanostructures on some selected biomedical alloys such as MP35N (Co-Ni-Cr-Mo base) alloys and Pt-Ir alloys. Unique nanostructured pillars evolved in high aspect ratio, with the microstructural features that vary with alloy compositions and processes. This plasma-induced nano texturing process provides unique advantages such as being of biocompatible metallic structure, exhibiting strong adhesion to the substrate, and enhancing cell adhesion and growth. Possible applications of the textured surface include all metallic drug-eluting-stents (DES) with reduced late stent thrombosis (LST), low impedance conducting electrodes for neural stimulation and neural signal collection, drug delivery reservoirs, and other applications benefited by nanostructures and high surface area. Different nanostructure morphologies can be obtained and manipulated corresponding to altered process parameters. Microstructural and compositional analysis results indicate a strong dependence of texturing on the material phase distribution of the alloys. Possible mechanisms for such behavior as well as the surface topological evolution will be discussed in relation to microstructures and physical properties of the materials involved.

Polymeric scaffolds are widely studied for repair and regeneration of damaged tissues in bone tissue engineering. In our previous work, we introduced a novel biomimetic scheme to mineralize hydroxyapatite (HAP) using organically modified nanoclay (OMMT) particles. Polymeric scaffolds are synthesized using this biomineralized HAP with polycaprolactone (PCL) for bone tissue engineering applications. These scaffolds have shown good biocompatibility, degradability and mechanical strength. Here we report molecular modeling approach to systematically construct representative models of mineralized HAP (OMMT-HAP). The results obtained from the simulations provide useful information about the mineralization mechanisms in OMMT-HAP. We also report a representative model of the polymeric scaffold consisting of OMMT, HAP and PCL. The representative model is based on the X-ray diffraction, density and minimum energy of the system. The OMMT, HAP and PCL have shown molecular interaction with each other in OMMT-HAP-PCL system. Various moieties of the OMMT-HAP-PCL system are individually analyzed in order to evaluate molecular interactions in the system. The interaction energy calculations indicate high attractive interactions of clay with PCL and HAP. Further, HAP shows a strong influence on the functional group and backbone of PCL chains. Our experiments also indicate that nanoclay based mineralized HAP with PCL has improved nanomechanical properties as compared to pristine PCL polymer. Steered molecular dynamics simulations have been performed to analyze the load-carrying behavior of the polymeric scaffold. The stress deformation response of the representative model of polymeric scaffold is studied. The molecular dynamics studies provide the insight into the overall mechanical properties of the polymeric scaffold at the molecular scale. These studies in quantitative interaction energy calculations provide an in silico methodology for design in biomedical engineering.

The applications of polymeric nanofibres and nanofibre mats are very extensive, expanding rapidly and covering many traditional and new areas such as filtration, protective clothing, devices/sensors, catalyst supports and biomedical constructs such as scaffolds used in tissue engineering, drug release vehicles, artificial organs and wound healing patches. However, there is still a lack of modern technologies to mass produce nanofibres more consistently, robustly, reliably and economically. In particular, tailoring the size and size-distribution of polymeric nanofibres to suite demand is an important area which requires further research on their innovative processing and forming methods.
This invited paper describes a novel method to mass process multi-functional nanofibres with competitive morphologies using the simultaneous application of rotation and pressure. A simple rotating vessel with millimetre-scale holes on its walls is used. The rotation of this vessel containing a polymer/monomer solution and the applied pressure amplify the tensile force to draw fibres to the nanoscale with a very high yield. The rotating speed, working pressure and polymer solution concentration changes offer a dramatic reduction in fibre diameter. Fibres as small as 60 nm diameter were formed. In addition fibres with a diameter of micro-nanoscale (1000-60 nm) could also be produced, suggesting that this technique is capable of tuning not only fibre diameter but its distribution. The rotating speed determines the fibre length. Therefore, our process couples the best features of the three main nanofibre production methods - electrospinning, centrifugal spinning and blowing, reported in the literature. Many types of biopolymeric fibres have been produced and several examples will be described in this presentation.

Manipulation of matter and material features at the nanoscopic length scale has become fundamental generators for innovations in nanomedicine and biomedical applications. Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) synthesized by a simple green chemical approach, in which natural nutrients (e.g., vitamin C) were used as reducing agent, whereby their oxidized products acted as capping agent to imparting excellent solubility and stability in water, PBS buffer and cell culture media. The as-prepared USPIONs have an average core size of ca. 5 nm and exhibit good crystallinity and high magnetization saturation value (47 emu.g-1). The remarkable biocompatibility of the magnetic quantum dots was verified using primary human immune-competent cells and the zebra fish embryo tests. Detailed NMR analysis of the suspensions provided insight into the magnetic order within the colloid and demonstrates the suitability of the materials as negative contrast agents for MRI. Phantom experiments on the contrast agent (clinical 3 T MRI scanner) reveal an enhanced r2/r1 ratio of 36.4 (r1= 5 s-1mM-1 and r2= 182 s-1mM-1) by comparing with the clinically approved SPIOs and USPIOs, which implies the DHAA-Fe3O4 NPs suspensions are expected to be a promising candidate for negative contrast applications. The cellular uptake studies showed that NIH 3T3 cells have much higher uptake of the DHAA-Fe3O4 NPs than Sinerem@.
This talk will discuss both the potential of nanomaterials for biomedical applications. The examples will include application of superparamagnetic iron oxide nanoparticles for drug delivery applications, plasma-enhanced chemical vapour deposition of biocompatible coatings and electrospinning of woven nanofiber mats for tissue engineering applications.

We demonstrate a 2×2 stretchable transistor active matrix which can conform to complex shapes and may lead to large-area stretchable sensors in the future. Here we have developed the robust stretchable substrate with modulus gradient from 9.1 GPa to 300 kPa utilizing a diffusion of molecule. Furthermore, we have developed printable elastic conductor made of fluorine rubber and silver flake and single walled carbon nanotubes, which conductivity is 100 S/cm and stretchability is 40%.
Stretchable electronics have the potential to enable electronics which can interface with highly deformable dynamic environments such as biological bodies. One method to realize stretchable electronics utilizes a robust stretchable substrate which can accommodate strain, and patterned rigid islands which can protect strain-sensitive devices from degradation. The fabrication process is as follows. An organic transistor array was first fabricated on a 25 µm thick polyimide (PI) islands, with a dinaphtho[2,3-b:2',3'-f]thieno[3,2-b]thiophene (DNTT)[1] semiconductor layer, a 4 nm thick aluminum oxide gate dielectric and a 2 nm thick self-assembled monolayer [2]. The transistors in this stretchable matrix exhibit a mobility of more than 1.6 cm²/Vs and on/off ratio exceeding 10⁵. Next, we cut out only transistors from films and embedded them in polydimethylsiloxane (PDMS). A modulus gradient was formed by altering the crosslink density of PDMS in discrete regions surrounding the rigid islands, which reduces strain concentrations in the substrate. Diffusion of polymethylhydrosiloxane in PDMS causes discrete boundaries to become a continuous modulus gradient [3]. Then, elastic conductors are formed with silver flakes and single walled carbon nanotubes. Stencil printing of them enables matrix addressing of the organic transistors.
[1] T. Yamamoto and K. Takimiya, Journal of the American Chemical Society 129, 2224-2225 (2007).
[2] H. Klauk et al., Nature 445, 745-8 (2007).
[3] D.-J. Guo, et al., Langmuir 21, 10487-10491 (2005).

Prevention of pathogen colonization of medical implants is a major medical and financial issue since infection by microorganisms constitutes one of the most serious complications after surgery or critical care. Immobilization of antimicrobial molecules on biomaterial surfaces is an efficient approach to prevent biofilm formation. We reported the first self-defensive coating against both bacteria and yeasts where the release of the antimicrobial peptide is triggered by enzymatic degradation of the film due to the pathogens themselves. Biocompatible and biodegradable polysaccharide multilayer films based on functionalized hyaluronic acid (HA) by cateslytin (CTL), an endogenous host-defensive antimicrobial peptide, and chitosan (CHI) were deposited on a planar surface. After 24 h of incubation, (HA-CTL-C/CHI) films fully inhibit the development of Gram-positive Staphylococcus aureus bacteria and Candida albicans yeasts, common and virulent pathogens agents encountered in care-associated diseases. Furthermore, the limited fibroblasts adhesion on (HA-CTL-C/CHI) films, without cytotoxicity, highlights a medically relevant application to prevent infections on catheters where fibrous tissue encapsulation is undesirable. Further developments are investigated to obtain a mechanically stable coating with complementary bioactivities.

Scientific publications on electrospinning and electrospun fibrous structures have been increasing exponentially since late 1990s, which testifies the worldwide interest in this “new” technology. It has been demonstrated over the past two decades that electrospun micro- and nanofibers can have diverse applications such as filtration, sensors, cosmetics, etc. There has been a huge interest in electrospun fibrous structures for biomedical applications, which stems primarily from the distinctive advantages of using fibrous structures in tissue engineering and controlled release. With advances in electrospinning technologies and associated disciplines, electrospinning will find increasingly wider applications in the biomedical field. The principle of electrospinning is simple, and the parameters for basic electrospinning are not many. But with innovative modifications of the basic electrospinning technology, new fiber collecting techniques, and developments of techniques such as emulsion electrospinning, possibilities for creating fibrous structures with distinctive and desirable properties appear to be boundless. Our research has investigated the effects of polymer solution properties and electrospinning parameters on fiber formation using a number of biopolymers and their composites. Negative voltage electrospinning (NVES) is rarely reported. Our studies pioneered NVES of tissue engineering scaffolds and have provided insights into electrospinning which can help to overcome some major technological problems in scaffold fabrication. Furthermore, our results showed that negative residual charges in scaffolds could significantly influence the cellular response. For making scaffolds with highly aligned fibers, we used a rotating-drum fiber collector together with arrayed electrodes. Not every polymer can be made into nanofibers via conventional electrospinning. We investigated using co-axial electrospinning to form nanofibers for several biopolymers. Developing bioceramic-polymer composite scaffolds for bone tissue engineering is a major area in our research and we investigated electrospinning of nanocomposite scaffolds. In vitro biological studies showed that composite fibers elicited enhanced response of osteoblastic cells. For the controlled release of biomolecules such as bone morphogenetic proteins, emulsion electrospinning was employed to form fibrous delivery systems. The emulsion composition was shown to be important for forming core-shell structured nanofibers, which affected the release behaviour of biomolecules. For the controlled, simultaneous or sequential release of two growth factors, we have developed new electrospinning technologies and fabricated bi- or tricomponent fibrous delivery vehicles. Biological assessments have affirmed the synergistic effects of dual delivery and controlled release of growth factors from novel scaffolds.

Ultramet has been developing and commercializing refractory metal and ceramic coatings and freestanding parts for corrosive and high temperature environments for more than 42 years. Tantalum has a long history of use as an implant material in both bone and soft tissue. Ultramet developed and licensed a process for fabricating open-cell tantalum metal foam that closely resembles the structure and properties of human bone for use as FDA-approved orthopedic bone implants. Under NASA funding Ultramet developed a process to diffuse highly corrosion-resistant tantalum metal into the surface of conventional stainless steels and superalloys to improve the acid corrosion resistance of components used in precision space shuttle orbiter propellant transfer valves. A thin, metallurgically bonded tantalum surface layer was established that graded from pure tantalum at the surface to a mixture of tantalum and the substrate elements. The tantalum surface layer precisely replicates intricate substrate features and does not require post-process machining or polishing. The potential exists to use this coating to increase the biocompatibility of conventional metal implant alloys, and the processing also allows the application of various surface textures. Ultramet's experience with tantalum foam structures and coatings will be reviewed.

There are countless ways that lasers can be used to process virtually every class of material and they all begin with a fundamental understanding of the laser-material interaction. Biological systems have enjoyed the benefits of lasers almost since their discovery and in almost every conceivable facet (tissue ablation, cellular and tissue imaging, micro- and nano-manipulation, ceramic cladding, etc.). And even now some 50 years later, lasers are still finding new applications in biological systems. What makes laser processing of biomaterials so unique and exciting is that laser radiation can take on such a wide range of qualities, e.g., wavelength, pulse width, energy/power, repetition rate, spot size, etc. and these qualities directly govern the properties of any subsequent material interaction. By understanding and exploiting the many unique qualities of lasers to control the interaction and similarly guiding the energy relaxation in materials we are then able to produce a material modification that is often unobtainable by conventional physical or chemical methods. Recently, our group and others have applied lasers to process biologic materials in novel ways for disparate applications ranging from the controlled self-assembly of biomacromolecules to be used for biomimetic nano-manufacturing by laser nano-manipulation to fundamental cell biology by CAD/CAM writing of patterns of different individual cells for studying cellular signaling in idealized co-cultures. As an example, for the latter the ability study embryonic stem cell differentiation in idealized 2D and 3D constructs is critical for their complete understanding and therapeutic utilization. This presentation will provide an overview of lasers interaction with biologic materials and the novel applications and fundamental studies that they enable.

This lecture provides an overview of recent developments in our laboratory for several plasmonic nanomaterials and biosensing technologies that allow biomedical diagnostics and therapy at the molecular and cellular level. Plasmonics refers to the research area of enhanced electromagnetic properties of metallic nanostructures that produce ultrasensitive and selective detection technologies. The technology involves interactions of laser radiation with metallic nanoparticles, inducing very strong enhancement of the electromagnetic field on the surface of the nanoparticles. These processes, often called ‘plasmonic enhancements&’, produce the surface-enhanced Raman scattering (SERS) effect. Unique metallic nanoprobes have been developed for sensitive and selective SERS biosensing of DNA/RNA biomarkers of various diseases. The SERS-based ‘Molecular Sentinel&’ (MS) nanoprobe technology exhibits the capability of multiplex detection using homogenous assays in solution. The unique label-free MS biosensor on plasmonic Nanowave chip is presented. The Nanowave chip fabrication is relatively simple and low-cost with high reproducibility based on depositing a thin shell of gold over close-packed arrays of nanospheres. Other plasmonic platforms, such as gold nanostars, offer plasmon properties that efficiently transduce photon energy into heat for photothermal therapy. Nanostars, with their small core size and multiple long thin branches, exhibit intense two-photon luminescence, and high absorption cross sections that are tunable in the near infrared region with relatively low scattering effect, rendering them efficient efficient photothermal agents in cancer therapy. A theranostic nanoplatform construct was created, allowing SERS imaging and photodynamic therapy. The use of the cell-penetrating peptide, TAT, greatly enhances particle uptake by the cells, allowing for the imaging and detection of a specific target, followed by localized release of therapeutic agents. Some examples will illustrate the combination of therapeutics and diagnostics into a single construct, i.e. theranostics, an emerging field of medical research that aims to further improve personalized medicine in a seamless fashion.
Acknowledgments. Supported in part by the Duke Exploratory Project Funds, the Defense Advanced Research Projects Agency (HR0011-13-2-0003), and the National Institutes of Health (T32 EB001040). The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred.

Molecular imprinted polymers have long been recognized for their potential in diagnostic devices. However, it has been difficult to obtain polymers that specifically bind larger than 1,500 D molecules that are often the target in diagnostic devices. In addition, some polymeric networks may be stable for a large spectrum of temperature and pH ranges, thus potentially simplifying diagnostics in low resource and remote settings. We present here data on West Nile (WN) antibody imprinted porous silica microparticles. The particles were prepared using the TEOS sol/gel method in the presence of the antibody, using carbon black as a pigment and SDS as a porogen. After rinsing the particles in a mixture of acetic acid and methanol until no absorbance at 290 nm could be detected in the supernatant, the resulting particles were able to aggregate upon the re-introduction of serum containing the West Nile antibody. Particles did not aggregate upon introduction of WN negative serum or control human chorionic gonadotropin (hCG) antibodies. Other particles imprinted using hCG as a template hCG, would aggregate in the presence of hCG, but not in the presence of serum containing the anti West Nile antibodies. Thus, we are speculating that the binding is indeed specific. A device was built to record the results of the agglomeration tests and to be able to determine the sensitivity and specificity of the WN antibody test. These types of microparticles may be useful in microfluidic lateral flow devices and might find applications in low resourced areas.

Cell migration is essential for both physiological and pathological processes, including wound healing, inflammation, and cancer invasion and metastasis. The high-throughput screening of the genes responsible for cell migration is therefore desired for the elucidation of the mechanisms underlying these processes [1]. Reserve transfection (RTF) has been utilized to transfect hundreds of genes to target cells on a single glass slide. However, a potential drawback of the system is an increased possibility of the cross-contamination of transgenes and/or cells between neighboring RTF spots as the spot density increases. This study describes a microdevice with RTF for cell migration assays. The microdevice consisted of multiple nanoliter scale chambers, which separate individual RTF spots to prevent cross-contamination of reagents and cells among neighboring RTF spots. The microdevice was designed to use forces generated by surface tension to seed cells in the chambers in a simple manner. To demonstrate that the microdevice can be used for cell migration assays, paxillin (pxn) was used as a model target gene. Pxn is a focal adhesion-associated adaptor protein and is essential for cell migration. We demonstrated that the migration of cells was significantly suppressed by the transfection of anti-paxillin siRNA using the microdevice. This approach may provide a promising platform for robust and reliable RTF assay systems for various biological analysis applications.
[1]R. Onuki-Nagasaki, A. Nagasaki, K. Hakamada, T. Q. P. Uyeda, S. Fujita, M. Miyake and J. Miyake, “On-chip screening method for cell migration genes based on a transfection microarray”, Lab chip, 1502-1506, 8, (2008).

Biofilms are communities of microorganisms that colonize and grow on surfaces. Although many biofilms are beneficial, some biofilms cause grave health concerns. Often when pathogens form on indwelling medical devices, the infection is difficult to eradicate; therefore, the device, for example, a catheter, intubation tube, implant, or shunt, must be removed. Thus, the development of new topological coatings that can prevent or treat biofilm formation is imperative. Chitosan, a copolymer of 2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose units joined by β(1→4) bonds, was chosen as one major constituent in our synthesized nanobiomaterials due to its inherent biocompatibility, antimicrobial activity, and biodegradability. In this study, chitosan-pectin nanoparticles were generated via an ionic suspension and characterized for their potential use in treating chronic wounds. Pectin, a weak polyanion was chosen because it is known to help prevent persistent acute inflammation. Varying ratios of chitosan and pectin were mixed to form particles of varying sizes. The morphology, size, and zeta potential of the particles were characterized using transmission electron microscopy and electrophoretic light scattering. Changes in size and zeta potential due to pH were investigated as were the effects of addition order and concentration. A second modality — flexible nanofibrous chitosan coatings — were engineered in an effort to delay biofilm formation by offering a broader range of cytotoxicity. Thus, the essential oil, cinnamaldehyde (CA), was chosen as a non-toxic agent that targets pathogens non-specifically. A Schiff base reaction was employed to reversibly conjugate CA to the chitosan so that the chitosan and the chitosan-CA derivatives could be electrospun into nanofiber mats having an average fiber diameter of ~50 nm. At physiological conditions, the Schiff base reversed, which released CA-liquid and CA-vapor from the chitosan-CA nanofiber mats. CA release was correlated to time-dependent bacterial cytotoxicity against two Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa. A complete inactivation (>99%) of E. coli was achieved after only 30 min using the “control” chitosan-CA (0%) nanofiber mats, while a high inactivation of P. aeruginosa, 81 ± 4.1%, was achieved using chitosan-CA (5.0%) nanofiber mats. These studies indicate that potentially, chitosan-based nanoparticles and nanofiber mats can aide in the treatment and prevention of biofilm formation.

Introduction: Hospital acquired (nosocomial) infections, a leading cause of death in the United States, affects at least 1.7 million patients, and causes 99,000 deaths per year [1]. In particular, infections of medical devices are a substantial setback, increasing the cost of patient treatment, and are a harbor for antibiotic resistant infections. Medical device surfaces once implanted are susceptible to the formation of bacterial biofilms; the biofilms can increase antibiotic resistances by 10-1,000 fold [2]. Free fatty acids (FFAs,) as found in human tissues, breast milk, and blood, are hydrophobic constituents of the innate immune system with bactericidal action [3]. In particular, lauric acid (LA), found in human breast milk, and oleic acid (OA), found in tissues of the body, each provides a low minimum inhibitory concentration (MIC; lowest values of 6.25 or 80 µg/ml for OA and LA respectively), natural abundance, and safety [3, 4, 5]; yet, poor water solubility is a barrier to delivery [4, 5]. Instead, we propose to incorporates FFAs in solid lipid nanoparticles (SLNs), and apply these as coatings.
Materials and Methods: This strategy combined FFAs including LA and OA with nanotechnology to develop infection resistant coatings on medical devices. FFAs were incorporated into SLNs, towards delivery of the antimicrobial on surfaces. Next, studies were carried out to prove the effectiveness of these materials for coating medical devices, using atomic force microscopy (AFM) and Transmission Electron Microscopy (TEM). SLNs antimicrobial activities were tested by measuring a zone of inhibition (ZOI), in relation to the concentration of added FFAs, demonstrating the ability of the SLNs inhibit bacterial growth.
Results and Discussion: Coatings were made with nanotopography for preventing infections using techniques of nanotechnology (AFM). Further investigations found that at specific effective combinations of FFAs, production of SLNs with a unique core-shell. ZOI were also established for SLNs combinations consisting of either LA or OA; when both FFAs were added, the greatest antimicrobial ZOI were observed with a threshold of 400 mg LA and 75 mg OA. The SLNs with FFAs were highly effective in reducing bacterial growth and killing bacteria on the surfaces of medical devices.
Conclusions: In conclusion, we produced unique nanostructures through the incorporation of multiple free fatty acids into solid lipid nanoparticles. These nanostructures produced coatings with antimicrobial properties that could be highly useful in the fight against drug resistant infections on medical devices in the nosocomial setting.
References: [1] Klevens et al. Public Health Rep. 2007; [2] O&’Toole et al. Nature 2003; [3] Chao-Hsuan et al. J. Microbiol. Biotechnol. 2011; [4] Yang et al. Biomaterials 2009; and [5 ] Huang et al. Biomaterials 2011.

Our laboratory has been developing various nanosystems for diagnostic applications. These include designing plasmonic nanocrystals for single nucleotide polymorphism (SNP) genotyping. The platform involves polymerase chain reaction (PCR) for target sequence amplification and colorimetric detection for pharmacogenomics applications. We have also established polymer-based lab-on-a-cartridge for automated sample preparation and PCR detection. The integrated all-in-one system, termed MicroKit, allows for the rapid and accurate typing and subtyping of influenza and other viral infections within 2 hours. We have created the silicon-based Microsieve system for rapid and selective isolation of circulating tumor cells (CTCs) from peripheral blood. This non-invasive, near real-time, inexpensive liquid biopsy approach allows for the enumeration and biomarker analysis of CTCs for cancer diagnosis, prognosis and monitoring.

The ability to pattern biomaterials in planar and three-dimensional forms is of critical importance for several applications, including 3D cell culture, tissue engineering, and organ mimics. Direct-write assembly enables one to rapidly design and fabricate soft materials in arbitrary patterns without the need for expensive tooling, dies, or lithographic masks. In this talk, the design of novel cell-laden, hydrogel, structural, and fugitive inks with tailored rheological properties will be described. Recent advances in microscale printing of 3D living tissues with embedded microvasculature will be discussed as well as our ongoing efforts to characterize these 3D constructs.

Seashells are natural nanocomposites with superior mechanical strength and eminent toughness. What is the secret recipe that Mother Nature uses to fabricate seashells? What roles do the nanoscale structures play in the strengthening and toughening of seashells? Can we learn from this to produce seashell-inspired nanocomposites? The recent discoveries of nanoparticles in nacre and conch shells are summarized, and the roles these nanoparticles play in seashell&’s strength and toughness are elucidated. It was found that rotation and deformation of aragonite nanoparticles are the two prominent mechanisms contributing to energy dissipation in seashells. The biopolymer spacing between nanoparticles facilitates the particle rotation process. Individual aragonite nanoparticles are deformable. Dislocation formation and deformation twinning were found to play important roles in the plastic deformation of individual nanoparticles, contributing remarkably to the strength and toughness of nacre and conch shells upon dynamic loading.

Biomaterials play pivotal roles in engineering tissue regeneration and repair. To fabricate an entire organ or a large piece of tissue for transplantation, a predesigned scaffold with the patient-specific anatomy is required. However, there are often irregular shaped defects and wounds that need to be filled and repaired. In such cases, injectable biomaterials are desirable because they allow for easy manipulation and minimally invasive tissue repair procedures. We hypothesized that the extracellular matrix (ECM)-mimicking nanofibrous architecture advantageously enhances cell-material interactions; channels and pores at multiple scales promote cell migration, proliferation and mass transport conditions, facilitating tissue regeneration and integration with host. We synthesized star-shaped biodegradable polymers and copolymers, proposed a theory of nanofibrous hollow structure formation, and developed technologies for such polymers to self-assemble into nanofibrous microspheres, nanofibrous hollow microspheres, nanofibrous hollow discs, and nanofibrous shells, and nanofibrous sponge-like microspheres. The nanofibrous microspheres were found to enhance osteogenic differentiation of mesenchymal stem cells and bone regeneration in a caldarial defect repair model over control microspheres. The nanofibrous hollow microspheres were found to efficiently accommodate cells and enhance cartilage regeneration in a critical-size osteochondral defect repair model over control microspheres, achieving equivalent biomechanical properties to those of native cartilage and smooth integration with the host cartilage tissue. The nanofibrous sponge-like microspheres were found to enhance the proliferation and odontogenic differentiation of dental pulp stem cells for tooth tissue regeneration. The above data have consistently demonstrated that these novel nanofibous microcarriers are excellent injectable carrier of various mammalian cells and stem cells for tissue regeneration.

Recent work has demonstrated the clinical utility of aligned muscular cell sheets for the treatment of heart failure. Current methods for producing aligned cell sheets rely on elastic substrates with micro- or nanoscale topographical features and the application of strains (~ 10% ) using external mechanical devices, but these techniques require large amounts of space, are inefficient and are difficult to scale-up. Here, we report the development of biocompatible monodomain liquid crystal elastomer (LCE) composite materials as electrically responsive substrates for cell sheet growth and alignment. Conductive monodomain LCEs are produced using a two-step crosslinking method along with the addition of carbon black particles both before and after crosslinking, resulting in conductive LCEs (resistivity ~ 4 - 5 Omega; m) with as little as 2 wt % carbon black nanoparticles. Surface wrinkling with defined wavelength and amplitude is achieved by layering a thin polystyrene film on the top surface. The resulting LCE bilayer composites exhibit rapid (~ 0.1 - 1 Hz) and reversible shape and topography changes in response to modest voltages (10 - 40 V). The response of the substrate can be controlled by variation of the pulse amplitude, duration, and frequency to achieve reversible strains from 1 - 20 % with a response time on the order of seconds. Electro-mechancial epxansion of conductive LCEs was completely reversible with no hysteresis, even after 12 hours of electrical stimulation. Neonatal rat ventricular myocytes remain viable on LCE-carbon black bilayer substrates, and aligned myocyte cell sheets were successfully grown on LCE-composite bilayers. Current work is focused on characterizing the alignment and force generation of cell sheets grown on LCE-bilayer composite substrates under periodic electrical stimulation. These materials potentially provide a straightforward and scalable route to aligned cell sheets and enable investigation of cell response to a dynamically changing surface pattern.

Paper-based microfluidic devices are a low cost, instrumentation free alternative to conventional open channel geometries. These compact chips range from simple lateral flow assays to complex three-dimensional networks that enable multistep analyses. Despite its many advantages, the small pore-size of paper has kept this approach from being extended to many applications of conventional microfluidics, such as whole cell and emulsion-based assays. To address this issue, we have demonstrated a hybrid open channel / paper microfluidic device. This hybrid chip relies on the capillary forces of paper to control fluid flow within an open-channel. In this way, the advantages of paper microfluidics can be extended to open-channel geometries. Open channels are connected in series to paper networks and flow rates within the open channel are controlled by varying the fluidic resistance of the paper. The hybrid chips are characterized by controlling laminar flows in the open channels, and the results agree well with Darcy&’s Law. To demonstrate the utility of this approach, a hybrid chip is demonstrated that hydrodynamically focuses micrometer-scale beads in an open channel without any external instrumentation
Open channel microfluidic devices were fabricated using plastic sheets (mylar, 50mu;m thick) with CO2 laser patterned (Universal Laser Systems, Scottsdale, Arizona) Whatman No. 1 filter paper (Whatman, Piscataway, NJ) in contact with the open-channel. Paper resistance was varied by applying a wick on the paper resistor using Kimwipe paper (Kimberly Clark, Roswell, Georgia). To demonstrate the control of the flow of discrete objects, 1mu;m fluorescent polystyrene beads (Life Technologies, Grand Island, NY) were utilized.
We were able to show that flow rates in an open-channel can be controlled by changing the resistance of the paper network to a wick. The change in flow rates showed a linear trend with the absolute flow rates decreasing as the resistance in the paper increased, in agreement with Darcy's Law. Secondly, we were able to demonstrate control of laminar flow patterns in an open channel. Two separate paper networks were connected to an open channel, each of which could pull at a different flow rate, and thus control the laminar flow pattern. These flow patterns were observed using fluorescent dyes and an epi-fluorescence microscope. Subsequently, a device demonstrated the focused polystyrene beads using sheath-flow hydrodynamic focusing. Single beads could be distinguished within the core, thus demonstrating that discrete particles, such as cells or droplets, could be controlled with these hybrid chips.
Hybrid open channel and paper microfluidic devices harness the advantages of paper-based microfluidics and extend them to conventional open channel geometries. As such, these hybrid chips are well suited to expand the reach of complex analytical and clinical analysis to point-of-care settings.

Modifying the nanostructure/chemistry of materials allows to optimize their properties [1]. Our strategy rests on creating nanopatterns that act as surface cues [2,3], affecting cell behavior. Chemical oxidation creates unique topographies [4], becoming a general strategy to improve biocompatibility. Our treatment selectively inhibits fibroblast growth while promoting osteogenic cell activity [5] in vitro. Enhancement of mechano-biocompatibility may occur by coating with spider silk [6, 7]. Improvement of antibacterial properties using laser and plasma strategies will also be discussed [8].
[1]F Rosei, J Phys Cond Matt 16, 1373 (2004)
[2]F Variola et al, Small 5, 996 (2009)
[3]F Variola et al, Biomaterials 29, 1285 (2008)
[4]F Vetrone et al, Nanolett 9, 659 (2009)
[5]L Richert et al, Adv Mater 20, 1488 (2008)
[6]C Brown et al, Nanoscale 3, 3805 (2011)
[7]C Brown et al, ACS Nano 6, 1961 (2012)
[8]O Seddiki et al, in preparation

An important trend in flexible personal electronics involves the development of materials, mechanical designs and manufacturing strategies that enable the use of unconventional substrates, such as polymer films, metal foils, paper sheets or rubber slabs. A particular challenge lies in the need for the entire system to accommodate not only bending but also stretching due to the time dynamic and curvilinear of human body. Here we introduce a set of materials and design concepts for a wearable and mechanically invisible device patch that exploits the rigid commercial off-the-shelf (COTS) chips. This design utilizes thin soft silicone elastomer with a hierarchical relief structure as the substrate, multilayered ‘self-similar&’ structures as interconnects, and a novel packaging scheme to isolate the strain at the hard/soft interface. The result enables a skin mounted multifunctional electronic system with reversible stretchability up to 70% and capable of wireless charging, multi-channel biological signal sensing, e.g. electrophysiological potential, temperature and motion, and RF communication to the backend receiver.

Histamine and serotonin are biogenic amines that are indispensable in the efficient functioning of various physiological systems. The traditional techniques to detect these target molecules are costly, lack in speed, require a lab-environment and sophisticated equipment. Therefore, a polymer-type sensor platform for the detection in biological fluids was designed. Molecularly Imprinted Polymers (MIPs) were used as polymer-type receptors since they are robust, can be produced at low-cost, and have a high affinity for their template molecules. MIPs with a high affinity for serotonin and histamine were developed and integrated into a sensor platform which can specifically detect small molecules with two techniques. The first is based on electrochemical impedance spectroscopy [1,2]; the second is a novel read-out technique which measures the heat-transfer resistance [3,4]. The methods can be measured simultaneously, allowing direct validation of the results. With a refined sensor setup for biological samples, native serotonin concentrations in human blood plasma were determined by impedance spectroscopy. The obtained results were nicely in agreement with High Performance Liquid Chromatography (HPLC) reference tests. With the same platform, histamine concentrations of mildly acidic bowel fluid samples of several test persons were analysed. We showed that this sensor provides reliable data in the relevant concentration regime, which was validated independently by Enzyme-Linked Immunosorbent Assay (ELISA) tests. The second technique based on heat-transfer resistance was previously applied for DNA mutation analysis and screening of cells, but this is the first time detection of small molecules with MIP-type receptors has been reported. This method allows straightforward detection of a variety of targets in buffer solutions. As a first proof-of-application, a calibration curve in saliva was constructed. Due to the speed, low-cost and high specificity of both developed methods, they can be considered as techniques with a high clinical and commercial potential for medical devices.
[1] M. Peeters, F.J. Troost, et al.,Sens. Actuators, B, 171, 602-610 (2012).
[2] M. Peeters, F.J. Troost et al., Anal. Chem. 85, 1475-83 (2013).
[3] B. van Grinsven, N. Vandenbon et al., ACS Nano., 6, 2712-2721 (2012).
[4] M. Peeters, P. Csipai et al., Anal. Bioanal. Chem., DOI: 10.1007/s00216-013-7024-9 (2013).

Stretchable electronics provide a foundation for new biomedical devices due to their ability to mechanically conform with soft materials. We show that thin metallic films patterned in fractal layouts are a basis for new device architectures that can combine useful function with stretchable mechanics. By using Peano, Greek cross, and box fractals as a design metric, we fabricate and characterize high precision temperature sensors, heaters, electrodes, and compact radio frequency antennas. These devices support conformal mounting on skin, and they display properties such as space-filling and invisibility under magnetic resonance imaging.
To characterize the mechanical properties of these devices, a hysteresis study is performed in which the fractal samples are cycled between a state of no stress and states of increasing amounts of maximum applied stress. By carefully correlating changes in electrical resistance with tensile strain, the transition from elastic to plastic deformation is quantified. Measurements indicate that third iterative order Peano structures stressed along both x- and y-axes undergo an elastic-to-plastic transition in the range of 27-32%. These values of uniaxial stretchability are consistent with three-dimensional FEM numerical analysis and confirm that such devices are suitable for most skin mounting applications.

The function and lifetime of an implantable CGM device are intimately linked with the stability of the glucose oxidase (GOx) enzyme, responsible for glucose detection. Enzyme denaturation, loss due to imperfect immobilization and biofouling-induced pore-clogging gradually decrease device stability. Poly(ethylene glycol) (PEG)-ylated hydrogels offer an opportune venue to minimize biofouling, while retaining their highly hydrated state to prevent enzyme denaturation. These hydrogels, not only retain enzyme activity but also provide good antifouling properties and ease of fabrication via traditional photo-lithography methods. In this contribution, we report the synthesis and characterization of a random copolymer based on PEGylated side chains together with cinnamyl ethyl methacrylate (CEMA) and glycidyl methacrylate (GMA) (namely poly(PEGMEM-CEMA-GMA)). The polymer was mixed with GOx enzyme and drop-casted on the electrode, followed by UV exposure for crosslinking and 24 hour incubation for reaction between the GOx-amine and hydrogel&’s GMA-epoxide group. GOx-grafted, poly(PEGMEM-CEMA-GMA) hydrogels showed excellent electrochemical stability for continuous glucose detection over a period of 30 days. By retaining a greater of 65% mole ratio in PEG functionality, these hydrogels provide a good biofouling resistance against serum albumin. The sensors exhibited a sensitivity of 320 nA mM-1mm-2 with an apparent Michealis-Menten constant of 25 mM of glucose and limit of detection of 1 µM.

In this paper we present a full innovative low cost, safe, disposable and self-sustainable diagnostic test for tuberculosis using the recent highly promising Lab-on-Paper technology, according the guidelines established by the WHO for the development of ASSURED tests for point of care applications. The devices are based on the definition of microchannels and test zones on hydrophilic paper via the patterning of walls of hydrophobic polymers, photoresist or wax. Movement of fluids within channels is by capillarity and independent multi-analyte assays can be conducted that do not require use of pumps or electric energy. Here, we report on a paper based platform capable of integrating a previously developed Au-nanoprobe based Mtb detection assay, and we call it “Gold on Paper”.
Veigas et al. 2012. Gold on paper-paper platform for Au-nanoprobe TB detection. Lab Chip. 12:4802

Hundred micrometers-sized porous hydroxyapatite globules have proved as successful tissue engineering strategy for bone defects in vivo, as was shown in studies on human mandibles. These granules need to provide enough porous space for bone ingrowth, while maintaining sufficient mechanical competence (stiffness and strength) in this highly load-bearing organ. This double challenge motivates us to scrutinize more deeply the micro and nanomechanical characteristics of such globules, as to identify possible optimization routes. Therefore, we imaged such a (pre-cracked) granule in a microCT scanner, transformed the attenuation coefficients into voxel-specific nanoporosities, from which we determined, via polycrystal micromechanics, voxel-specific (heterogeneous) strength and elastic properties. The importance of the latter and of the presence of one to several hundred micrometers-sized cracks for realistically estimating the load-carrying behavior of the globule under a typical two-point compressive loading (as in a "splitting" test) is shown through results of large-scale Finite Element analyses, in comparison to analytical results for a sphere loaded at its poles: Use of homogeneous instead of heterogeneous elastic properties would overestimate the structure's stiffness by 5% (when employing a micromechanics-based process as to attain homogeneous properties) - the cracks, in comparison, weaken the structure by one to two orders of magnitudes. Furthermore, the implementation of a highly parallelized computer-system, facilitating efficient processing of the tremendous computational workload posed by detailed strength analyses, allowed for voxel-specific safety assessment with respect to local structural failure.

We have proposed a novel concept and technology of the formation “Growing Integration Layer” [GIL] method,where GIL(s) can be prepared via integration of ceramic film formation from a component of the metallic materials by chemical and /or electrochemical reactions in a solution at low temperature of RT-200 C. They have particular features:1)Widely diffused interface(s),2)Continuously graded layers grown from the bulk(substrate),3)Low temperature process,etc. They are quite different from Layer-by-Layer[LBL] strategy,where every layer is deposited from the Top. BaTiO3 or SrTiO3/TiOx GIL films on Ti plates formed by hydrothermal-electrochemical method showed good adhesion. CaTiO3/Al2O3/Ti2Al GIL films on TiAl exhibited excellent adhesion and anti-oxidation performances. On a Ti-base Bulk Metallic Glass, we could succeed to prepare bioactive titanate nano-mesh layer by hydrothermal-electrochemical techniques at 90-120 C.
The GIL method can be applicable for wide variety of applications like thermal barrier, mechanical parts, environmental and/or chemical coating, conducting and or insulating
films, biological and/or medical coating,etc. We may include Zirconia coating on SUS materials.

Poly(2-oxazoline)s (POx) and linear poly(ethylene imine)s (LPEI) represent two interesting polymer classes for a multitude of biomedical applications. While PEI is mainly used for gene delivery applications due to its high content of amine groups and cationic charges,POx are characterized by an excellent biocompatibility. Moreover hydrophilic POx, such as poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethy-2-oxazoline) (PEtOx), reveal a stealth effect similar to PEG making them suitable for drug delivery and anti-fouling purposes. The microwave assisted cationic ring-opening polymerization (CROP) of 2-oxazolines is fundamental for the synthesis of both kinds of material. Using this approach, it is possible to work at high temperature and high pressure, resulting in significantly reduced reaction times in comparison to conventional heating.[1] Furthermore, the living character of the reaction leads to an excellent control over the polymer length and the polydispersity index (PDI) values. It is also possible to vary the structure utilizing different (functional) start-, end-, and side groups yielding systems which posses beneficial properties for a wide range of bio-applications.[2] In an additional step, the side chains of the polymer can be cleaved in a controlled and fast manner using microwave irradiation to create LPEI or partially cleaved POx.[3] In this way, a series of poly(2-ethyl-2-oxazoline-co-ethylene imine)s has been synthesized with different degrees of hydrolysis depending on the time of microwave irradiation. Besides their application for gene delivery, the copolymers can be used as starting material for the formation of hydrogel networks via a bis-functional crosslinker. An enormous swelling behavior of the resulting hydrogels is obtained using water-soluble polymer fragments (i.e. ethyl side chains) or crosslinkers. The number of remaining secondary amines, responsible for subsequent binding and release of DNA, can be adjusted by variation of the amount of crosslinker.
Another example for the synthesis of biomaterials using CROP is the polymerization of an oxazoline monomer with a Boc protected amine group in 2-position, yielding polymers which, after deprotection, possess primary amine groups.[4] These functionalities can be used in several ways, e.g. hydrogel formation by cross-linking as described above, attachment to surfaces or other polymers, labeling, and interaction with genetic material for the purpose of detection or delivery.
1 K. Kempe, C. R. Becer, U. S. Schubert, Macromolecules 2011, 44, 5825. 2 N. Adams and U. S. Schubert, Adv. Drug Del. Rev., 2007, 59, 1504. 3 L. Tauhardt, K. Kempe, K. Knop, E. Altuntas, M. Jäger, S. Schubert, D. Fischer, U. S. Schubert, Macromol. Chem. Phys. 2011, 212, 1918. 4 M. Hartlieb, D. Pretzel, K. Kempe, C. Fritzsche, R. M. Paulus, M. Gottschaldt, U. S. Schubert, Soft Matter 2013, 9, 4693.

Electrophysiology of cell is usually measured by inserting delicate microelectrodes that record the potential, or the current, across the membrane. The process often damages the cell. In the recent years electrochemical field effect transistors (eFET) made from graphene, carbon nanotubes and Si nanowires have emerged as potential non-invasive methods to measure membrane potential modulation during these ion exocytosis processes. We will describe a single electron eFET made from 10 nm Au nanoparticle necklace array to measure subtle modulation in membrane potential. The gain of the eFET is an order of magnitude better than structures made from other integrable nanostructures. Two applications will be discussed: (a) Measurement of internal membrane potential modulation during photosynthesis where there is no exocytosis of ions. (b) Probing membrane potential modulation due to single ion channel activity.

The efficient sorting of rare biological targets from complex biological backgrounds has numerous clinical and research applications. To address this challenge, we have developed a microfluidic chip, the Track-Etched Magnetic micro-POre (TEMPO) filter, to efficiently separate magnetically tagged cells from suspension. The TEMPO filter consists of a track-etched polycarbonate membrane coated with a paramagnetic film (NiFe) integrated into a microfluidic chip. On this chip, targets of interest ranging in size from tumor cells (~10 µm) to viruses (100 nm) can be efficiently separated from a suspension for subsequent downstream analysis. Due to its exceptionally high magnetic forces and the use of columnar flow, the TEMPO filter offers extremely high throughput (> 10 mL/hr) and very pure enrichment (1:10⁵). To demonstrate the utility of this chip, a 5 µm pore size TEMPO was used to efficiently isolate immunomagnetically labeled E. Coli from a vast background of similarly sized bacteria for subsequent downstream analysis.
Track etching allows pore sizes to be scaled from 100nm - 100 µm to optimally trap a wide range of biological targets (mammalian cells, bacteria, virus, microvesicles). We coat the polycarbonate filter with a 200 nm thick film of magnetically soft NiFe and a passivation layer of Au to protect the magnetic film from the biological fluid and oxidation. The samples flow vertically through the porous membrane, bringing each cell into close proximity of the trapping region. The TEMPO chip sits in a strong applied field Bo ~ 0.5 T, provided by a ~ 1 inch² NdFeB magnet, to magnetize the NiFe film and the MNP coated pathogens. The filter is integrated into laminate sheet microfluidics constructed from Mylar plastic. Characterization is done by using fluoresecnetly labeled non-magnetic (polystyrene) and magnetic (Iron Oxide) beads.
We characterized the efficiency of the magnetic filter by sorting non-magnetic beads from the magnetic beads, and measuring the output with flow cytometry. Enrichment of the non-magnetic beads by xi; > 10⁵ was demosntrated at >10 mL/hr . The beads could be subsequently released at a recovery rate of 90%. Multiple layers of TEMPO filters could be be connected in series for enhanced enrichment, giving exponential increase in sorting efficiency for each additional filter. Furthermore, the TEMPO chip was utilized to efficiently separate immunomagnetically labeled E. Coli (xi; = 300) from a hetrogeneous mixture of bacteria (S. Aurus., E. Coli), demonstrating the capability to isolate rare pathogens in unprocessed clinical (e.g. blood) or environmental (e.g. drinking water) samples for subsequent molecular analysis.
Acknowledgements: University of Pennsylvania, The UPenn Wolf Nanofabrication Facility

Tissue engineering provides an opportunity to develop patient specific implants comprised of scaffolds seeded with donor cells. Conventional methods such as solvent-casting with particulate leaching, gas foaming, phase separation, melt molding, freeze drying, and solution casting are inadequate due to their inability to produce an engineered microarchitecture. Scaffolds with engineered pores and internal channels can direct cell growth and provide transport to and from seeded cells. Solid freeform fabrication (SFF) methods developed to engineer microarchitecture include 3D printing, stereolithography, fused deposition modeling, and phase-change jet printing, however most SFF methods require an internal support structure that must be dissolved with solvents, where residuals pose a risk of toxicity to seeded cells.
Scaffold engineering through multi-layer construction is a relatively new field where microarchitecture is created by stacking porous 2D membranes into 3D structures. In general, these methods and the aforementioned SFF methods are time consuming, which is to say it takes hours rather than seconds to produce a single scaffold layer. One underlying reason for manufacturing inefficiency is the pursuit of scaffold material production at the same time as hole creation by rapid prototyping methodologies. The novel approach presented in this paper is to separate these manufacturing processes by first pursuing high efficiency methods of material film production and then fabricating hole patterns via micromechanical punching.
A press capable of micro-alignment and progressive punching was developed such that dies with different geometries could be interchanged. The micro-scale male and female die sets were produced based on techniques commonly used to fabricate microelectromechanical systems, allowing for custom designed punch geometries. Punching was performed on 35-50 micron thick poly(caprolactone) (PCL) films, themselves produced by hot melt extrusion. It was found that the elastomeric nature of PCL made it impossible to punch at room temperature. However, cooling the PCL below its glass transition temperature made it brittle enough to fracture during punching.
One of the most important parameters in tissue scaffold engineering is optimization of porosity. Porosity is dictated by the minimum web size between holes which, for the micro-stamping approach, is limited by the stresses induced during punching. Since punching stress is largely affected by punch-to-die clearance, the effect of punching clearance on minimum web size was investigated.

Chronic pain is an issue that affects more than 70 million Americans, with greater annual cost to healthcare than heart disease, cancer, or diabetes. Common strategies in pain treatment include frequent dosages of analgesics including NSAIDs and opioids. Systemic delivery of the former has been shown to have gastrointestinal, liver, and kidney side effects, which are especially problematic with the elderly while the opioid and related drugs can lead to debilitating addictions. Localized delivery of NSAIDs can yield positive analgesic effect while minimizing the toxic effects from systemic delivery.
Herein we generated polyelectrolyte multilayer thin films utilizing a polymer-attached prodrug form of a small molecule NSAID; here, diclofenac is selected as a representative NSAID in this work. Utilizing degradable linkages, we have found a sustained long-term release of diclofenac over a period of several months at significant payloads sufficient to maintain therapeutic levels during these extended timeframes. With a localized delivery approach, high efficacy can be retained within proximity of the film, with a significant dilution systemically that considerably reduces the side effects in comparison to oral or intravenous dosing. This approach has relevance for any pendant small molecule release, and can potentially be used as a strategy for mutli-therapeutic release.

As one of the biosensing approaches, saccharide sensing is one of a key technology in medical and food fields. For instance, higher sensitive and more rapid detection methods are required in glucose sensing for diabetic patients from the aspect of non-invasive diagnosis. In addition, higher sensitive sensors of food saccharides such as fructose and sucrose are needed for measuring sugar concentration of foods like fruits in the field of food, in which the demand for higher safety and quality monitoring is increasing.
From these backgrounds, we have studied about a novel concept of glucose sensor by use of a semiconductor-based field effect transistor (FET), which was chemically-modified by Phenyl Boronic Acid (PBA). Because the mechanism of glucose sensing using this FET biosensor was based on the specific binding of boronic acid and diol structure of glucose, it was expected that boronic acid could bind other kinds of saccharides as well. In this study, we tried to detect other kinds of saccharides by means of the specific binding characteristic of PBA, and clarify the difference of its affinity to each saccharide for widespread application using the saccharide-based FET.
The concept of extended-gate FET was used as a detection device for the saccharide-based FET, and concentration changes for each saccharide at the electrode surface were detected by converting ionic and molecular charges to electric signals using a gold thin film as a gate electrode. In detail, saccharide recognition device was fabricated by forming self-assembled monolayer (SAM) on the surface of gold gate electrode using 4-Mercaptophenylboronic acid (MPBA), and captured the changes of negative charge derived from specific diol binding between PBA and saccharides using the detection device. The electrical detection of saccharides were carried out by calculating the changes in threshold voltage (VT) using semiconductor parameter, and monitoring surface potential changes on the gate electrode using the real-time analyzer.
In this study, we found the possibility of sensing for various kinds of saccharides using the saccharide-based FET with the gold film gate, on which PBA-SAM was modified chemically as sugar recognition sites. Among that result, the saccharide-based FET was able to detect not glucose but other monosaccharides such as fructose and galactose, and furthermore, disaccharides such as lactose and sucrose. For the measurements of these various saccharides, the surface potential changes of saccharide-based FET were found to differ depending on the kind of saccharides. This result indicates the diverse affinity for each monosaccharide with boronic acid owing to the structural difference of their hydroxyl groups of 1,2-cis-diol or 1,3-cis-diol. In this talk, we would like to show the results about the detection sensitivity of saccharide-based FET based on the binding of PBA to some sorts of saccharide, and discuss about the potential applications of saccharide-based FET.

Owing to numerous advantages such as high specific surface area, excellent wicking properties, compatibility with conventional printing approaches, significant cost reduction and easy disposability, paper substrates are gaining increased attention in biodiagnostics, food quality testing, environmental monitoring, flexible energy and electronic devices. Recent surge in the activity related to paper-based diagnostic devices is primarily focused on realizing microfluidic paper-based analytical devices (µPADs) for point-of-care assays and inexpensive diagnostic tools for resource-limited environments. Most of these developments rely on labour, time and resource-intensive patterning techniques such as photolithography, wax printing, inkjet printing of polydimethylsiloxane (PDMS), to create fluidic pathways and/or differential functionalization of predetermined regions for site-selective adsorption of the biochemical reagents.
Here we demonstrate simple yet powerful “pen-on-paper” approach for realizing multiplexed label-free bioassays using a regular ball pen filled with gold nanorods or biofunctionalized gold nanorods as (bio)plasmonic ink. Pen-on-paper plasmonic biosensors offer two distinct advantages over plasmonic paper substrates obtained using immersion method as reported previously. Firstly, pen-on-paper serves as a facile method to miniaturize the test domain size to few mm^2, which significantly improves the sensitivity of the plasmonic biosensor compared to bioplasmonic paper fabricated using immersion approach. The localized surface plasmon resonance (LSPR) wavelength shift was improved by ~58% higher by reducing test domain area by ~50%. Secondly, pen-on-paper using bioplasmonic ink enables simple and efficient multiplexed biodetection of paper substrates thus leading to multi-marker biochips. Two target proteins (goat anti-human IgG, and goat anti-mouse IgG) were tested to have stable and reproducible response by writing two distinct test domains comprised of AuNR with corresponding antibodies without cross-contaminating the test domains. In this study, we demonstrate these two aspects using gold nanorods as plasmonic nanotransducers.

In recent years, the number of diabetic patients is drastically increasing in the world and many approaches have emerged to establish diagnostic glucose sensors and systems. These glucose-sensing devices usually utilize enzymatic reaction between glucose oxidase (GOD) and glucose due to high selectivity, convenient form and low-cost. However, this method has several disadvantages; the commercially available glucose sensors need blood sampling invasively in order to measure blood glucose level, accompanied by pain, and the detection sensitivity of this principle is insufficient for glucose sensing when glucose concentration in biological fluids is utilized for diagnosis instead of blood glucose level. In our research, we focus on the field effect transistor (FET)-based biosensing technique because of some advantages such as nonuse of enzymes, low cost and high sensitivity. Our research aims for noninvasive monitoring of glucose concentration included in biological fluids by use of the FET-based glucose sensor (glucose transistor).
The use of phenylboronic acid (PBA) derivatives can provide an attractive approach to obtain glucose transistor. The PBA derivative of 1 mM in the ethanol, 4-mercaptophenylboronic acid, was introduced onto the Au sensing electrode utilizing Au-thiol bond and formed self-assembled monolayers (SAMs). The glucose sensitivity in the range of about 70 uM to 2 mM was evaluated for the prepared glucose transistor using the semiconductor parameter analyzer. Furthermore, the glucose transistor which enables protein blocking was fabricated utilizing oligoethyleneglycol (OEG). PBA and OEG were mixed in some ratios and formed hybrid SAMs for enhancement of S/N ratio. To confirm the effect of protein blocking, the change of electrical properties of glucose transistor such as the gate voltage (Vg) and drain current (Id) was compared by adding PBS solution including 4mg/l albumin onto each glucose transistor with or without OEG-SAMs, respectively. Moreover, the glucose sensitivity was evaluated for the change of glucose concentration including the albumin by use of the OEG-based glucose transistor.
In this study, we found the correlation between the logarithm of glucose concentration from the low concentration of less than 100 uM and the Vg change, and the sensitivity to glucose was about 60mV/decade. This result showed that the quantitative and highly sensitive detection of glucose concentration was possible using the glucose transistor. Furthermore, the sensitivity to glucose was kept to some extent even in the solution including albumin with the OEG-based glucose transistor, compared with the sensitivity lost using the glucose transistor without OEG. Thus, the effect of OEG to reduce the protein adsorption was demonstrated for glucose sensing in some biological fluids including proteins.

Molecular detection of biomarkers is a promising approach to diagnosis and monitoring of numerous diseases, as well as the key to molecular medicine and drug discovery. Current clinical applications of biomarkers are limited by the sensitivity, complexity and low selectivity of available detection methods. Electronic nano-materials such as graphene and carbon nanotubes are favorable as sensor platforms for simple, fast and ultrasensitive detection of biomolecular binding; however, no versatile method has so far been developed to date for simultaneous functionalization and passivation of the sensor surface, which would allow for enhanced detection and selectivity of the device. In the present study we demonstrate selective detection of a model protein against a background of serum protein using a graphene sensor functionalized via co-assembled, multifunctional, self-assembling peptides, which simultaneously display the probe and prevent non-specific adsorption. In particular we demonstrate a graphene field effect transistor sensor which can detect streptavidin against a background of serum bovine albumin at less than 50ng/ml. We have also developed a regeneration protocol, which allows us to utilize each sensor for over many experiments. This peptide based functionalization platform is particularly well suited to biological applications due to the ease of fusion of peptides with proteinaceous probes, and biocompatible nature. This system can be applied to a variety of bio-sensing problems, such as the detection of cancer markers under clinical conditions.

Alzheimer&’s disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-β protein and tau deposition in the brain. Hyperphosphorylated form of collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. However, the role of phosphorylation of CRMP2 for AD is poorly understood. Recently, we have demonstrated the relationship between amyloid-β neurotoxicity and the phosphorylation of CRMP2 by using CRMP2 phosphorylation-deficient knock-in mice. Interestingly, the increased phosphorylation occurs prior to the onset of pathology in the mouse model. The phosphorylated form of CRMP2 attracts much attention as a promising biomarker of AD.
We have developed a novel biosensor using photonic crystal nanolasers, which enable label-free detection. The nanolaser sensor detects the adsorption of marker proteins as its lasing wavelength shift. In this study, we demonstrate the specific detection of low-concentration CRMP2 by using the nanolaser sensor.
A nanolaser sensor was first immobilized by anti-CRMP2 antibody. Then, bovine serum albumin was adsorbed as a blocking to prevent non-specific adsorption. As a solution of CRMP2, we prepared CRMP2-overexpressed cell lysate through transfection to HEK293T cells and diluted it with pure water to 10 pM. After the nanolaser was immersed in the solution, clear wavelength shift was observed. The same process was carried out for another nanolaser sensor, which was first immobilized by anti-CRMP1 antibody. In this case, the wavelength shift was not observed. These results indicate that specific binding with CRMP2 was successfully detected in the solution. The next target is the specific detection of marker proteins in human blood.
This work was supported by the Grant-in-Aid of MEXT, #24226003.

In this contribution we report on a novel technique for the specific identification of a variety of viable cells including macrophages and the human cancer-cell lines MCF-7 (breast cancer) and Jurkat (leukemia). The receptors for these cells are prepared by surface imprinting of template cells into semi-cured polyurethane (PU) layers on aluminum chips. After curing of the PU layers and washing off the templates, the imprints in the polymer layer are able to rebind cells selectively. This selectivity is based on geometrical matching and, even more important, on non-covalent interactions between functional groups on the cell membranes and the PU layers [1]. The areal density and morphology of the cell imprints were characterized by optical- and atomic-force microscopy. The detection of cell-binding events with surface imprints was reported earlier utilizing quartz microbalances as a readout technique (QCM), however we will show here that the novel heat-transfer method (HTM) is also perfectly suited for this analytical task. The heat-transfer method was published recently in the context of DNA mutation analysis and the detection of neurotransmitters using molecularly imprinted polymers as synthetic receptors [2,3].
To adapt the method for cell recognition, a thermal current is generated passing through the cell-imprinted PU layer. This can easily be accomplished by keeping the sensor chip at a fixed temperature (e.g. 37°C) and by monitoring the resulting temperature inside the liquid compartment of the sensor setup above the imprinted layer [4]. Binding of the cells to the imprints causes a substantial, precisely measurable increase of the heat-transfer resistance Rth at the interface between the solid PU layer and the liquid cell medium. Currently, we achieve a detection limit in the order of 10,000 cells per ml and, after lysis and releasing of the bound cells, the sensor chips are reusable for at least 6 subsequent measuring cycles. Interestingly enough, also cells with identical dimensions such as MCF-7 and Jurkat cells can be perfectly discriminated by applying a mild shear flow, proving that the recognition of a specific cell type depends equally on geometrical and chemical complementary. Reference tests with a mixed cell population of a healthy volunteer confirmed that this new cell-identification method is insensitive to false-positive signals.
[1] O. Hayden et al., Sensors and Actuators B - Chemical 91, 316-319 (2003).
[2] B. van Grinsven et al., ACS Nano 6, 2712-2721 (2012).
[3] M. Peeters et al., Anal. Bioanal. Chem. DOI 10.1007/s00216-013-7024-9 (2013).
[4] Patent application EP 13157264.6: "Heat-transfer-resistance-based analysis of bioparticles".

Burn injuries, trauma, and surgical procedures result in complex skin wounds that can lead to inflammation, infection, and scarring. Management of complex wounds remains a critical challenge for the medical community, because effective, localized treatments that provide sustained drug release are limited. Solid lipid nanoparticles (SLNs) are a promising platform for delivering therapeutics topically to skin due to their biocompatibility, stability, ability to accept a wide range of active ingredients, and, most importantly, their ability to penetrate the epidermis. In this study, we synthesized sub-10 nm SLNs using the Phase Inversion Temperature (PIT) method. The one-pot synthesis occurs in less than one minute by simply cooling the mixture to room temperature while vortexing. As a model system, we demonstrated the delivery of fluorescent dyes to a porcine skin model. Also demonstrated was the delivery of an immunosuppressant drug, rapamycin, through approximately two-thirds of the porcine epidermis. A systematic Design of Experiments (DOE) approach investigated the role of synthesis parameters on SLN form and function. Particle size and polydispersity were measured as a function of dye solubility, phase inversion temperature, melting point, and latent heat of melting. The measurements indicate that the particle size distribution is largely invariant to these processing parameters, the implication of which is that SLNs synthesized in this fashion can serve as a vehicle for the delivery of a wide range of therapeutic compounds. Inspection of the porcine skin samples further demonstrates the penetration of dyes across the entire viable epidermis. For comparison, the control samples exhibited no penetration due to their spare solubility in water.

Endowing polymeric micro- and nanoparticles with functionality - whether optical, magnetic, acoustic, or otherwise - is a cornerstone of a wide range of medical applications in bioimaging, sensing, and drug delivery. The ability to multiplex different functionalities in the same particle is of paramount importance in multi-modal imaging and guided surgery1. The controllable incorporation of multiple active agents within a single particle remains to date a major challenge. In typical top-down approaches, the polymer is impregnated with the active agents, which limits the combinations of functionalities accessible. Bottom-up approaches, on the other hand, are complicated by the necessity of synthesizing multifunctional particles in a sequential manner.
Here we address this challenge by developing a general methodology for controlling the geometric distribution of multiple functionalities within the three-dimensional volume of a polymeric micro- or nanoparticle. We make use of a recent strategy we have developed2-4 for the scalable production of size-tunable particles based on fluid instabilities induced thermally at the heterogeneous interfaces within a multimaterial fiber. We first construct a macroscopic ‘preform&’ consisting of a cylindrical core made of the particle material of choice surrounded by a cylindrical polymer cladding matrix. The preform is heated and drawn into an extended fiber with reduced transverse scale. The drawn fiber is then thermally treated to induce the Plateau-Rayleigh capillary instability (PRI), which leads to the transformation of the intact core into a necklace of spherical particles held in isolation from each other, which are then harvested by selective dissolution of the sacrificial cladding. Our approach here to produce multifunctional particles is to strategically design the preform by doping different axial segments of the core with active materials of choice that endow it with the desired functionality. Specifically, we identify a wide range of functionalities compatible with this process, including optical (fluorescence from organic dyes or quantum dots), magnetic, or Raman functionalities incorporated into particles derived from a wide range of biocompatible and biodegradable polymers. Next we demonstrate that each one of these functionalities may be confined geometrically within any desired radial or azimuthal segment of the particle. Finally, we combine these two capabilities by fabricating particles with up to four distinct functionalities controllably distributed within the particle volume.
Our in-fiber approach paves the way to three-dimensional digital design of multi-functional polymeric particles for nano-medicine, where the desired functionalities and their geometric placement within the particle are controlled by simply changing the structure of a macroscopic preform from which the fiber is drawn.
References:
1. M.F. Kircher, A. de la Zerda, J.V. Jokerst, C.L. Zavaleta, P.J. Kempen, E. Mittra, K. Pitter, R. Huang, C. Campos, F. Habte, R. Sinclair, C.W. Brennan, I.K. Mellinghoff, E.C. Holland, and S.S. Gambhir, “A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle,” Nature Medicine, 18, 829-834 (2012).
2. S. Shabahang, J. J. Kaufman, D. S. Deng, and A. F. Abouraddy, “Observation of the Plateau-Rayleigh capillary instability in multi-material optical fibers,” Appl. Phys. Lett. 99, 161909 (2011).
3. J. J. Kaufman, G. Tao, S. Shabahang, E.-H. Banaei, D. S. Deng, X. Liang, S. G. Johnson, Y. Fink, and A. F. Abouraddy, “Structured spheres generated by an in-fibre fluid instability,” Nature 487, 463-467 (2012).
4. J. J. Kaufman, R. Ottman, G. Tao, S. Shabahang, E.-H. Banaei, X. Liang, S. G. Johnson, Y. Fink, R. Chakrabarti, and A. F. Abouraddy, “In-fiber production of polymeric particles for biosensing and encapsulation,” Proc. Natl. Acad. Science USA, submitted (2013).

The alternation of positively and negatively charged groups by adsorption can yield nanometer scale thin films that present charged groups in dense layers on surfaces. We use this simple water based approach to generate finely tuned release surfaces that can release small molecule, proteins, nucleic acids and other biologic drugs over sustained time periods, and with significant control of release characteristics. Using as few as 3 or 4 bilayers to tens of layers, we can generate thin films with demonstrated therapeutic efficacy over a broad range of efficacy. Furthermore, the use of ultrathin barrier layers in between sets of multilayers can yield staged release of therapeutics from large, macroscopic surfaces such as biomedical devices and implants to microstructured arrays and nanoparticle surfaces. Examples that will be discussed include the release of growth factors and siRNA from scaffolds for tissue engineering and wound healing applications with release sustained over multiple days to weeks, and microneedles for transdermal delivery of DNA vaccines. Furthermore, key collaborations have led to demonstrations of the potential to translate this highly adaptable technique to mass-scale processing methods and rapid assay capabilities. Means of adapting these methods toward new, potentially disruptive applications and the potential of these methods for commercial development will also be discussed.

Pure titanium and titanium alloys are widely used in orthopedic and dental implants because on their desirable mechanical properties and biocompatibility. However, these materials suffer from electrochemical corrosion when implanted in the mouth, which induces release of metallic particles from the titanium surface, promoting inflammation around the implant, and implant failure, which was associated to the presence of macrophages in peri-implant soft tissue [1]. A novel ultrananocrystalline diamond (UNCD) thin film developed and patented in recent years exhibits excellent bionert/biocompatible properties and strong resistance to chemical attach as coating for implantable biomedical devices, as recently shown for encapsulation of a Si microchip implantable in the eye to restore sight to people blinded by retina degeneration [2]. Therefore, we are investigating UNCD coatings for dental implants, because high resistance to chemical corrosion strong mechanical resistance and biocompatibility. The UNCD coating acts as a protective barrier between the implant and the biological environment, which would prevent the release of metallic ions from titanium implants into the body. In order to assess the potential of UNCD as a coating for titanium dental implants, we investigated the osteointegration rate of titanium, UNCD-coated tinanium, and UNCD/W-coated titanium implants using the rat diaphyseal tibia as a model to study osteointegration, relevant to dental implants. Titanium laminar implants (Implant-Vel, Buenos Aires, Argentina) were coated with the three type of layers indicated above and used to study the osteointegration rate in the diaphysis of the tibia bone of rats, simulating the alveoli bone of the mouth cavity. All animal experiments were carried out following NIH guidelines for the care and use of laboratory animals. Macroscopic observations revealed that wound healing was occurring satisfactorily in all specimens and all implants remained in situ in the diaphyseal areas. Subsequently, the implants were extracted after 14 days of implantatin and histological sections were prepared and analyzed via Optical Microscopy. The optical pictures showed laminar bone formation on the surfaces of the three groups studied. Histological results, from short term experiments, indicated that UNCD and UNCD/W coatings of dental implants are well tolerated by the surrounding tissue and did not elicit any inflammatory reactions, similarly to Ti implants without coating. However, the corrosion observed in long-term Ti dental implants and the total inertness of UNCD coatings, as shown in long term in vivo animal eyes studies, indicate that UNCD-coated dental implants will perform much better than bare Ti implants. Work is in progress to perform long term in vivo studies of UNCD-coated dental implants.
1. D. Olmedo et al, Imp Dent vol 12 (2003) 75.
2. X. Xiao / O. Auciello et al., J. Biomedical Materials 77B (2006) 273.

Carbon nanotubes (CNTs) have recently attracted considerable interest in a variety of disciplines, amongst which is biomedicine. Tunable mechanical, electrical, and chemical properties of CNTs-through functionalization-make them viable actors in a diversity of scenarios within the biomedical sciences: from neuroscience to tissue engineering and towards the treatment of medical conditions. Indeed, CNTs ability to promote neural stem cell differentiation has been recently demonstrated, and they have been proposed as targeted drug carriers, either through sidewall functionalization, or by encapsulating drug molecules within the internal channel of the tube.
Currently, a thorough understanding of bonding and interaction dynamics between CNTs and functionalizing agents is an elusive building block in this bottom-up nanomaterial assembly paradigm. It is through those interfaces that surface modification is accomplished and through those interaction dynamics how functionality develops. Further, in a biomedical scenario, modified CNTs ultimately interact with biomolecules. Details of the latter interaction appear equally elusive. As a consequence, CNT biocompatibility and cytotoxicity remain under considerable debate. Health concerns arise from nanometre scale dimensions, potentially allowing undesirable penetration of cells or subcellular structures, and from potential effects from impurities along graphitic walls, and growth residuals within CNTs.
Synchrotron spectroscopic studies have been conducted on CNTs dispersed in biomedically relevant solvents to elucidate the ultimate interface between CNTs and biomolecules. CNTs coated with commonly used dyes have been examined to understand the bonding dynamics and resulting surface modification. Our spectroscopy results suggest successful surface modification. Several models are proposed to explain interactions between CNTs and solvent/dye, including non-covalent CH-π interactions. High-resolution electron microscopy imaging supports spectral findings, showing partial conformational arrangement of dye molecules and residual solvent on CNTs.

Biocompatible integrated nanopattern requires the fabrication of appropriately designed nanomatrix for high sensitivity homogenous assays, which are capable of ultimately mimic the physiological environment. Recently, we observed that the nanowell arrays (NWA) can enhance electrochemical responses for the binding event of molecule to molecule through the binding site control of captured molecule. By using these characteristics, we could detect small molecule such as liposome or aptamer with infinite concentration by square wave voltammetry.
In this report, we describe a highly sensitive impedimetric immunosensor based on NWA for detection of stress-induced-phosphoprotein-1 (STIP-1), which is an ovarian cancer biomarker. 57 uniformed NWA using an entire 6 inch Si wafer by one process was fabricated to develop NWA sensor through the cost effective combination of high-throughput nano pattern. We obtained a single NWA sensor with 21 x 10 mm2 area, and the NWA structure had 500 nm diameter with 200 nm interwell spacing. Quantification of antigen binding to the immunoaffinity layer of the NWA was performed by impedance analysis. We calculated the change of the charge transfer resistance (Rct) on 0.1-100 Hz frequency range and also estimated the limit of detection (LOD). LOD of the NWA was below 10 pg/mL for the STIP-1, representing a 100-fold improvement, compared with bare electrodes without an NWA. In addition, the LOD of NWA (7.4 pg/mL) is much lower than the cut off value.
The developed impedimetric NWA immunosensor obtained the high sensitivity/selectivity, compared with conventional immunosensors and can be suggested for label free detection without non-specific binding by means of the selective docking of immobilized antibodies.

In previous studies we have shown, both in skin and peripheral nerves, that severe wounds in adults that injure the connective tissue (stroma wounds) heal by regeneration, rather than by contraction and scar formation, provided that an appropriately structured collagen scaffold has been grafted on the wounds. The studies were based on use of libraries of collagen scaffolds comprising members with closely related structural features (pore size, degradation rate, surface chemistry), including scaffold members that had strong or weak regenerative activity. It was shown that, in both organs, scaffolds with demonstrated regenerative activity also inhibited strongly the process of wound contraction. Inhibition of contraction was associated in both organs with a sharp decrease in the density and loss of axial orientation of contractile cells (myofibroblasts). However, it has not been clear so far how an active scaffold affects contractile cell function in this strongly inhibitory manner.
Recent studies at the molecular scale have been based on a new optical method that quantifies the density of adhesion ligands for the two major collagen-binding integrins, α1β1 and α2β1, on the surface of 3D matrices such as porous collagen scaffolds. The method is an in situ binding assay that utilizes fluorescently labeled recombinant proteins that emulate α1β1 and α2β1. The binding specificity of these fluorescent markers is validated by several biochemical assays. Results from this assay provide the first in situ quantification of the surface chemistry of porous collagen scaffolds and show that scaffolds known to be regeneratively active have a much higher density of ligands compared to inactive scaffolds.
Contact with the biologically active surface of the scaffold clearly downregulates the expression of contractile phenotypes of cells during healing. One possible mechanism of scaffold activity is downregulation of contractile phenotypes due to integrin-specific adhesion of cells on the surface. Another possible mechanism is cell-surface interaction that regulates the expression of cytokines involved in contractile cell differentiation. The latter is supported by another recent set of data, which show that active collagen scaffolds are associated with downregulation of pro-contractile TGFβ1 and upregulation of TGFβ3.
Although the study is continuing the available evidence strongly supports a theoretical mechanism at the molecular scale for the observed organ regeneration in adults in the presence of the collagen-based scaffold. The available regeneration data are explained best by hypothesizing that a biologically active surface dramatically modifies the adult healing process via specific interaction with cells involved in healing. Specific integrin-ligand binding interactions lead to a bypass of normal healing in stroma wounds and direct instead the healing process toward regeneration.

Freeze-casting, a process that uses the solidification of a liquid carrier such as water for templating, has in recent years been discovered as a route to create highly porous hybrid materials with complex, hierarchical architectures. Freeze-casting is highly attractive for the manufacture of materials for applications that range from scaffolds for tissue engineering to structures for energy generation because it offers several advantages over other techniques. One advantage is that all classes of materials---polymers, ceramics, metals and their composites---can be shaped with it; another is that materials can be processed with benign, biocompatible liquid carriers; a third is that the resulting hierarchical microstructures can be carefully controlled by both the physical and chemical properties of the components used and the processing parameters such as the cooling rate; finally advantage can be taken of component self-assembly during solidification. The amount, type, size and geometry of the particles and the type of liquid carrier determine the slurry&’s viscosity and amount of sedimentation as well as the slurry&’s thermal properties and freezing behavior. In combination with the freezing front velocity and additives, they also determine pore connectivity and morphometry. The thickness and spacing of the cell walls and the size and the number of the material bridges between them can be controlled, as can be the cell wall&’s bulk and surface properties, and thus the materials interaction with a second phase. This is important for the manufacture of composites by infiltration or for the optimization of the interaction between scaffold and native tissue in biomedical applications. As a result, the freeze-casting process is ideally suited for the custom-designed manufacture of complex, hybrid materials with that emulate in synthetic materials multi-level hierarchical composite structures, which are thought to be the origin of the mechanical property amplification which is frequently observed in biological materials and highly desirable in biomaterials.

Repair and replacement of skeletal tissue by synthetic materials remains one of the great challenges in healthcare today. Although metallic orthopedic and dental implants enjoy widespread use and success in modern clinical practice, they remain poor substitutes for the tissues they are intended to replace. New design concepts and fabrication techniques are urgently needed to develop novel scaffolds for bone regeneration. In particular, more research is required to uncover the relationships linking composition and materials architecture at multiple-length scales with macroscopic mechanical behavior and the capability for osteogenesis. In this presentation we describe attempts to develop a range of bone- and nacre-like structural materials using freeze-casting technique, which utilizes the intricate structure of ice to create hybrid materials with complex lamellar and/or mortar and brick structures modeled across several length-scales. The challenge is to develop a new generation of implant materials that will combine the advantages of both ceramics and polymers, specifically their inertness, with a mechanical response comparable to those of implant alloys. The unique properties of ceramic materials, including their outstanding corrosion resistance and excellent aesthetics, make them appealing candidates for many implant applications. However, their inferior mechanical properties, particularly fracture toughness, until now have hampered their commercial use, especially in load-bearing situations. This is certainly a problem when designing porous implants or implants with rough and porous surfaces for better osseointegration. While the porosity will improve bone regeneration, increased porosity can easily result in diminished strength and fracture at relatively low loads. Through new fabrication technologies, these limitations may be overcome. Our results show ceramic-polymer hybrid materials with toughness well in excess of those expected from a rule of mixtures construction. The architecture and properties of the synthetic materials are compared to their natural counterparts in order to identify the mechanisms that control mechanical behavior over multiple dimensions and propose new design concepts to guide the synthesis of hybrid/hierarchical structural materials with unique mechanical responses. The ultimate goal is to produce materials and therapies that will bring state-of-the-art technology to the bedside and improve quality of life and current standards of care. This work was supported by the National Institute of Health under grant number NIH/NIDCR R01 DE015633

Two photon polymerization is a rapid prototyping technology that uses spatial and temporal overlap of photons for photopolymerization of photosensitive resins within well-defined volumes. One major advantage of photon polymerization is that this process can be done in a conventional environment; no specialized facilities such as cleanroom facilities are needed. Many types of photosensitive resins, including polymers and inorganic-organic hybrid materials, have been processed into structures for medical device applications using two photon polymerization. We are using two photon polymerization to prepare microneedles, tissue engineering scaffolds, and other medical devices. In addition, we are using an indirect rapid prototyping approach involving micromolding to prepare small-scale medical devices.

Acetone is one of the most abundant volatile compounds in the human breath and might be important for monitoring diabetic patients [1, 2]. Here, a portable acetone sensor [3] consisting of flame-made, nanostructured, Si-doped WO3 sensing films [4] was used to analyse the end tidal fraction of the breath (collected in Tedlar bags) from eight healthy volunteers after overnight fasting (morning) and after lunch (afternoon). After breath sampling, the gaseous components were also analysed by proton transfer reaction time-of-flight mass spectrometry (PTR-TOF-MS), and each person&’s blood glucose level was measured. The portable sensor accurately detected the presence of acetone with fast response/recovery times (< 12 s) and a high signal-to-noise ratio. Statistical analysis of the relationship between the PTR-TOF-MS measurements of breath gases (e.g., acetone, isoprene, ethanol and methanol), sensor response and the blood glucose level was performed for both sampling periods. The best correlations were found after overnight fasting (morning): in particular, between blood glucose level and breath acetone (Pearson&’s 0.98 & Spearman&’s 0.93). Whereas the portable sensor response correlated best with the blood glucose (Pearson&’s 0.96 & Spearman&’s 0.81) and breath acetone (Pearson&’s 0.92 & Spearman&’s 0.69).
[1] Cao W Q and Duan Y X 2006 Breath analysis: Potential for clinical diagnosis and exposure assessment, Clin. Chem. 52 800-811.
[2] Pleil J D and Lindstrom A B 1997 Exhaled human breath measurement method for assessing exposure to halogenated volatile organic compounds, Clin. Chem. 43 723-730.
[3] Righettoni M, Tricoli A, Gass S, Schmid A, Amann A and Pratsinis S E 2012 Breath acetone monitoring by portable Si:WO3 gas sensors, Anal. Chim. Acta. 738 69-75.
[4] Righettoni M, Tricoli A and Pratsinis S E 2010 Si:WO3 Sensors for Highly Selective Detection of Acetone for Easy Diagnosis of Diabetes by Breath Analysis, Anal. Chem. 82 3581-3587.

Capture and isolation of proteins, flowing cells and particulates from body fluids has enormous implications in diagnosis, monitoring, drug testing, and sorting for cell-based therapeutics. Devices that are capable of mimicking the biological environment, responding to chemical cues, without employing energy-consuming electrical or magnetic fields as in existing systems, would be highly desired for point-of-care clinical applications. Here we utilize adaptive, integrative materials to sort biologically relevant target molecules from a mixture solution in a single device and demonstrated real-time and non-disruptive sorting, concentrating and purification. We show the ability of our adaptively reconfigurable dynamic system, which utilizes aptamer-functionalized microstructure tips embedded in a responsive hydrogel material within a biphasic microdevice, to capture and release target biomolecules from solution in constant laminar flow. Our chemo-mechanically driven system uniquely couples chemo-mechanical energy transduction with the re-configuration of the attached capturing entity, affecting the binding characteristics of biomolecules and allowing for the separation of specific molecules from a solution mixture in a smoothly concerted action by the integrated material. With the enzyme thrombin, we show that simply by recycling the solution mixture through our dynamic microfluidic system, we can obtain a capture and release efficiency of 95.5% of the target molecule. Further investigation with cancer cells and bacteria opens up the versatility of our “smart” integrative material for applications in biomolecule separation, purification, concentration, and isolation. Further, with the high stability and specificity for a diverse array of target molecules by functionalization with various capturing entities, such a “smart” device can readily find use as a biosensor, a simple diagnostic tool of diseased indicators in solution, or even as a tool to quantitatively determine the extent of chirality achieved in the synthesis of a complex molecule.

The accurate annotation of human pathological tissues and fluids for early detection, prognosis and therapeutic response prediction is a clinico-biological priority. However, the intrinsic complexity of the samples limits the detection of disease markers present in trace amounts within a background of abundant and non-relevant molecules. Additionally, the detection of low abundant markers and low molecular weight (LMW) species remains a critical challenge due to experimental variability, long sample handling procedures, stability during sample processing, and generation of artifacts as a consequence of unreliable experimental procedures. Standardized and adequate sample preparation strategies and reliable fractionation approaches remain an extraordinary technical challenge. To address these bottlenecks that have impeded the use of mass spectrometry (MS) in clinical biomarker discovery, we have developed a fast, efficient, and reliable system based on nanoporous organosilicates to improve the detection of specific metabolites.
Nanoporous SiOCH thin films with tunable features at the nanoscale were fabricated on Si substrates by plasma enhanced chemical vapor deposition. The porosity was introduced via the co-deposition of an organo-silicon matrix and thermally labile organic species that are finally removed by a UV assisted thermal annealing. SiOCH with 0 - 40% porosity were obtained, the porosity being open and interconnected with mean pores diameters 1.3 nm. Due to their crosslinked Si-O-Si backbone with methyl groups bonded to silicon, these materials are highly hydrophobic whatever the porosity. By using different plasma treatments various hydrophilic behaviors, porosity, pore sizes, and chemical compositions were obtained. Strong oxidizing plasmas led to a sharp carbon depletion resulting in a strong decrease of the contact angle and porosity. Reducing plasmas has a softer effect on the carbon depletion and lead to an increase of the porosity and of the pore radius up to 2 nm. He plasma have a limited impact on the bulk material.
These different layers were used for selective recovery and enrichment of LMW metabolites from biological fluids. Using MS we demonstrated the correlation between the physicochemical characteristics of the SiOCH films and the efficiency of specific metabolites harvesting from urea samples. We further evaluated the selective harvesting capacity of our surfaces for tissue analysis. Using MS profiling and FTIR spectrometry, we clearly demonstrated the feasibility of our technique to discriminate pathological from normal samples. This immediate tissue profiling approach would be a useful complement to the extemporaneous anatomopathological diagnosis supporting the surgeon&’s decisions at the bedside.
Our study demonstrates that the ability to tune the physicochemical properties of nanoporous SiOCH film can provide enormous enhancement of low abundant disease marker discovery from complex biological fluids and tissues.

Magnetic medical microrobots have the potential to revolutionize current methods of diagnosis and treatment of many diseases [1]. These microagents can be wirelessly controlled to perform tasks such as drug delivery, surgery or sensing in confined regions of the body , thus improving clinical efficiency and reducing the complexity of certain therapies[2]. One method of enhancing the performance of these microdevices consists of integrating materials with intrinsic functionalities into the fabrication process. To investigate this approach, we coated our microrobots with chitosan, a smart polysaccharide that changes its solubilized state to a gel when the surrounding pH overcomes its pKa=6.3. By electrolyzing chitosan aqueous solutions, a pH gradient can be generated from the cathode surface due to the hydrogen evolution reaction [3, 4]. This local condition results in the controlled deposition of chitosan gels on the substrate. Electrodeposition was employed to functionalize cylindrical shaped microdevices with drug loaded chitosan matrices for targeted drug delivery. The process was investigated and optimized to provide a homogeneous coating. The resulting gels were analyzed in terms of porosity, pH sensitivity and drug release in simulated pathological and physiological conditions. Multiple modifications of the layer properties could be achieved by simply dipping the matrices into different ionic solutions, such as sodium hydroxide (NaOH), or sodium trypolyphosphate (TPP), or by slightly modifying the chitosan electrolyte[5]. These steps provide additional control on the design of the release kinetics of the embedded drugs.
[1] B. J. Nelson, I. K. Kaliakatsos, and J. J. Abbott, "Microrobots for Minimally Invasive Medicine," Annual Review of Biomedical Engineering, Vol 12, vol. 12, pp. 55-85, 2010.
[2] M. P. Kummer, J. J. Abbott, B. E. Kratochvil, R. Borer, A. Sengul, and B. J. Nelson, "OctoMag: An Electromagnetic System for 5-DOF Wireless Micromanipulation," IEEE Transactions on Robotics, vol. 26, pp. 1006-1017, Dec 2010.
[3] Y. Cheng, X. L. Luo, J. Betz, S. Buckhout-White, O. Bekdash, G. F. Payne, W. E. Bentley, and G. W. Rubloff, "In situ quantitative visualization and characterization of chitosan electrodeposition with paired sidewall electrodes," Soft Matter, vol. 6, pp. 3177-3183, 2010.
[4] S. T. Koev, P. H. Dykstra, X. Luo, G. W. Rubloff, W. E. Bentley, G. F. Payne, and R. Ghodssi, "Chitosan: an integrative biomaterial for lab-on-a-chip devices," Lab on a Chip, vol. 10, pp. 3026-3042, 2010.
[5] K. M. Gray, B. D. Liba, Y. F. Wang, Y. Cheng, G. W. Rubloff, W. E. Bentley, A. Montembault, I. Royaud, L. David, and G. F. Payne, "Electrodeposition of a Biopolymeric Hydrogel: Potential for One-Step Protein Electroaddressing," Biomacromolecules, vol. 13, pp. 1181-1189, Apr 2012.

Ultrasensitive biosensing is one of the main driving forces behind the dynamic research field of plasmonics. One may divide the experimental techniques into single nanoparticle schemes, where the ultimate limit of detection is single molecules, and studies of arrays of nanostructures that are not individually resolved but may instead form so-called metasurfaces with specific transmission and reflection coefficients, where the aim is rather to detect ultra-low surface coverage.
The sensitivity of single nanoparticle plasmon spectroscopy can be greatly enhanced by enzymatic amplification of the refractive index footprint of individual adsorbed protein molecules1. However, the plasmonic response induced by a single enzyme can vary for several reasons. Here, we discuss how such inhomogeneities affect the quantification of molecules bound to a single nanoparticle, based on simulations and measurements of large arrays of individual gold nanoparticles2. Although the intrinsic single-molecule sensitivity of the technique is confirmed, the exact number of adsorbed molecules per single particle was not resolvable. The main sources of uncertainty come from variations in sensitivity across the surface of individual particles and between different particles.
The more macroscopic metasurface-based sensing may benefit from using experimental techniques that measure parameters with larger refractive index sensitivity. The phase of reflected light changes very rapidly compared with the intensity as the Fresnel reflection coefficient of an interface traverses zero. The rapid phase jump occurs, for example, at the Brewster angle of a dielectric interface or from a carefully designed array of plasmonic nanoparticles3, 4. Here, we show and discuss the phase response to local refractive index changes upon molecular adsorption and how this compares to the more standard spectroscopic interrogation methodologies5.
1. Chen, S.; Svedendahl, M.; Van Duyne, R. P.; Kall, M. Nano Letters 2011, 11, (4), 1826-1830.
2. Chen, S.; Svedendahl, M.; Antosiewicz, T. J.; Käll, M. ACS Nano 2013.
3. Svedendahl, M.; Käll, M. ACS Nano 2012, 6, (8), 7533-7539.
4. Svedendahl, M.; Johansson, P.; Kall, M. Nano Letters 2013, 13, (7), 3053-3058.
5. Svedendahl, M.; Käll, M. In Manuscript

PEOlated silica-shell magnetic nanobeads with an inner core of superparamagnetic iron oxide nanocrystals and size of ~50 nm have been successfully prepared via interfacial template condensation at room temperature and near-neutral pH. The core-shell structure and the size of the nanobeads were verified by TEM and DLS. The surface of nanobeads was decorated with carboxylic acid groups to allow immobilization of highly specific ligands such as proteins, peptides, carbohydrates or other target specific molecules through amide-bond formation. In addition, the exterior surface of the nanobeads was covered by a layer of hydrophilic PEO chains instinctively, which endow the nanobeads to be highly colloidally stable in aqueous medium, allowing fast binding kinetics and short labeling procedures without the trouble of dealing with clumps.

We describe a rapid, sensitive, and label-free method to detect interferon -gamma (IFN-gamma), which is a biomarker of latent tuberculosis infection (LTBI). IFN-gamma is detected by measuring the capacitance change caused by the binding of IFN-gamma to anti-IFN-gamma antibody immobilized on the surface of an anodized aluminum oxide (AAO)-based capacitive sensor. The concentration of IFN-gamma in clinical samples was measured using the AAO-based capacitive sensor and compared with the results of the commercial QuantiFERON-TB Gold ELISA kit. Similar results were obtained with the two measurement strategies, demonstrating the applicability of the AAO-based capacitive sensor to the diagnosis of LTBI.

Organisms are intricately structured to provide life&’s functions. Nature provides the inspiration for engineering structural- and processing-design criteria for materials and systems. With a growing understanding of the molecular processes involved, bio-inspired strategies are increasingly explored to obtain hierarchical structures with unique properties and bio-enabled materials. The challenges in biomimetic strategies include controlling the self-organization at a molecular level and thus to provide control over the biological and inorganic interfaces under environmentally benign and biologically compatible conditions. In biological systems, proteins conduct various functions through their unique molecular recognition, self-assembly and templating properties. In addition to their role in biomineralization, proteins perform a wide spectrum of additional functions ranging from catalysis to self-repair. Our impetus in research has been to decode the peptide-material interactions, and using these foundations to develop highly organized and multifunctional hybrid structures. Using combinatorial biology, and rational design principles, we design, synthesize and utilize material-binding short peptides as building blocks in a wide range of applications. Building upon the modularity of protein domains, we design single to multi-functional peptides or recombinant proteins and enzymes. Here, we will describe bio-enabled, single step processes as a key route in simplifying the assembly and synthesis of multi-material nano-systems in environmentally benign conditions. Our specific examples will include the design and use of peptides conjugated to functional enzymes and proteins. We will next demonstrate their self-organization on a variety of materials in forming controlled assemblies from single to multiple layer organization. Using an array of multifunctional molecular units, we address different challenges including designing organic-inorganic interfaces, biological surface functionalization including bioactive and antimicrobial surfaces, multifunctional protein/peptide based hybrid nanoprobes for sensing and cell targeting as well as bionano fabrication of inorganic materials. The presentation will provide an overview of peptide based functional molecular materials and the approaches carried out in our collaborative groups. The project supported by GEMSEC-UW, an NSF-MRSEC at the UW, NSF Biomater, NIH.

Last several decades nanofibers demonstrated themselves as a great drug delivery system which can protect the therapeutic agent from in vivo environment and release them in sustain fashion for the extended period of time. Considering these properties of the nanofibers in this study we prepared a nanofiber sheets incorporated anticancer compound for prevention of the locoregional cancer reoccurrence and metastasis for highly malignant diseases such as pancreatic, gastric, colorectal cancers and etc. These diseases have very high rate of recurrence after curative resection. The nanofiber sheets that we propose here will be beneficial for the anastomosis healing, serving as a dressing with good mechanical properties. In addition, nanofiber sheets will deliver the natural herbal anticancer compound, Taiwanin A, that specifically targets cancer cells suppressing their proliferation, but it will have least cytotoxicity toward neighboring normal cells and will exhibit no systemic toxicity. In this study the nanofiber sheets carrying Taiwanin A were prepared from 12.5 w/v% polyurethane (PU, MW = 60,000) dissolved in DMF:THF (1:9) by using simple electrospinning process. We obtained ultrafine nanofibers with diameter of approximately 520 nm. SEM observations showed incorporation of the drug slightly increased the fiber diameter to 530 nm but did not affect surface morphology. Different cell viability tests confirmed that nanofiber sheets can inhibit ~73% and ~92% of cancer cells at 24 hrs and 48 hrs, respectively. Our study showed that PU nanofiber sheets have a good compatibility with drug Taiwanin A and it also demonstrated high cytotoxicity toward cancer cells releasing the drug in sustained fashion indicating these nanofiber sheets can serve as a good remedy for prevention of the locoregional cancer recurrence.

Amorphous nanosilica is the most common nanomaterial found nearly everywhere like in varnishes, paints, as drug excipient and even to cutting edge applications in medicine and biology. Understanding, however, the potential toxicity mechanisms of nanosilica has gained increasing interest over the past years while the need of studies with standardized materials is stressed frequently [1]. Here the cytotoxicity induced on mammalian cells, RAW 264.7 murine macrophages [2], by commercial flame-made nanosilica with 7 - 30 nm average primary particle diameter (by nitrogen adsorption and transmission electron microscopy) is investigated systematically. Ultrasonication at various intensities is applied to prepare aqueous suspensions of such nanosilicas with similar hydrodynamic size distributions, as determined by dynamic light scattering. Cell viability after 24 hour treatment with these suspensions is evaluated by a tetrazolium salt reduction assay [2]. A focus is on associating the cytotoxicity of nanosilica with the relative population of strained three-membered siloxane rings (3MRs) formed at high temperatures and their potential to generate hydroxyl radicals in aqueous suspensions [3]. Raman spectroscopy is used to determine the 3MR population at 605 cm^-1 [4], while free radical release is evaluated by electron paramagnetic resonance spectroscopy using a spin trapping technique. For the same mass concentration, nanosilicas of larger average primary particle size induce higher reduction in cell viability than the smaller ones in contrast to wet-made non-aggregated silica nanoparticles [5]. On the basis of the same surface area concentration, the same trend of increasing cell viability with decreasing primary particle size is even more pronounced. This strongly indicates a surprising difference in the surface properties and synthesis conditions of the investigated nanosilicas.
1. Napierska D, Thomassen LCJ, Lison D, Martens JA, Hoet PH. The nanosilica hazard: another variable entity. Particle and Fibre Toxicology. 2010;7:39.
2. Pratsinis A, Hervella P, Leroux J-C, Pratsinis SE, Sotiriou GA. Toxicity of silver nanoparticles in macrophages. Small. 2013. DOI: 10.1002/smll.201202120.
3. Zhang HY, Dunphy DR, Jiang XM, Meng H, Sun BB, Tarn D, Xue M, Wang X, Lin SJ, Ji ZX, Li RB, Garcia FL, Yang J, Kirk ML, Xia T, Zink JI, Nel A, Brinker CJ. Processing pathway dependence of amorphous silica nanoparticle toxicity: Colloidal vs Pyrolytic. Journal of the American Chemical Society. 2012;134:15790-15804.
4. Vaccaro G, Agnello S, Buscarino G, Gelardi FM. Thermally induced structural modification of silica nanoparticles investigated by Raman and infrared absorption spectroscopies. Journal of Physical Chemistry C. 2010;114:13991-13997.
5. Napierska D, Thomassen LCJ, Rabolli V, Lison D, Gonzalez L, Kirsch-Volders M, Martens JA, Hoet PH. Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells. Small. 2009;5:846-853.

Since methicillin-resistant Staphylococcus aureus (MRSA) was firstly isolated in 1961, many patients have been frozen with fear by life-threatening infection by multi-drug resistant bacteria in hospital. Although various antibiotics have been developed as a countermeasure of existing antibiotics such as methicillin or vancomycin, nitric oxide (NO) has been recently highlighted due to its broad spectrum of antibacterial activity and biocompatibility. It is reported that NO and its intermediates, such as nitrogen dioxide (NO₂#9679;), dinitrogen trioxide (N₂O₃) and peroxynitrite (ONOO-), contribute to host defense system against pathogens via DNA deamination and peroxidation of membrane lipid. Especially, the NO-mediated antibacterial agents have been considered as the next generation of antibacterial drugs because NO is a naturally occurring antibiotic synthesized by nitric oxide synthase (NOS) in vivo.
Therefore, many researchers have tried to develop materials for delivering exogenous NO into the bacterial-infected sites by using NO-donors which can store and release nitric oxide under proper conditions. Among a variety of NO-donors such as S-nitrosothiol, 1-substituted diazen-1-ium-1,2-diolates (NONOates), nitrite/nitrates, metal-complex and so on, NONOates, which can be formed at secondary amine, has been attracted great attentions due to its simple synthetic method and spontaneous release in physiological condition.
Herein, we would like to report NO-releasing antibacterial agent composed of biocompatible Pluronic F68, NONOates and BPEI as a precursor of NONOates. The successful synthesis was confirmed by ¹H NMR, FT-IR, elemental analysis, UV/vis spectra, and Zeta potential. The amount of NO release was monitored by Sievers 280i Nitric Oxide Analyzer. The bactericidal activity of F68-BPEI-NONOates and negative controls was evaluated against Gram negative Escherichia coli, Gram positive Staphylococcus aureus and MRSA through the colony forming unit (CFU) assay and the Live/dead assay. F68-BPEI-NONOates showed not only better antibacterial activity aganist all types of bacteria but also better biocompatibility than controls. In addition, through the TEM images, we could conclude that high antibacterial activity of F68-BPEI-NONOates is originated from the ability of NO which induces the destruction of bacteria membrane.

The surface nitric oxide (NO) delivery garners paramount importance in various biomedical applications such as anti-restenosis, wound healing, anticancer, and antibacterial. Despite numerous reports for NO-releasing materials which include polymeric micelles, inorganic nanoparticles, dendrimers, peptides, hydrogels, and xerogels, methods to immobilize the NO-releasing materials are under developed. Most previous approaches utilize direct physical adsorption of NO-releasing materials or adsorption of matrices that contain NO-releasing materials. However, the stability of the adsorbed materials in physiological conditions is the major problem of these types of approaches. Another frequently employed strategy for surface NO-delivery involves aminosilane-based sol-gel chemistry, which is generally difficult to make the precursors for NO-immobilization and provides thick coating (mu;m-mm). Formation of NO-releasing self-assembled monolayer (SAM) was reported, but the SAM approach is limited to noble metals that are not commonly used in medical devices. Thus, developing a general strategy of surface chemistry for NO-immobilization is critical and necessary in future development of NO-releasing medical devices.
The key point to achieve these purposes is to find the special surface chemistry which has precursors for NO-releasing moieties and can be formed on a variety of substrates. It has been reported that poly(dopamine) (pDA) and poly(norepinephrine) (pNE), which form chemically active adherent thin films through oxidative self-polymerization, can show the material-independent coating ability with secondary amino groups. We hypothesized that this secondary amino group could be functionalized by 1-substituted diazen-1-ium-1,2-diolates (diazeniumdiolates) which is generally installed at secondary amino groups and spontaneously dissociates under physiological conditions to release two equivalents of NO from each functional group.
To sum up, we present the first report of a facile, versatile, and biocompatible strategy for surface NO delivery utilizing catecholamines, functionalized with diazeniumdiolates. Furthermore, resulting surfaces can efficiently inhibit the bacterial adhesion, and kill adhered bacteria cells and yet demonstrate excellent biocompatibility. This localized NO-delivery strategy offers a versatile platform which could be applied to various other catecholamines and pave way for the advancement of NO-mediated biomedical domains comprising of wound healing or anti-restenosis as well as prevention of implant-associated infection.

1. Introduction
Nano and microparticles (NP/MP) prepared from poly(DL-lactide-co-glycolide) (PLGA) have unique biocompatibility and biodegradability properties. Drugs and/or other compounds can be encapsulated into these nanoparticles, or may be chemically attached or physically absorbed into their surface allowing targeted delivery and timed release. Cresyl Violet (CV) is a recognized synthetic hydrophilic dye that is widely utilized to stain neuronal tissues. CV is also used as a reference fluorescent probe due to its high fluorescence quantum yield value (Phi;f=0.54). The spectral characteristics of CV make it particularly attractive for in-vivo analysis using hyperspectral fluorescence and two-photon microscopy methods. Here, we exploit these characteristics to assess the in-vivo biodistribution of different polymeric nanoparticles loaded with cresyl violet.
2. Methods
PLGA particles encapsulating CV were prepared using the solvent evaporation technique. Prepared particles were then evaluated in relation to size, zeta potential, drug encapsulation efficiency, release profile, and external morphology and spectroscopic properties. Two different sizes of particle were used in this study, classified as NP-nano and MP-micro range (CV-PLGA-NP and CV-PLGA-MP). Prepared particles (0.2mL) were injected into the tail vein of nude mice and 30 minutes of injection, the animals were sacrificed and the organ-specific localization of each particle type was assessed using wide-field hyperspectral fluorescence imaging and fresh-tissue two-photon microscopy.
3. Results
The entrapment efficiency of CV in each type of particle was 47% for CV-PLGA-NP and 55% for CV-PLGA-MP. The particles had hydrodynamic diameters of 368.30±1.40 nm and 961.61±1.8, respectively. Negative data with Laser Doppler Electrophoresis (LDE) were acquired for zeta potential (-10,9±0.2mV to VC-PLGA-NP and -35.5 ±0.45mV to VC-PLGA-MP) showing low aggregation index. These results indicate that the both particles are well-suited for in vivo applications. Our in-vivo analysis showed that the VC-PLGA-MP localized mostly to the lungs (as expected) and that VC-PLGA-NP were found in the lung, kidney, heart and liver. Intact, spherical particles containing CV were identified in fresh-tissue tow-photon microscopy measurements (exc = 700 nm (350 nm) and em = 640 nm)
4. Conclusion
We report the successful preparation and in-vivo characterization of PLGA encapsulated cresyl violet. We applied novel optical imaging and microscopy techniques to assess and validate the biodistribution of these particles in-vivo. Our in vivo results suggest that smaller PLGA nanoparticles can reach a range of organs, making them a promising vehicle for diagnostics or the targeted delivery and timed release of drugs.
Acknowledgments
CNPq, FINATEC, FAPDF,CAPES Fundaccedil;atilde;o Lemann, IANAS, NIH, NSF

Atherosclerosis, characterized by the thickening of the arterial wall to form an atherosclerotic plaque, is prevalent and leading cause of mortality worldwide. Macrophages are key cellular mediators in early plaque development, plaque rupture and thrombosis. Thus, accurate detection of macrophages in the plaques is a primary goal in cardiovascular research. Magnetic resonance (MR) imaging has been widely adopted for non-invasive monitoring and detecting of diseases with high spatial resolution; however, the technique lacks sufficient sensitivity to detect vascular lesions without aid of signal enhancement in target lesion. To improve sensitivity of MR imaging and make clear distinctions in MR signal between lesions and normal tissues, development of sensitively detectable MR contrast agents targeting specific disease lesion is essential. Superparamagnetic iron oxide magnetic nanocrystals (MNCs) have been extensively employed as T2 MR imaging contrast agents because they are biocompatible and eminently sensitive to magnetic field. In recent researches, uniform and high crystalline MNCs with nanosize were developed using thermal decomposition method. Preceding research shows that the T2 relaxivity of MNCs is increased with the size of MNCs. However, this strategy possesses critical limitation because single MNC larger than 30 nm exhibits ferromagnetic properties, which cause poor colloidal stability resulting from magnetic dipole interaction and agglomeration. In addition, these MNCs are synthesized in organic solvents with adhesion of hydrophobic ligands on their surface so that they are not soluble in aqueous phase. Therefore, development of water-dispersible assembly of superparamagnetic MNCs performing high MR sensitivity is essentially important.
In this study, we developed and engineered magnetic nanoprobes facilitating specific detection of atherosclerotic plaques via T2-weighted MR imaging. Monodisperse MNCs were synthesized by thermal decomposition method. Oleyl dextran (oleic acid-conjugated dextran, ODex) was prepared by conjugating oleic acid to dextran through the esterification for targeting of macrophages. ODex-modified magnetic nanoclusters (ODMCs) were fabricated by micellar encapsulation of MNCs with ODex. The magnetic properties of ODMCs were engineered to enhance MR imaging sensitivity by controlling degree of substitution (DS) of oleyl group in ODex and the concentration of ODex used for coating of MNCs. Engineered ODMCs exhibited increased T2 relaxivity and in vitro targeting ability against macrophages without cytotoxicity. In vivo T2-weighted MR imaging after intravenous injection of ODMCs into a rat artery balloon injury model showed considerable MR contrast enhancement with a decrease of MR signal intensity at the plaques of the injured carotid artery. From these results, we verified that ODMCs have sufficient potential as a MR imaging probe for diagnosis of atherosclerosis.

Materials responsive to stimuli, such as temperature, pH, applied magnetic field, and mechanical stress are important in biomedicine because they allow spatiotemporal control over drug release. The focus of this study is the development of a tension-responsive drug delivery device by exploiting a difference in mechanical properties between a drug-loaded core material and a superhydrophobic barrier coating consisting of interconnected micro- and nano-sized particles fabricated by electrospraying a mixture of two biocompatible polymers: poly(ε-caprolactone) [PCL] and poly(glycerol stearate-co-ε-caprolactone). The textured superhydrophobic (contact angle > 167°) surface employs microscopic structural topographical features commonly observed in nature to increase surface roughness and prohibit water infiltration. In the absence of tension, this superhydrophobic coating maintains a stable air layer that protects against wetting into the underlying cisplatin-loaded core. However, upon the application of force, this protective barrier breaks down through crack propagation to afford rapid water infiltration and subsequent release of cisplatin from the core. The amount of drug released is dependent on the tensile strain applied to break the superhydrophobic coating, and in vitro cell assays on cancer cell lines demonstrate release of a therapeutic cisplatin dose. In summary, we have shown the ability to release a drug payload upon mechanical stimulation and provide a broad-based approach to design mechano-responsive drug delivery depots.

Ion-channel proteins are membrane proteins that are of crucial physiological importance and are major targets for drug design. Reconstitution of ion channels in free-standing bilayer lipid membranes (BLMs) provides an excellent system for screening the effect of drug candidates. Recently, we reported on the formation of mechanically stable BLMs by preparing membranes in micropores fabricated in silicon chips (1). In the present study, we report on the reconstitution of the human ether-a-go-go-related gene (hERG) channels in the stable BLMs formed in the micropores (2). The hERG channel has been drawing considerable attention, because it has been found to be related to serious arrhythmic side effects following drug treatment. Ion currents of hERG channels were investigated in terms of single-channel conductance, voltage dependence, and sensitivity to typical drugs.
The hERG channels (Kv11.1) were isolated from Chinese hamster ovary cell lines expressing the channels. Micropores with a diameter of 20minus;60 mu;m were fabricated in Si chips, and the surface of the chip was coated with Teflon-AF and thermal oxide (3). BLMs were prepared by the monolayer-folding method. The incorporation of hERG channels into BLMs was performed by fusing hERG-containing proteoliposomes. Current recordings were performed with an Axopatch 200B patch-clamp amplifier (Molecular Devices).
We first examined recordings of the hERG single-channel currents after incorporation of the hERG channel into BLMs. Stepwise currents were evident, with an average single-channel chord conductance of 11 ± 1.2 pS. This conductance level is similar to a previously reported value (12 pS) obtained using the patch-clamp method. Channel activity was only observed at negative applied potentials, which is in agreement with reported patch-clamp observations for hERG channels. The channel current was completely blocked by E-4031, a specific channel blocker, and astemizole, an antihistamine that has been withdrawn from the market due to its side effect on the hERG channel. Thus, the sensitivity of the hERG channel to most typical drugs has been successfully reproduced in the present BLM reconstitution system. The BLM with hERG channels incorporated exhibited a lifetime of sim;65 h and a tolerance to repetitive solution exchanges, and were not broken by applying a constant voltage of ±1 V. Such stable BLMs containing biological channels have the potential for use in a variety of applications, including high-throughput drug screening for various ion-channel proteins.
References
(1) A. Hirano-Iwata, K. Aoto, A. Oshima, T. Taira, R. Yamaguchi, Y. Kimura, M. Niwano, Langmuir, 26, 1949-1952 (2010).
(2) A. Oshima, A. Hirano-Iwata, H. Mozumi, Y. Ishinari, Y. Kimura, and M. Niwano, Anal. Chem., 85, 4363-4369 (2013).
(3) A. Oshima, A. Hirano-Iwata, T. Nasu, Y. Kimura, and M. Niwano, Micro and Nanosystems, 4, 2-7 (2012).

L-Glutamate is a major excitatory neurotransmitter in the central neuronal systems and plays a vital role in brain development, synaptic plasticity and neuropathology. Detecting local changes in L-glutamate concentration induced by physiologically related stimuli, such as weak electrical stimuli, is particularly important, because glutamate responses can be related to various neurological disorders. Recently, a glass capillary-based enzyme sensor has been shown to detect glutamate released by local electrical stimuli in mouse hippocampal slices (1). However, no study carefully discussed the influence of electrical stimuli on sensor signals. In the present study, we compare responses of glutamate electrodes induced by weak electric stimuli with those of blank electrodes which were inactive to glutamate, and propose a method for extracting glutamate faradaic signals from observed current ones induced by electrical stimuli.
L-Glutamate sensor was prepared by inserting three electrodes in a pulled glass capillary filled with artificial cerebrospinal fluid (ACSF) containing ascorbate oxidase. A gold wire coated with glutamate oxidase served as an underlying working electrode. Teflon-coated Pt and Ag/AgCl wires served as counter and reference electrodes, respectively. Transverse hippocampal slices were taken from adult male mice and placed in an interface recording chamber maintained at 28°C. Electrical stimulation was applied via a monopolar stimulating electrode and evoked field excitatory postsynaptic potentials (fEPSPs) were recorded with a glass microelectrodes. A glutamate sensor, together with stimulating and recording electrodes, was placed in a hippocampal slice, and glutamate currents and fEPSPs following electric stimuli were evaluated.
We first examined a current vs. time profile when electric stimuli were applied for glutamate and blank sensors placed in hippocampal slices. In the case of the glutamate sensor, application of a current pulse induced a sharp negative-going current, followed by a positive-going current which gradually decreased to a basal level. However, similar time-profile was also observed with the blank sensor, indicating that a faradaic signal at the glutamate sensor may be obscured by the non-faradaic current transient. Our approach to extract a faradaic signal from the observed current was to take a current signal when the capacitive transient dropped virtually to zero. By using this analysis, a clear response was observed following 2 Hz electric stimulation at the glutamate sensor, while no significant response was observed at the blank sensor. The validity of this approach was examined using various stimulation protocols. In the presentation, application to evaluation of glutamate release during synaptic plasticity is also discussed.
References
(1) S. Hozumi, K. Ikezawa, A. Shoji, A. Hirano-Iwata, T. Bliss and M. Sugawara, Biosens. Bioelectron., 26, , 2975-2980 (2011).

We propose the use of a potentiometric biosensor that incorporates the efficient and specific molecular imprinting (MI) method with a self-assembled monolayer (SAM). We first tested the biosensor using carcinoembryonic antigen, CEA, a biomarker associated with pancreatic cancer. The specificity and sensitivity of CEA sensor was evaluated through cross test. In addition, we use biosensor to discriminate normal fibrinogen and damaged fibrinogen, which is critical for the detection of bleeding disorder. To further study the mechanism of MI biosensor, computer simulations of the hemoglobin structure under different pH value were performed. Through the computer simulation results, the sensitivity of biosensor was evaluated. We found that even small changes in pH can generate rotation of the surface functional groups. Yet, the results show that only when the detection and imprinting conditions are similar can robust signals occurs, which is consists with our experiment data. Therefore we concluded that both morphology and surface chemistry play a role in the process of recognition.

The direct laser writing by two-photon polymerization has been used to manufacture microdevices for applications from optics to biology. This technique has several advantages, among which we can mention the resolution above the diffraction limit and high spatial selectivity, which enables the fabrication of complex three-dimensional structures. The doping of the host resin used in the two-photon polymerization (2PP) with compounds of interest (organic dyes and bioactive agents) allows the fabrication of devices with specific features tailored for different purposes.
In this work, we present two types of microenvironments produced using 2PP to evaluate the motion and development of bacteria. The first microenvironments consist of micro-fences, which were used to trap the bacteria Escherichia coli ATCC 25922 (E. coli), allowing monitoring bacterial growth and motion. The second type of environment developed here consists of an array of cylinders doped in a specific site with ciprofloxacin, an antibiotic used in the treatment of bacterial infection. In this case we have evaluated the inhibition of bacterial growth around the doped site of the microenvironment. The microenvironments were produced by 2PP using 150-fs laser pulses (Ti:sapphire laser oscillator operating at 780 nm) focused through a microscope objective (0.25-NA or 0.85-NA) in the volume of a polymeric resin containning a photoinitiator, responsible for triggering the polymerization. The average laser power is approximately 40 mW and the fabrication speed used is 10 µm/s.
Bacteria E. coli inoculated in the microenvironment have all necessary conditions for their development. To monitor the bacterial growth in the microenvironment optical microscopy image was performed hourly. For the first micro-fences we were able to observe an increase in the number of trapped bacteria as function of time. The bacterial density in such traps seems to saturate after approximately 1 hour, which is attributed to the fact that some entrapped bacterial leave the micro-trap through the top, that is open to aid visualization. For the microenvironment doped at the specific site with ciprofloxacin, inhibition of the bacterial growth was observed around the doped site. In this case we have been able to evaluate the range of antibiotics action in this microenvironment.
The approach presented here is a promising alternative for the fabrication of scaffolds using biocompatible polymers, to study bacterial growth and migration, opening new possibilities to study advanced drug delivery systems.

The present investigation involves the synthesis of chitosan based composite sponges in view of their applications in wound dressing, antibacterial and haemostatic. Chitosan-PVA composite sponges were prepared by a modified freeze-drying method using acid-base reaction initiated bubbles as templates for the porous structure. This described technical process allows fabricating porous material with controllable morphology and porosity. Effects of the content of cross-linking agent and PVA on morphology, mechanical properties and moisture permeability were examined. Improved strength and flexibility of the chitosan sponges were observed with the presence of PVA. Further, the antibacterial and haemostatic activities have been demonstrated. The Chitosan/PVA sponges have appropriate moisture permeability, excellent antimicrobial property and unique haemostatic behavior for wound dressing applications.

Nitinol (nickel-titanium alloy) is one of the best candidates for shape-memory related applications, for example coronary, biliary, esophageal and tracheobronchial stents. Nitinol stents, called self-expansion stents, have the shape-memory effect and super elasticity. However, the elution of nickel ions from the nitinol stents often leads to allergic reaction and need to be solved. The electrochemical deposition of hydroxyapatite (Ca10(PO4)6OH2; HAp) is gaining increased attention because of ease process control and suitability for complex geometry such as a stent. Although Ban et al. has already described calcium phosphates deposition on various metal substrates by an electrolytic deposition (ELD) technique, there have been few investigations on nitinol. In this study, we focused on HAp deposition on the nitinol stents by ELD. ELD were conducted on the nitinol stents as a cathode in a 25 mM Ca(H2PO4)2_2H2O (MCP) solution at 75 degreesC under the application of pulsed direct current. A constant pulsed on or off time (duration) of Ton=1 s or Toff = 1 - 10 s was applied at a current density of Jon = 1.59 mA/cm^2. After ELD, the specimens were treated in 0.1 M NaOH solution at 60 degreesC for 3days. From X-ray diffraction measurements, calcium phosphate layers were mixed phases of Ca8H2(PO4)6_5H2O (OCP) and CaHPO4 (DCPA). With the increase of Toff, OCP decreased and the layers deposited under Toff of 10 s were a single phase of DCPA. The NaOH treatment completely converted OCP and DCPA phases into a single phase of HAp. From scanning electron microscope observation, fine or large plate-like crystals of OCP or DCPD transformed to fine needle-like crystals of HAp. The metal ions elution was examined in 0.01 M phosphate buffered solution (PBS) at 37.5 degreesC for 7 days. From ICP-MS analysis, the bare nitinol eluted 3.81x10^-7 M of titanium ions and 15.5x10^-7 M of nickel ions. The OCP and DCPA layers completely prevented the titanium ions elution and reduced the nickel ions elution to. Compared 1/2 with the OCP and DCPA layers, the HAp layers reduced the calcium ions elution to 1/4 in addition to equal reduction in titanium and nickel ions elution. We will discuss the effect of the conditions of HAp deposition on the reduction of metal ions elution.

Immunotherapy is a non-invasive treatment method controlling the immune response, and is usually conducted via vaccination. Injected vaccines are recognized and bound to antigen presenting cells (APC) in the immune system. However the vaccine recognition to APC is generally inefficient. One of the solutions to enhance the recognition is to use adjuvants, which carry the injected vaccines into APC. Thus, a novel vaccine carrier with controlled-release to induce adjuvant action has been investigated. In this study, hydroxyapatite-chondroitin sulfate (HAp/ChS) nanocomposite microparticles were fabricated as a carrier of model vaccines. The HAp/ChS microparticles show the high adsorption ability of proteins and high biocompatibility.
The HAp/ChS nanocomposite was synthesized through a precipitation method with a calcium hydroxide suspension and phosphoric acid solution containing ChS. Then, the HAp/ChS microparticles were fabricated by a spray dry method and characterized by XRD, FT-IR and SEM.
The specific surface area of HAp/ChS microparticles was about 160 square meters / gram, which was 1.5 times larger than that of normal HAp particles. HAp/ChS microparticles had a characteristic adsorption property for proteins, that is, an increase of the amount of ChS in the nanocomposite led to the favorable adsorption of positively charged proteins such as lysozyme compared with negatively charged ones such as ovalbumin. This indicated that ChS in the nanocomposite controls the electrostatic interaction with proteins. Since the vaccines in clinical practice are often positively charged, the HAp/ChS particles prepared could have an advantage for their adjuvant. The loading and release properties of model-tumor antigens such as ovalbumin 323-339 peptide on the particles will be discussed.

Many biomedical devices, such as partial dentures, are combining of acrylic and metallic parts that are bonded together. These devices often present catastrophic mechanical failures due to lack of bonding between their acrylic and metallic components; in fact, chemical bonding between alloys and polymers (i.e. poly-methyl methacrylate, PMMA) does not occur spontaneously. The objective of this research was to develop an efficient and effective method of creating a strong chemical bond between alloys and polymers for dental prostheses based on diazonium chemistry. The bond between metals and polymers was achieved in two steps. In the first step (primer), the aryldiazonium was chemically reduced to form the aryl radical which spontaneously grafted onto the metallic surfaces. The second diazotisation step (adhesive) was optimized to achieve covalent binding between the grafted layer and poly-methyl methacrylate. The chemical composition of the treated surfaces was analyzed with X-ray photoelectron spectroscopy (XPS), and the tensile bonding strength between metals and poly-methyl methacrylate was measured. XPS characterization confirmed the presence of a polymer coat on the treated metallic surfaces while the mechanical test results showed the bond strength between poly-methyl methacrylate and treated titanium or stainless steel wire was increased by 5.2 and 2.5 folds, respectively, compare to untreated control samples (P<0.05). Diazonium chemistry provides an effective way of achieving a strong chemical bond between alloys and poly-methyl methacrylate that can be used to improve the properties of dental devices.

We wish to propose here an impedance spectroscopic method for the quality control of contact lenses that has not been unraveled before. The silicone hydrogels are excellent materials for use as contact lenses and their utilization is dependent on salt intrusion characteristics which are related to the pore resistance and water uptake. When the contact lenses are used in the eye, they are expected to permeate ions and molecules for active health. The hydrogel pores control the permeability as a quality control parameter. Two models are considered here; in one the contact lenses are considered as strong rigid films with no permeability. In another, the hydrogels are having ionic permeability. We designed a silicone hydrogel contact lens attachment holder that is amenable for the electrochemical impedance measurements. The electrochemical impedance measurements were carried out in inert medium of 0.1 M Na₂SO₄. The experimental parameters used for the impedance measurement here are a) AC potential 10 mV rms b) frequency range 0.1-210 kHz and c) open circuit potential 0.207 V. The impedance variation with frequency was constructed for a number of hydrogels. The ideally acceptable silicone hydrogel contact lenses showed an impedance change with frequency in a sigmoidal fashion with a characteristic phase angle (acceptable in the range of 70-75^o ). The hydrogel pore resistances for the acceptable contact lenses are in the range of 4.5-11 kOmega;. If the impedance showed a linear decrease or no sharp peak for phase angle, the contact lens is considered acting as an insulator and is to be rejected. A test of the model was done with several acceptable contact lenses in the market. This study revealed interesting aspects of the influence of pulsating electric field on the silicone hydrogels that would be presented.

Several methods for drug-incorporation into polymer matrices are currently practiced, but they frequently lack efficiency and cost effectiveness or lack scalability. Spray-assisted LbL film assembly has recently been developed to reduce assembly time from days to minutes (in comparison to traditional dip-assisted LbL) by aerosolizing polymer/drug solutions onto solid or porous substrates. While this allows for rapid and scalable film assembly, a large percentage of the solutions are lost making this process prohibitively costly for use with expensive biologics.
We have developed a strategy for spray LbL, which greatly reduces the amount of costly biomaterial necessary to build a film and significantly reduces the loss of solution. By collecting and recycling the sprayed solutions, we minimized the material used. A comparison of the recycling system to a non-recycling system was done using a (Poly2/heparin/lysozyme/heparin)n tetralayer system to monitor the solution recovery, change in film growth, lysozyme loading efficiency, and lysozyme release profiles for the two systems. This recycling process for Spray LbL has many potential applications for film assembly and could greatly reduce both the amount of necessary time needed for film assembly and the amount of costly materials necessary as well.

Introduction: It is now recognized that surface nanostructures on biomaterials greatly influence cellular behaviors as well as surface biochemical properties[1]. Nano-featured surfaces might be used as an excellent bioactive interface for implantable materials. Stainless steel is one of the most important alloys for biomedical applications. However, their bioinert surface characteristics are disadvantage for numerous applications. The aim of this study was to fabricate nano-structured surfaces on 316L stainless steel by an anodization method and examine the effects of these anodized nano-surfaces on the attachment and proliferation of fibroblasts.
Materials & Methods: 316L stainless steel (25 mm × 25 mm × 0.5 mm) (Goodfellow Cambridge Ltd. England) was anodized at 30 and 40V for 10 min in ethylene glycol containing 5 vol. % perchloric acid to create bioactive surfaces. The surface morphology and elemental composition of 316 L after anodization were characterized by scanning electron microscopy (SEM, Hitachi S-4800, Tokyo, Japan) and energy-dispersive X-ray analysis (EDAX). Fibroblasts were seeded on the samples to evaluate initial cellular responses to the nano-structured surfaces. The anodized surfaces were assessed for their in vitro cell-material interactions using fibroblast cells for 4 h, 1, 3 and 7days. The attachment and proliferation of fibroblasts were determined by a methyl thiazolyl tetrazolium (MTT) assay. Smoothly polished 316L was used as control surface. Light microscopy and SEM were used to observe the morphology of cells. Five specimens of each material were tested for each incubation time and each test was performed in triplicate. Data were analyzed statistically using student t-test.
Results and Discussion: Nano-porous structures on 316L stainless steel were prepared by the anodization technique. The pore size could be adjusted and ranged from 30 to 100 nm. After 4 hours of culture, the fibroblasts adhered well on the surfaces of all the stainless steel specimens. SEM images showed that fibroblast exhibited normal morphology and were able to penetrate into pores by long filopodia. MTT tests indicated that the anodization of 316 L stainless steel significantly promoted a higher degree of fibroblast attachment and proliferation as compared to the polished 316L stainless steel (p<0.05).
Conclusions: The findings in this study showed that anodization significantly improved the initial cell-material interactions on the 316L stainless steel. Although further work is required to determine the long-term effects of the anodized surfaces on the growth of cells, the results of this study indicated that the anodized surfaces of the 316L stainless steel had great potential for biomedical applications and might be designed as bioactive interfaces for numerous applications, including vascular stents.
References
1. Martínez E, Engel E, Planell, JA, Samitier, J. Ann Anat 2009; 191: 126-135.

Atherosclerosis is a condition in which an artery wall thickens as a result of the accumulation of cholesterol, macrophage, and low density lipoprotein (LDL) accompanying inflammatory response. The ruptures of atherosclerotic plaques causing the formation of a thrombus will lead to myocardial infarction or stroke. Thus, the early detection of atherosclerosis is important issue for the effective therapy. Herein we developed ultra-sensitive nanoprobe hybridizing macrophage targetable dextran with magnetic nanoclusters for atherosclerosis detection via MRI.
Magnetic nanoparticles (MNPs) were synthesized using thermal decomposition method for contrast agent. Pyrenyl dextran (PyDex), a macrophage-targetable surfactant, was synthesized by conjugating the dextran with the 1-pyrenebutyric acid. MNPs were encapsulated by a PyDex using nanoemulsion method to formulate dextran coated magnetic nanoclusters (DMNCs). We investigated magnetic properties, colloidal stability, biocompatibility and macrophage binding affinities of DMNCs. We studied in vivo MRI of Watanabe heritable hyperlipidemic (WHHL) rabbit, representative atherosclerosis animal model, after intravenous injection of DMNCs. For multimodal MR imaging of atherosclerosis, Ultra-short TE (UTE) and T2-wieghted turbo spin-echo (T2 TSE) sequences were used. Histological analysis of WHHL rabbit artery was performed using oil red o (ORO) staining and Prussian blue staining.
TEM images of DMNCs showed clustered shape of nanoemulsion without aggregation. DLS/Zeta results showed that DMNCs were stably dispersed in water during 15 days and the surface was maintained as negatively charged. The inverse spinel structure of DMNCs was confirmed by XRD diffraction pattern and superparamagnetic behavior of DMNCs was investigated using vibration sample magnetometers. From the solution MRI of DMNCs, brightened images with increased signal and darkened images with decreased signal was found in UTE and T2 TSE sequences, respectively. DMNCs showed target ability to macrophage (raw264.7) without cytotoxicity. In vivo MRI of WHHL rabbit after DMNCs injection showed significant contrast enhancement of artery wall due to the accumulation of DMNCs at atherosclerotic plaque. DMNCs showed brightened plaque in UTE MRI and darkened plaque in T2 TSE MRI. Ex vivo MRI of WHHL rabbit artery was well agreed with in vivo MRI and showed sensitive contrast enhancement in plaque. Histological analysis showed that inflammatory plaque was sufficiently developed in WHHL rabbit artery wall and DMNCs were accumulated in plaque.
DMNCs exhibited excellent atherosclerosis targeting ability with no cytotoxicity. Macrophage-mediated delivery of DMNCs to plaques contributed to sensitive MRI of atherosclerosis. DMNCs were used in both positive and negative contrast agent for atherosclerosis MRI. Consequently, these advantageous features of DMNCs allowed us to detect early stage of atherosclerosis via MRI and future clinical applications.

The objective of the study was to comparison tests the biocompatibility of 3D porous biopolymer matrices (tissue-cellular scaffolds), made of biocompatible polymers (polycarbonate, polyetheretherketone /PEEK/), including the materials with biocompatible oxide ceramics additive (TiO2, Al2O3, ZrO2 and hydroxyapatite) of micro and nano sizes, for implantology and tissue-engineering purposes. The porous samples were prepared via a layer-by-layer SLS method. The surface microstructures and their roughness were analyzed by the optical microscopy equipped with the cell analysis software. The cellular morphology, proliferative activity and adhesion of the macro- and nano ceramic-polymer matrices were the subjects for comparison. The study showed that all the tested materials possessed biocompatible properties.

Biocompatible nanooxide ceramics (TiO2, Al2O3, ZrO2 and hydroxyapatite) were added to bioresorbable polymer (polycarbonate and/or polyetherketone) powders for the layer-by-layer laser-assisted manufacturing of the porous tissue engineering scaffolds. The optimal regime comparison for laser sintering on the CO2 and Nd+3YAG lasers were carried out, as an influence of pre- and post thermal anealing. Results of microstructural evaluation of the nano corundum ceramic samples were conducted using the scanning electron microscopy (SEM) equipped with the energy-dispersive x-ray (EDX) analysis and evaluated with results of the X-ray analysis. The observations showed that after a successful laser sintering the increase of the nano ceramic particles sizes was insignificantly. The results confirm the medical resource of the SLS-fabricated tissue engineering scaffolds.
Keywords: selective laser sintering (SLS); nanooxide ceramics, bioresorbable polymers; polyetheretherketone (PEEK); polycarbonate (PC).

Separation of a homogeneous cell population from heterogeneously distributed various cells is an essential process for stem cell therapy, sensing of disease biomarkers such as cancer cells and bacteria. Development of high throughput with precision cell separation technology has still been a great issue to accomplish albeit many remarkable technical evolutions have been made for cutting-edge cell separation so far. Particularly, magnetically activated cell sorters (MACS) have received much attention because of its rapid separation potential of homogeneous cell population from various types as well as causing less cell damage with high purity, recovery and low cost. However, only bimodal isolation has yet been available because the separation moiety relies on the difference between magnetically tagged and untagged cells. Therefore, multi-fractionation of diverse types of cell populations in the sample but still maintaining high throughput is demanded for minimizing both separation time and associated cost per assay compared with conventional magnetic separation.
For the separation of various cell types simultaneously, we herein investigated the mobility of the cells tagged with magnetic nanoclusters possessing various magnetic intensity under external magnetic field. First, we synthesized three kinds of magnetic nanoclusters with equal diameter (80 nm) by changing their metal compositions (MFe2O4, M=Fe, Mn, Co) in order to have different magnetization values under same external magnetic field. When the magnetic field (2000 G) was applied, three different types of magnetic nanoclusters with different compositions represented different magnetization values. Precisely, their specific magnetization values with 2000G regarding on magnetic ion concentration were 74 emu/g Fe, 83 emu/g Fe+Mn and 68 emu/g Fe+Co, respectively and the values based on clusters (emu/cluster) were corresponded with 1.609 * 10-17, 1.805 * 10-17 and 1.479 * 10-17. Subsequently, we tagged these magnetic nanoclusters on the human epithelial cancer cell line (A-431 cell) to verify the potential of multi-cell sorting which could be achieved by difference in cell mobility due to different magnetic susceptibility. In addition, we calculated the Lagrangian trajectories of cells treated with different types of magnetic nanoclusters for comparison. We used the constitutive model equation which accounts for the magnetic force and Stokes-Cunningham drag force under magnetic field to predict the mobility of the magnetically tagged cells and we confirmed that experimental results were in good agreement with the predicted ones. Consequently, the cells tagged with MnFe2O4 magnetic nanoclusters revealed the strongest response, and followed by Fe3O4 and CoFeO4 in order, showing that the marked potential for the multi-fractionation of the target cells according to the magnetic susceptibility based cell mobility difference.

We present a polymerase chain reaction (PCR) chip incorporating graphene heater. Transparency of the graphene heater allowed the realtime fluorescence detection of PCR product using a light passing through a reaction chamber. Low-power consumption as well as rapid convection-based thermo-cycling enabled a highly-miniaturized PCR, which would blaze a trail to a smart-phone-based DNA detection.
The number of DNA copy can be exponentially amplified through thermal cycling of PCR, thus, PCR becomes a general tool for detecting DNA molecules without a highly-sensitive sensor. Many groups have developed various PCR chips in pursuit of convenient, fast, efficient DNA detection. Our group presented a palmtop-sized PCR system capable of fast thermal cycling applying a thermosiphon effect (Lab Chip, 10, 202, 2010). In our system, PCR reactant was filled in a loop channel of a polymer chip, and the chip was inserted in a slot made by metal blocks maintained at three different temperatures. The PCR reactant was moved along the loop channel via thermal convection, and we obtained sufficient PCR products in less than 10 minutes.
However, the heated metal blocks provided very narrow windows open to the loop channel, which demanded a tiny fluorescence detector for realtime PCR. Moreover, the metal blocks needed not small power to maintain their temperatures. Here, as a novel trial, we adopted graphene heater replacing the previous bulky metal blocks. Due to grapheme&’s transparency, fluorescence detection using a light passing through a chamber becomes possible. Also, the PCR chip does not need additional components except graphene layers, which realizes a highly-miniaturized PCR apparatus.
A figernail-sized PCR chip (15x15x1.4 mm3) was fabricated. A polycarbonate plate with a chamber (10 mu;l, 5x2x1 mm3) was bonded with two cover glasses on its top and bottom sides using a UV adhesive. A multilayered graphene (5x4 mm2, 2 k#8486;) was prepared by CVD growth on nickel sheet and transferred on the outside surface of the PCR chip. Finally, a silver paste was applied on the two ends of the graphene pattern for electric connection. A mechanical jig with pin connectors was utilized for external power supply. One side of the chip was heated up to 95~105 oC for denaturation and the opposite side was heated to 50~65 oC for annealing and extension steps. The present graphene heater was very stable in room temperature, and the temperature at a fixed power was maintained with little variation, thus, the temperature sensor was eliminated for simplicity. We conducted numerical simulations to obtain an optimized chip geometry for reliable heat transfer as well as for secure convection speed. The PCR reactant circulated the microchamer in less than 8 seconds, which means total PCR time for 30 cycles corresponds to 4 min. NAT2 (470bp) and BRCA1(127 bp) genes were utilized for the PCR runs. The PCR results under various conditions will be presented.

Recent advances in nanotechnology have allowed the development of highly sensitive and selective detection methods. So, nanoparticles have been used in several areas including analytical becoming an important alternative to conventional dye-based techniques. Silver nanoparticles (AgNPs) have emerged as a powerful tool in sensing applications due to their optical properties. In this work, we developed a paper-based sensor impregnated with AgNPs for colorimetric detection of ascorbic acid by measuring the mean intensity of the color of digital images and by a homemade transmittance colorimeter. The results showed quantitative colorimetric detection with this sensor in the concentration range from 1.00 to 4.00 mmol L-1 (LOD = 0.3 mmol L-1) and 0 to 3.50 mmol L-1 (LOD = 0.2 mmol L-1) using a scanner and portable transmittance colorimeter, respectively. Small Angle X-ray Scattering and Scanning Electron Microscopy were used to characterize the sensor. In the process, vitamin C reduces the silver ions on the surface of the AgNPs, which acts such as seed particles, resulting in increased size favoring the formation of clusters distributed uniformly on the surface coverage of the paper-based sensor. These clusters appear gray and progressively become darker in color with increasing concentration of analyte. Nowadays, rapid test strips for ascorbic acid determination are commercialized in a semi-quantitative visual colorimetric sensor. However, the proposed method is able to monitor lower concentrations in a quantitative way. Thus, our device should be useful for determining, for example, minimum ascorbic acid contend in fruit juices according to some worldwide legislations

The synthesis of stable copper-based nanopartiles with controllable size and compositions is challenging because of the propensity copper oxidation, but has a wide range of applications because of their functional properties for biological and chemical sensing applications. In this work, the synthesis of several copper alloy nanoparticles such as AuCu, AgCu, and PdCu nanoparticles is investigated using a modified two-phase method. The compositions of the as-synthesized alloy nanoparticles were analyzed using ICP-OES technique. Preliminary results showed a linear relationship between the composition in the nanoparticles and the feeding composition for some of the alloy annoparticles. The sizes of the nanoparticles were also demonstrated to be controllable in the range of 2~6 nm. The surface plasmon resonance characteristics of the alloy nanoparticles were shown to be dependent on the bimetallic composition. These findings are significant because the controllability of the bimetallic composition would enable the tunability of the electrical and optical properties of the nanoparticle derived thin film assembly for biological and chemical sensing applications.

Organic monolayer-capped metal nanoparticles have attracted a great deal of interests as chemical sensing nanomaterials on various sensor platforms for detection of toxic gases or vapors. One intriguing sensor platform involves flexible chemical sensor devices which are not only low cost in fabrication, but also have versatile functionalities for applications that demand fast and sensitive detection under conditions such as conformal wrapping/bending over curvilinear surfaces, high tolerance toward repeated bending, mechanical shock resistance, and special sensing environment. This report describes recent results of an investigation of a flexible chemiresistor device with printed metal nanoparticles as sensing materials, focusing on how the device bending and wrapping affect the electrical response characteristics of the nanoparticle materials to different chemical species including small gas molecules and volatile organic compounds. The results provide new fundamental insights into the effects of the device strain and interparticle structural properties on the sensitivity and selectivity of the flexible sensors in response to thermally induced structural evolution and sintering. Comparisons of the results with theoretical modeling of the electrical properties of the nanoparticle thin films and the flexible devices will also be discussed.

Enzyme inhibitors are as ubiquitous as enzymes and are central to wide spectrum of clinical and technological problems. Enzyme inhibitors are typically viewed as organic molecules with inter-molecular lock-and-key matches to sites of target substrates. Unconventional approaches to new enzyme inhibitor designs are required to circumvent biodegradation as well as to develop new momentum and impart new findings to the field. Based on the general nanoparticle-protein analogy, we hypothesized inorganic nanoparticles with specific shapes can be efficient enzyme inhibitor surrogates. We verified the hypothesis through inhibition activity and kinetics of β-galactosidase (GAL) by ZnO nanoparticles of different shapes. Amongst the ZnO nanoparticles, which include nanospheres, nanoplates and nanopyramids, the inhibition was the strongest for ZnO nanopyramids. Such originated from the geometrical and chemical match between their apexes and grooves on GAL&’s surface thereby mimicking biomacromolecular inhibitors. These findings open the road for nanoscale engineering of NPs to fit specific enzymes with biotechnological or biomedical relevance.

The detection of hydrogen peroxide has been shown to be very important in recent years due to its relevant role in many industrial applications as well as biological reactions. We are interested in it as a quantitative marker for oxidase-based biosensor applications where it is produced when substrate (e.g., glucose, cholesterol) is catalysed by its respective oxidase enzyme.
Previously, a commercial silver flake-based screen-printing ink (PF-410, Acheson®), when treated with surfactant and salt (dodecylbenzenesulfonic acid (DBSA) and KCl) was shown to significantly enhance the electrochemical reduction of hydrogen peroxide up to 80-fold over non-treated inks1.
In this study, the size and morphology of the silver in the screen-printing ink is investigated for its effect on the electrocatalysis of hydrogen peroxide. In order to do this, inks loaded with various silver materials (e.g., micron-sized flakes and silver nanopowder) were prepared using a binder solution extracted from the Acheson® PF-410 ink. This binder solution was obtained by a simple centrifugation of the ink to remove the silver flakes. The binder was then blended with different types of silver materials (75% w/w) and their electrocatalysis was studied on glassy carbon electrodes.
The findings of this study show that the electrocatalytic behaviour of the inks towards hydrogen peroxide was greatly dependent on the type of silver used. For example, enhancements of up to 250-fold were observed for the electrocatalysis of hydrogen peroxide on the silver nanopowder when compared to that of the original Acheson® silver. Modification with DBSA and KCl has also shown some interesting electrocatalytic effects, depending on the type of silver used.
This presentation will use electrochemical, microscopic and spectroscopic techniques to explore why there is such variability across the different silver types used. It will be explored if there is a dependence on silver flake size, morphology, impurity levels, dispersability, etc. Once this is understood, the challenge will be to develop highly conductive screen-printable ink formulations based on a tailored silver that can provide the high conductivity and efficient electrocatalytic behaviour required for the development of a fully printable cholesterol biosensor applicable for clinical use.
The research leading to these results has received funding from the European Community&’s Seventh Framework Programme [FP7/2007-2013] under grant agreement no 257372.

The design and fabrication of miniaturized, implantable, low-power wireless sensors for continuous glucose monitoring hold great promise for diabetes mellitus inflicted patients. This involves addressing a variety of issues including robust electrochemical sensors, extreme circuit miniaturization, as well as counteracting negative tissue response and biofouling following sensor implantation. In this contribution we present a highly miniaturized sensor design that fits through a hypodermic needle and holistically addresses all aforementioned tribulations. For this a high performance amperometric electrochemical glucose sensor is integrated with a custom designed complementary metal-oxide-semiconductor (CMOS) electronic device employing 0.35 mu;m design rule. The fabricated electrochemical sensor is based on Clark&’s first generation devices, and utilizes the stratification of five functional layers resulting in linear amperometric response within the physiological range of glucose concentration (2 - 22 mM). The overall sensor is encased with a thick polyvinyl alcohol (PVA) hydrogel containing poly(lactic-co-glycolic acid) (PLGA) microspheres which provides continuous, localized delivery of dexamethasone utilized to combat inflammation and fibrosis subsequent to implantation. In vivo evaluation in a rat model has shown that this sensor reproducibly tracks glycemic events. Such miniature size and low power operation (0.665 mm2 and 140 µW, respectively) of the electronic system render it an ideal platform for continuous glucose monitoring and other metabolic sensing applications.

Introduction: Inflammation and infection of catheters is a significant problem. It is hypothesized that nanotextured and nanotubular surfaces can be carefully manipulated to inhibit immune cell (specifically, macrophages) and bacteria responses due to their unique surface energy properties. The objective of this study was to create polydimethylsiloxane (PDMS) molds of titanium (Ti) anodized to possess nanotubes and test inflammatory cell and bacteria responses on such substrates.
Materials and Methods: 99.2% pure Ti foils were cleaned with acetone, 70% ethanol, deionized water and then etched for 2min with a solution of 1.5% nitric acid and 1.5% hydrofluoric acid (HF). The cleaned Ti sample was used as an anode, while a platinum mesh served as a cathode. Both were immersed in an electrolyte solution consisting of 1.5% HF and were connected to a DC power supply. A constant voltage of 20V was applied for 10 minutes.
Next, the PDMS monomer and cross-linking agents were mixed at 10:1 and were then placed in a vacuum chamber for 30min to remove air bubbles. The mixture was cast onto the nanotubular Ti master mold and then was placed into a vacuum chamber for 1h. The PDMS was cured at 60 °C for 2h followed by cooling and was gently peeled away from the Ti template.
Staphylococcus aureus (American Type Culture Collection 25923) was used in this study. Sterile samples were rinsed with phosphate buffered saline (PBS) prior to seeding. Then, 1mL of the 106 bacteria/mL stock solution was dispensed into each well. Once seeded with bacteria, the samples were then incubated in a 37°C, 5% CO2, humidified environment for 1h. At the same time, serial dilutions of the seeding density were also conducted and then streaked on a sterile PBS agar plate for specific seeding density values.
Macrophages (TIB-186; ATCC, Manassas, VA) were seeded at 3500 cell cm2 per substrate and were cultured in a 37°C, 5% CO2, humidified environment for 24 hours. Then, nonadherent macrophages were removed by rinsing in PBS. Macrophages adherent on the substrates were fixed with 4% formalin in PBS and stained with both rhodamine phalloidin and DAPI.
Results and Discussion: As expected, nano-sized tubes were distributed uniformly on the Ti surface after anodization and the uniform pores were estimated to have a diameter of 60-70nm and a depth of 200nm. It was found that the surface modification of Ti significantly decreased adhesion of both S. aureus and macrophage, specifically for the 80 nm nanotube diameter. After pouring the PDMS slurry onto the surface of the nanotubular Ti and peeling off the PDMS template, a nanopatterned structure was observed. Most importantly, the PDMS replica showed similar nanostructures and also demonstrated promise on decreasing bacteria responses.
Conclusions: A PDMS replica was obtained from anodized nanotubular Ti and demonstrated a promising ability to inhibit bacteria and inflammatory cell functions.

Immune disorders targeting the destruction of red blood cells can occur in three basic situations: autoimmune hemolytic anemia (AIHA), hemolytic transfusion reaction(HTR) and hemolytic disease of the newborn (HDN). AIHA is a rare disease caused by autoantibody-induced hemolysis leading to premature destruction of circulating red blood cells (RBC). HTR and HDN are caused by alloantibodies. The first is a caused by transfusion of incompatible blood and the second by the passage of maternal antibodies through placental barrier, destroying antigens expressed on the erythrocyte membrane of the fetus. It is usually diagnosed by detecting blood coagulation in the presence of the antibody antiglobulin, in the so-called Coombs test, which is prone to many false positives since is involves visual observation. In this study, we developed a specific IHA immunosensor produced with a one-bilayer layer-by-layer (LbL) film containing monoclonal antibody against human immunoglobulin (mAbIMUG) deposited on a layer of silk fibroin (SF) derived from Bombyx mori cocoons. Adsorption of mAbIMUG on a SF layer was confirmed by appearance of the emission band at 326 nm. Immunosensors were prepared with LbL films deposited on interdigitated gold electrodes for impedance spectroscopy and on screen printed carbon electrodes for cyclic voltammetry measurements. When the SF/mAbIMUG LbL film was exposed to healthy RBCs, no cell attachment could be noted on optical microscopy images, and the voltammogram and the impedance spectrum were barely altered. In contrast, the electrochemical signal was significantly reduced and the impedance spectrum shifted for the sensing units containing RBCs with the antibody attached on surface (“sick cells”). Furthermore, cell attachment was so strong that optical images still showed covered electrodes even after washing in PBS buffer. The detection with two distinct methods seems promising for an effective diagnosis of IHA.

Taking advantage of conventional UV-photolithography and micro-molding technology, we are able to fabricate ultra-cheap microfluidic based geometries that enable microbead-assisted preconcentration and electrochemical signal amplification. Choosing physical barriers for filtering purposes, we have successfully obtained sub-5 µm patterned gaps that can effectively localize microbeads down to 5 microns onto electrode&’s surface. This enables for an effective antigen pre-concentration as well as efficient electron transfer for enzyme-linked immunoassays. The latter is attained using nanostructured gold electrodes that were decorated with Pt nanoparticles to increase their electrocatalytic activity. By appropriately positioning these electrodes within the PDMS microfluidic channel, effective signal amplification was achieved via multi-label secondary antibody-nanotube bioconjugates for highly sensitive prostate specific antigen (PSA) detection in serum and tissue lysates. This presentation focuses on the synergy in combining high amplification, low interference and ease of automation for the design of cheap and reliable immunoassays for Point of Care (PoC) diagnostics.

The design of physiological measurement systems has been a growing research interest in the last decade due to the potential applications in the medical industry. Multi-parameter monitoring systems are more reliable and useful, compared to single parameter monitoring devices. Applications, such as monitoring patient&’s physiological levels in intensive care or continuous monitoring of patients, demand a miniature sensor with faster response time. The active area of our sensor is <10 micron2 paving the way for in-vivo mode applications, and two-electrode configuration is used to allow continuous resistance response monitoring. One of the major areas of study associated with multi biosensors is the use of enzyme-immobilized semiconductor SWNTs. This conductance based sensor led to a good detection of D-glucose, L-lactate, and urea with its high repeatability (10, 9, and 9 times), stability (14, 22, and 14 days) and sensitivity (0.005 mu;M, 0.001 mM, and 0.001 mg/dL of detection limit), respectively. Also, the presence of other biomolecules had no impact on the detection capabilities. This study shows that the developed biosensor enables the detection of D-glucose, L-lactate, and urea over a large range (0~300 mu;M, 0~100 mM, and 0~100 mg/dL, respectively) that can be sufficiently covered for physiological monitoring in blood. The electronic properties of the SWNTs had a significant impact on the performance of these sensors. Use of a different immobilized enzyme can potentially lead to sensors for detection of other targeted biomolecules.

Biomedical devices implanted in human body induce foreign-body reactions, which lead to the collagenous capsulation of the implants, impair the device function and can cause tissue distortion and pain. The foreign-body reactions are triggered by non-specific surface adsorption of proteins. Zwitterionic hydrogels have been synthesized and implanted in mice, which resist the foreign-body reaction by eliminating protein adsorption during the in vivo tests(1). Compared to the prevalent polyethylene glycol (PEG) or poly(2-hydroxyethyl methacrylate) (PHEMA) surface chemistries, zwitterionic hydrogels exhibited better long-term stability and resistance to collagenous capsulation(2).
However, there are technical challenges to apply the current synthesis scheme to implantable biomedical devices. First, the solution synthesis scheme involves various lethal solvents such as chloroform, which can become toxic residue and pose health threats. Secondly, crosslinker has to be incorporated to render the zwitterionic polymers insoluble in aqueous environment. The fouling resistance decreases significantly because the hydrophobic segments in the copolymer tend to migrate to the outermost surface during solvent evaporation to minimize interface energy. In addition, coatings with micrometer thickness are the common products of substrate-independent solution synthesis methods, which can obstruct the mass transport and/or electric communication between the implant and the host body and disable the implanted device.
Here, we report an all-dry synthesis scheme that produces surface-concentrated zwitterionic moieties with sub-hundred-nanometer thickness for implantable biomedical devices. A precursor film containing tertiary amine is deposited via initiated chemical vapor deposition (iCVD) in a single step using commercially available monomers. Then a vapor phase reaction with 1,3-propanesultone is carried out under diffusion-limited conditions to convert the tertiary amine to zwitterion(3). The diffusion-controlled conditions are the key to the high surface concentration of zwitterionic moieties. This allows for the tuning of bulk film properties independently of the surface fouling resistance. An in situ Interferometer was used to obtain the desirable coating thickness precisely(4). Solvent is eliminated from this process; the residue of toxic compounds is thus minimized. Virtually any surface can be rendered antifouling with this substrate-independent method and therefore various implantable biomedical devices can be fabricated.
1. Zhang, L.; Cao, Z.; Bai, T.; Carr, L.; Ella-Menye, J.-R.; Irvin, C.; Ratner, B. D.; Jiang, S. Nature Biotechnology 2013, 31, 553-556.
2. Yang, R.; Asatekin, A.; Gleason, K. K. Soft Matter 2012, 8, 31-43.
3. Yang, R.; Gleason, K. K. Langmuir 2012, 28, 12266-12274.
4. Yang, R.; Xu, J.; Ozaydin-Ince, G.; Wong, S. Y.; Gleason, K. K. Chem. Mater. 2011, 23, 1263-1272.

Magnetic-core plasmonic shell nanoparticles possess the capability of magnetic separation coupled with plasmonic properties. For example, iron nanoparticles have been used as contrast agents in magnetic resonance imaging (MRI) and biological molecule separation. Plasmonic nanoparticles such as gold or silver have been used for the sensitive detection of biomolecular analytes due to their plasmonic properties. Gold nanoparticles are also biocompatible and can be easily bioconjugated through the Au-S bond. They have the potential to act as a tool for medical diagnostic applications. As a result, interest in hybrid magnetic-core plasmonic shell nanoparticles has been increasing because of the bifunctional nature of the material. To study these properties, we first synthesized FePt core-plasmonic shell nanoparticles (using gold and/or silver as the shell material), which possess both magnetic and plasmonic properties. The synthesized nanoparticles were then modified with lactose which is inherently biocompatible. The particles were then characterized by transmission electron microscopy, X-ray diffraction and energy dispersive X-ray analysis. The lactose conjugated nanoparticles will act as a cellular probe for imaging of biological events and will demonstrate the ability to separate cellular organelle from the cell by utilizing the magnetic properties of the core@shell nanoparticles.

Graphene, a two-dimensional sheet structure of sp2 conjugated atomic carbon, has attracted considerable attention in a myriad of researches due to its outstanding electronic, electrochemical, and thermal properties. As a result, its novel characteristics have led to a broad range of device applications. Among various applications, graphene-based biosensors have been extensively investigated to selectively detect various analytes at high sensitivity due to their outstanding sensing capability and economic viability. In this work, we utilize thermally reduced graphene oxide (RGO) Layer-by-Layer (LbL) assembled thin films as a working electrode in electrochemical impedance sensors for inducing π-stacking interactions between denatured protein probes and RGO surface. To achieve this goal, protein molecules used as a probe were denatured to render their hydrophobic residues exposed in order to facilitate their direct π-stacking interactions with the surface of RGO nanosheets. As a model probe, bovine serum albumin (BSA) is used following denaturation via chemical or thermal treatment to enable its direct adsorption onto the RGO surface by π-stacking interactions. A successful attachment of BSA probes is confirmed by monitoring the topological change on the surface by atomic force microscopy (AFM), Raman spectra and EIS data. Further binding on the immobilized BSA probes through an antigen-antibody reaction with anti-BSA antibody was also confirmed by EIS measurement, resulting in a high sensitivity with tens of femtomole regime at the minimum. As the final outcome, this approach was implemented in the electrochemical sensor detecting endocrine-disrupting chemicals, Bisphenol A (BPA), using activated LacI proteins as the probe which are anticipated that the obtained advantages as a robust operability in sensing platform and high precision in sensitivity are widely accepted for the conventional biosensors and bio-devices.

Thermoplastic composites are ideal candidates for orthopedic implants, as their mechanical properties can be tailored to those of the surrounding bone. Poly (ether ether ketone) (PEEK) is a biocompatible polymer due to its inert nature and is a preferred composite matrix for load bearing applications, such as bone replacement and scaffolding. PEEK is a high engineering-grade, semicrystalline thermoplastic, featuring excellent mechanical properties that are retained at high temperatures and solvent resistance. Previous studies into the biocompatibility of carbon reinforced polymer composites yielded positive results, including carbon fiber (CF), carbon nano-fiber (CNF), and multi-walled carbon nanotubes (MWCNT). Carbon reinforcement of PEEK is sought to further enhance the mechanical properties for high load bearing bio-medical applications, specifically to provide superior dynamic failure behavior.
Typically, thermoplastic composites are processed in two steps, using twin-screw extrusion followed by injection molding, to achieve acceptable compounding and part fabrication. To fabricate orthopedic implants, polymer composites can be compression molded or injection molded into large blocks, which are then machined to final part dimensions. These products however can suffer from non-uniform particle distribution of the reinforcing agent within the polymer matrix, resulting in non-uniform mechanical properties and quality control issues. In this work, a novel, one-step injection molding method that incorporates axial fluted extensional mixing elements (AFEM) in the screw design, is utilized to provide optimal mixing, consistent mechanical properties, and final part fabrication of carbon reinforced PEEK composites. Additionally, this novel process reduces thermal degradation of the polymer matrix by eliminating the pre-compounding step and can be used to easily fabricate intricate parts for orthopedic implants that require minimal to no subsequent machining.
To determine the viability of micro and nano-scale carbon reinforcement of PEEK for bone replacement and scaffolding, the mechanical properties and morphology of novel, one-step injection molded CF-PEEK, CNF-PEEK, and MWCNT-PEEK blends are presented.

Clostridium difficile is a frequent cause of infectious colitis, usually occurring as a complication of antibiotic therapy, in elderly hospitalized patients. The diagnosis of C. difficile infection still remains a challenge. Searching for a selective and specific technique for determination of its level in biological samples is crucial for regular clinical tests. The current diagnostic modalities mainly consist of the detection of the C. difficile organisms and of their toxins in fecal samples. However, these methods are often time consuming and require sophisticated equipment. With recent advances in nanotechnology, various nanoparticles in different structures, shapes and composites provide good potential for their application in diagnostics and therapy. Nowadays, electrochemical biosensors based on nanoparticles have attracted extensive interest for sensing the formation of antigen-antibody, because of their fast and precise response, high sensitivity and simple pretreatment procedures. Herein, we developed an electrochemical impedance biosensor with gold nanoparticles labeled antibody as the amplifying probe for detecting clostridium difficile toxins. Initially capture antibody was immobilized on gold electrode using conventional amine coupling chemistry. After clostridium difficile toxins were attached on the capture antibody, gold nanoparticle labeled detect antibody as the amplifying probe was used to optimize the immunosensing performance by a sandwich immunoassay. Compared with the traditional method, our method can be operated very simply and rapidly, thus it may possess great potential for clinical applications in the future.

Rapid, accurate, and low cost techniques to identify target DNA sequences are needed to diagnose diseases in point of care settings. Traditional assays like quantitative PCR or microarray technologies require both amplification and labeling steps to identify disease specific DNA sequences. We have developed a novel solid-state nanopore (NP) coated with an electrospun nanofiber mesh (NFM) to address this need which detects target nucleic acid sequences without amplification or labeling. These NP-NFMs are designed to first capture specific nucleic acid sequences from a complex sample onto the NFM, allow washing to remove non-specific components, and finally release the target sequence from the mesh for subsequent single-molecule detection using sub 10 nm in diameter nanopores. Our NFMs consist of blends of high molecular weight poly (ε-caprolactone) (PCL) (70,000-90,000 g/mol) to control the physical properties of the meshes (fiber diameter, mesh density, etc.) and poly (glycerol-co-ε-caprolactone) (PGC), which is functionalized with a poly(ethylene glycol) (PEG) terminated with a maleimide group to facilitate tethering to a thiol-functionalized oligonucleotide capture probe. The high surface to volume ratio afforded by our 3-dimensional nanofiber meshes provides our system with a significant advantage over capturing an analyte on a smooth 2-dimensional surface. The NFM is electrospun onto the surface of the NP before the capture probes are tethered onto the NFM surface. Once the capture probes are tethered to the NFM, the NP-NFM is exposed to a solution containing the target sequence which hybridizes with the tethered capture probes. The non-specifically bound materials are washed from the device, leaving only the specifically bound target sequence on the NFM. The target sequence is then released from the NFM and electrophoretically pulled through the solid-state nanopore and detected. Since the solid-state nanopore detects individual DNA molecules and has a sub-pico molar detection limit, this technique does not require sample labeling or amplification to identify the presence of a target sequence. The combined NP-NFM device designed to capture and release specific sequences of DNA will provide a novel platform for label-free and amplification-free nucleic acid detection with applications in many areas.

Genosensors are advanced analytical devices that exhibit high specificity and may be applied for early detection of genetic diseases. Typical genosensors are based on the use of sensing electrodes containing immobilized DNA probes capable of hybridizing with specific target DNA sequences. Here we show that upon an appropriate design of the biorecognition platform, efficient sandwich-type genosensors based upon DNA-AuNPs nanocomplexes can be efficiently applied to the detection of a Systemic Arterial Hypertension (SAH) polymorphism located in intron 16 of the Angiotensin-converter enzyme (ACE) gene. Since SAH is intimately related to heart diseases, especially blood hypertension, its early detection is of great biomedical interest. The biorecognition platforms were assembled using mixed self-assembled monolayers (SAMmix), which provided the immobilization of organized architectures with molecular control. Detection of the DNA target sequence at concentrations down to 1 nM was carried out using electrochemical impedance spectroscopy (EIS). The use of EIS combined with specific nanobiocomplexes represents an efficient method for the unambiguous detection of complementary DNA hybridization for preventive nanomedicine applications.

Nanotechnology provides new tools to study biological mechanisms, especially at the cellular and molecular levels, because of the similar length scales that nanomaterials and biomolecules share. Studying biology in living cells is one of the most challenging tasks in cell and molecular biology. Micro and nanoneedle technologies offer opportunities for biological studies of microenvironments such as single-cell analysis. We have been developing a minimally invasive cell manipulation and analysis technology, which uses an atomic force microscope (AFM) and an ultra-thin AFM probe sharpened to a diameter of 200 nm and high aspect ratio of over 50 (“nanoneedle”). In previous studies, we reported nanoneedle was probed to be useful for single cell analysis (1). Further, nanoneedles with functionalized surfaces used to monitor mRNA expression in living cells (2), discrimination of cell type and cell diagnosis (3,4). However, the use of single nanoneedle limits the throughput. To overcome this limitation, we propose a method to fabricate Si-nanoneedle array device to investigate a large number of cells simultaneously. This study broadly addresses fabrication of a highly dense silicon nanoneedle array with high-aspect-ratios for analyzing cellular signals at single cell level in a large single-cell population.
We fabricated nanoneedle arrays from Si wafers by MEMS technique. The Si chip contains several ten thousands of needles with diameters of 200-300 nm and a length of 10-20 mu;m with aspect ratios over 50. Initially, micron pillar arrays were made by photolithography and ICP dry etching (Bosch Process). Then, from these pillars, nanoneedles are produced by thermal oxidization and removal of the oxidized layer (SiO2) with HF. Si-wafer was then diced into half-centimeter to yield individual devices, having 100 x 100 nanoneedles. We have also built the customized piezo motor system to attach Si-nanoneedle array and to approach cells on Petri dishes. To manipulate large number of cells using a nanoneedle array, we developed a cell array in which each cells were settled beneath each nanoneedles precisely. Cell adherent spots were formed on a dish by micro contact printing using micron pillar array and biocompatible anchor for membrane (BAM, OE-020CS, NOF, Japan) as adhesive. The cells seeded on the substrate were tethered on the BAM spots and were arrayed successfully. We are currently working on simultaneous insertion of nanoneedles into cells on the substrate.
References
(1) Obataya et al. Nano Letters, 5, 27-30, 2005
(2) Kihara et al. Biosens. Bioelect. 26, 1449-1454, 2010
(3) Mieda et al. Biosens. Bioelect. 31, 323-329, 2012
(4) Silberberg et al. Biosens. Bioelect. 40, 3-9, 2013
Acknowledgement
This research is funded by JSPS through the “Funding Program for Next Generation World-Leading Researchers”, initiated by the CSTP.

Detection of biomarkers is critical, especially in case of cancer and cardiovascular diseases. For instance, monitoring carcinoembryonic antigen (CEA), a biomarker for colorectal cancer (CRC) is crucial for CRC patients in remission. Unfortunately, the CEA levels fall below the limit of detection of currently available conventional tests, as a result, the recurrence is diagnosed at later stages that could be fatal. Hence, monitoring biomarkers in these patients demands for new biosensor with high sensitivity and high specificity to detect recurrences. In case of cardiovascular diseases, cardiac myosin is used as a biomarker for myocardial infarction (MI), the normal level is < 200 pg/mL and can be elevated up to abnormal levels > 2000 pg/mL. The challenge is early detection of cardiac myosin with high sensitivity and high specificity. To overcome these challenges, we have developed a microscale biosensor platform device for simultaneous detection of multiple biomarkers. This polymer-based biosensor incorporates multiple active isolated areas, as small as 70 µm x 70 µm, for antigen detection. The fabrication approach involved conventional micro- and nano-fabrication processes followed by site-specific electrophoresis directed assembly of antibody-functionalized nanoparticles. Using a simple fluorescence-based enzyme-linked immunosorbent assay (ELISA) technique, the biosensors exhibit high sensitivity, detecting 31.25 pg/mL of CEA and 500 pg/mL of cardiac myosin. This demonstrates the potential for a biosensor device for early detection and monitoring recurrences of CRC and detection of MI.

Polymer micro- and nanoparticles are now a mainstay in biomedicine, medical diagnostics, and therapeutics. They are utilized in these areas as imaging-contrast agents, in sensing, and in drug delivery. Current fabrication approaches are typically tuned to specific polymer species, range of sizes, and particle structures. Here, we describe a general methodology for producing uniformly sized spherical polymer particles that is scalable and yields particles with complex architectures and continuously tunable diameters - from the millimeter scale down to 50 nm, which enables broad applications in cancer treatment, immunology, and vaccines. We harness thermally induced, predictable fluid instabilities in composite core/cladding polymer fibers drawn from a macroscopic scaled-up model called a ‘preform&’. The instability leads to the breakup of the initially intact cores - independently of the polymer chemistry - into necklaces of spherical particles held in isolation inside the cladding matrix along the whole fiber length. This process is independent of the specific polymer chemistry and relies only on their rheology. Using this process, we generate particles from the following polymers: cyclic olefin polymer (COP), polyethersulfone (PES), polyetherimide (PEI), polycarbonate (PC), polystyrene (PS), and acrylonitrile butadiene styrene (ABS). Through a stack-and-draw process, we produce fibers containing a multiplicity of identical cylindrical cores made of the polymer of choice embedded in a polymer cladding. Multi-core fibers coupled with the extremely long lengths produced by traditional fiber drawing establish an efficient and potentially mass-producible pathway for polymer particles.
Using the polymeric particles produced in this fashion, we demonstrate surface functionalization for bio-detection through protein-protein specific interactions, volumetric encapsulation of a biomaterial (collagen) in spherical COP polymeric shells, and combining both surface and volumetric functionalities in the same COP particle. We also demonstrate quantitative control over the amount of protein attached to (and retrieved from) the particle surface. These different particle structures used in distinct modalities may be produced using the same biocompatible polymer of choice by changing only the geometry of the macroscopic preform from which the fiber is drawn.

Recent results on thermally induced fluid instabilities in multi-material fibers have led to the development of a novel approach to efficiently producing size-tunable micro-and nano-particles. The process starts by constructing a macroscopic (centimeter-scale) model called a preform, typically in the form of a cylindrical core embedded in a cylindrical cladding. The preform is thermally drawn into extended lengths of uniform fiber with reduced transverse scale. Thermal treatment of the drawn fiber induces the Plateau-Rayleigh capillary instability (PRI) at the heterogeneous core/cladding interface thereby transforming the initially intact, extended cylindrical core into a necklace of periodically spaced, uniformly sized spheres whose diameters are proportional to the intact core diameter. To date, the materials used to demonstrate this process are characterized with softening temperatures typically above 100 °C. While a wide range of materials are thus amenable to this processing route, most biodegradable polymers are excluded, thereby limiting the utility of particles produced in this manner to drug delivery.
Current efforts in nanoparticle-based drug delivery are utilizing biodegradable polymers such as polyethylene glycol (PEG), polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), and PLGA, the copolymer of PLA and PGA; all of which have a relatively low melting temperature (< 90 °C). Here we extend the in-fiber PRI-based methodology to become a pathway to the scalable fabrication of micro-and nano-particles from low-melting-temperature biodegradable polymers. For the first time, we use the telecommunications-fiber fabrication process of thermal fiber drawing from a preform to draw extended lengths of fiber with a biodegradable polymer (from those listed above) as the core embedded in a thermally matched polymer cladding. After thermal treatment of the drawn fiber, the core breaks up into a sequence of spherical particles shaped under the influence of surface tension, which are then released by selective dissolution of the cladding. As a result, we produce biodegradable particles of controllable diameter independently of the chemistry of the polymer used. Furthermore, inducing the PRI process below 90 °C opens up the possibility of encapsulating or impregnating the particles with biological agents. Challenges of thermally extruding and drawing materials at such a low temperature will be discussed. In addition, we will introduce the design and construction of scaled-down versions of extruders and draw towers typically utilized in fiber mass-production to a scale that may be used in a materials or chemistry research laboratory setting.

Adhesion is an integral part of all cellular functions in adherent cells. Signals are transmitted through focal adhesions on the cell surface to the cystosol, following phosphorylation of tyrosin residues on focal adhesion kinase (FAK). Cytoskeletal elements connect to nuclear lamina by multimeric SUN protein of the nuclear envelope through Nesprin family of proteins and are involved in the deformation of the nucleus. Aim of this study was to test the effects of different materials and microfabricated surfaces. Four different micropatterned templates with different micro-level pillar dimensions were reproduced on PLGA poly(lactic acid-co-glycolic acid) and collagen films. Micropatterned areas on the films contain pillars with 4x4, 8x8 and 16x16 µm squares with 6 µm and 10 µm heights and 4, 8 and 16 µm gaps between the pillars. The films were seeded with human osteosarcoma cells (Saos 2) with a cell density of 5000 cells/ film. Fluorescence micrographs were obtained after staining for cytoskeleton and nucleic acids. CLSM images were obtained after immunostaining for LMNA nuclear lamina together with actin cytoskeleton and DNA. DAPI images were used for analysis of morphometric changes of nucleus morphology with NIH ImageJ software NMA toolbox. Results show that most significant nuclei deformations are observed with pillars with the smallest gaps and also the smaller area pillars tended to bend under cell bodies. On surfaces with of 16 µm gaps the cells tended to locate within the gaps rather than spreading over the tops of the pillars. On surfaces with narrower (4 µm) pillar gaps, cell cytoskeletons were observed to align within the gaps. LMNA immune staining of the nuclear lamina showed that the nuclei bend within the gaps and the DNA content is distributed within these nuclei. This observation supports that SUN protein of the nuclear membrane is connected to the nuclear lamina and the underlying chromatin network. Image analysis showed polydispersity among several parameters of nuclei including surface area, perimeter and aspect ratio in both unpatterned and patterned surfaces. These engineered surfaces coupled with chemical functionalization appears to have a potential for use in the determination of neoplastic potential of cells.

A new, fast, cheap and facile approach was proposed to be able to assemble zeolites on silicon wafer by the help of electron beam lithography (EBL) and direct attachment methods for the first time. Zeolites were attached onto the surface of silicon wafer with a close packed monolayer form by direct attachment method without using any chemical. The novel part of this study is the ability to pattern the surfaces with e-beam lithography system without using electron beam resist. The patterns are directly applied onto the zeolite monolayers by utilizing EBL system. In the patterning process, the energy, which is transferred form electrons onto zeolites, forms a linkage between zeolite and silicon wafer and this is the main concept of electron beam assisted direct zeolite patterning. Substrates were ultrasonicated in ethanol, washed with distilled water and dried under N2 gas. Well defined zeolite patterns were obtained with desired dimensions with this method. Minimum feature size generated in this study was approximately 500 nm which was the dimension of two single zeolite crystals. Patterning different zeolites on surfaces leads different hydrophilic/hydrophobic properties, surface charge densities and etc. on these substrates. The ultimately obtained differences of combined hydrophobic interactions and charge repulsion on a single surface were shown to be two critical forces responsible for the observed adsorption differences of protein molecules, which can be applied for various biomedical applications.

Conventional methods of fabrication of microfluidic devices have centered on silicon and glass. Recently polymer based microfluidic devices have gained popularity due to its low cost, fastness and ease of processing, as well as compatibility with analyses. As an emerging manufacturing technique, nanoimprint lithography (NIL) can fabricate micro and nanoscale features at very high accuracy and reliability. However, the high cost and low throughput of batch mode nanoimprint lithography (NIL) became the limiting factors in meeting industry manufacturing requirements. The roll-to-roll (R2R) NIL technology is emerged as a solution to this issue. In this research, we developed an innovative R2R T-NIL machine for the fabrication of paper based microfluidic devices. The R2R system allows control of imprinting parameters-pressure, imprinting temperature, pre-heating, and feed rate of the web. Microfluidic devices are fabricated on Para-Methoxy-N-methylamphetamine(PMMA) with paper as the substrate material. The mold for this R2R process is based on Polydimethylsiloxane(PDMS) in the aim of producing microfluidic structures at high rate. An innovative air pressure device (APD) was developed and integrated with R2R machine. The APD replaces the conventional 2-roll line contact pressure approach and can cover one third of the surface of the imprinting roller with a uniform pressure (1-3 psi). During the imprinting experiment, a mixture of PMMA (20w %) and 2-Ethoxyethyl acetate is applied on the paper substrate by an inking roller using capillary force and an IR heater is used for pre-heating and drying of polymer layers before it is fed into the imprinting module. Two 500-Watt cartridge heaters are installed on the roller and provide the heat to raise the PMMA film temperature during the imprinting. The effects of process parameters such as imprinting temperature, imprinting pressure, and imprinting time on feature fidelity and uniformity of microfluidic device are investigated systematically.

Fragmented nucleic acids from apoptotic cells can activate immune cells such as macrophages as well as induce cytokines. However, the inappropriate activation of these immune cells can cause a variety of inflammatory and autoimmune disease. Thus, it is important to developing materials that can inhibit the proinflammatory effects of any nucleic acid. Electrospun nanofibrous mesh is a promising material for scavenging because of its enormous surface-to-volume ratio in a limited space. Therefore, in this study, we fabricated super cationic nanofibrous mesh as a molecular scavengers for free nucleic acid. Poly(ε-caproloactone)-branched polyethyleneimine (PCL-BPEI) block copolymer was synthesized by conjugating the primary amines of BPEI to the terminal carboxylic acid groups of PCL. For electrospinning with PCL-BPEI, PCL or PCL-BPEI in mixture of methanol and chloroform was injected at 15kV through 25G needle. In order to manipulate the charge density of the mesh, the surface exposed amine groups of PCL-BPEI nanofibrous mesh were replaced to tri-methyl group. Briefly, the PCL-BPEI nanofibers were swollen by methanol and hydrated in phosphate buffered saline (pH 7.4). Iodomethane in methanol (50mM) was added to the nanofibrous mesh and reacted at 37`C for 48h. We confirmed that PCL and BPEI were conjugated by 1 to 0.5 molar ratios by 1H-NMR. The degree of tri-methylation was quantified by measuring the residual primary amines on the surface of the nanofibrous mesh. After tri-methylation, the amount of residual amine groups was 0.18nmol per device, suggesting that 87.5% of surface-exposed amine group was replaced to tri-methyl group compared to the initial amount of surface-exposed amine groups on the PCL-BPEI nanofiber (4.71nmol/device). We also characterized the surface of tri-methylation nanofiber by Raman spectroscopy. The band of tri-methyl group, at 3062cm-1, appeared result from tri-methylation of amine group on PCL-BPEI nanofiber. Additionally, the water contact angle of tri-methylaiton nanofiber was decreased to 43.2` compared to PCL-BPEI nanofiber (113.9`). We thus speculated that the tri-methyl group of nanofibrous mesh can significantly increase the cationic charges on the nanofiber and that electrostatic interactions toward anionic molecules such as nucleic acids can be also enhanced. In order to confirm the scavenging effect for nucleic acids, various amount of DNA was added to the nanofibrous mesh and the incorporation efficiency was calculated. The binding efficiency of DNA was 3.5%, 28.3% and 54.6% in PCL nanofiber, PCL-BPEI nanofiber and tri-methylation nanofiber, respectively. Thus, the charge density on the nanofibrous mesh could be controlled by tri-methylation and this super cationic nanofibrous mesh can maximize the binding efficiency of nucleic acids.

In the hospital environment, hand washing has been identified as the most significant manner towards preventing the spread of microbial infections, with hand drying as the critical last stage of the hand washing process. In some circumstances, such as for paper towels hanging in sink splash zones or those used to clean surfaces, they have been considered as potential sources of bacteria contamination. In this study, for the first time, selenium nanoparticles were coated on normal paper towel surfaces through a quick precipitation method. In addition, their effectiveness at preventing biofilm formation was tested in bacterial assays involving Staphyloccocus aureus and Pseudomonas aeruginosa. The results of bacteria tests showed high effectiveness for the selenium coated paper towels at inhibiting bacteria growth on the paper towel surfaces. The selenium coatings significantly inhibited S. aureus growth by about 90% after 24, 48 or 72 hours and also successfully inhibited P. aeruginosa growth after 48 or 72 hours by 55% and 84%, respectively, on the surface of paper towels. Moreover, from the 24 hour culture time to the 48 hour culture time, there was an increase in bacteria numbers on uncoated paper towel samples, but was constant to the 72 hour culture time, implying that the uncoated paper towel was saturated by bacteria after 48 hours of treatment. In contrast, bacteria numbers on the selenium coated paper towels remained at a lower level not increasing from 24 to 48 to 72 hours, indicating successful inhibition of bacterial growth. Overall, the effectiveness of selenium coated paper towels inhibiting bacterial growth reached about 80-90% after 3 days compared with the uncoated paper towels. Importantly, this was accomplished without using antibiotics. This study suggests that selenium nanoparticle coatings could be used as an effective way to decrease bacterial infections (specifically, by S. aureus and P. aeruginosa) on paper products, which might have potentially important applications in the food packaging industry, medicine, and in clinical environments.

Ag is one important metal which has novel optical properties in the nanoscale size regime. Ag nanoparticles show a wide range of colors corresponding to their localized surface plasmon resonance. In addition, Ag has an exceptionally high extinction coefficient associated with very high enhancement ability in Raman spectroscopy. These characteristics have made it an ideal candidate for use as a probe in sensing and biodiagnostics applications. Combination of Ag and other precious metals in a single particle has become an attractive trend to limit its disadvantages, such as easy oxidation, difficulty in aqueous synthesis, sensitive to ionic environment, etc. In addition, heterostructuring could modify and enhance characteristics of Ag in diagnosis and treatment for biomedical sensors. One of the primary highlights for this combination is Pt-Ag system for not only SERS and plasmonic properties but also electro-catalytic activity. In our recent results in the study of Pt@Ag core@shell nanoparticles with controllable size and shell thickness, the characteristics of NPs are induced by unique electron-transfer effect which will provide a useful tool for processing and designing materials. In addition, SERS and plasmonic properties of Pt@Ag core@shell nanoparticles are interesting and promising for a tunable nature that can be used in diagnostic and biosensing applications. Results are discussed in terms of UV-Vis, XRD, TEM, HR-TEM, EDS, XPS, Raman scattering and HAADF-STEM.

Electrospun nanofiber matrices have been produced using natural and synthetic polymers for a variety of biomedical applications. However, electrospinning of water soluble polymers still remains as a major challenge. Polysaccharides are difficult to spin and often mixed with other synthetic polymers to produce electrospun nanofiber matrices. Chitosan, the deacetylated form of chitin, is reported to be biocompatible, biodegradable, antimicrobial and non-toxic polysaccharide and thus used for a variety of biomedical applications including tissue engineering, drug delivery device and wound healing. Chitosan electrospinning remains challenging due to limited solubility and a rigid crystalline structure which does not allow sufficient polymer concentrations required for successful fiber formation by electrospinning. Here we present a novel and “smart” chitosan modification methodology that allows direct electrospinning of high molecular weight neat chitosan at very high solution concentrations. In brief, to facilitate chitosan electrospinning a new 2-nitrobenzyl-chitosan derivative was synthesized and electrospun nanofibers were produced by dissolving the derivative in TFA solvent. In this derivative 2-nitrobenzyl aldehyde is used as a photolytic removal group to block the amino group of chitosan and subsequently reduce its structural rigidity. The electrospun nanofiber matrices produced by 2-nitrobenzyl-chitosan was exposed to UV light to produce pure chitosan nanofiber matrix. Electrospinning parameters were optimized to produce defect free cylindrical nanofibers at 15% (wt/v) chitosan solution concentration and 1 kV/cm electrical potential. In this work we report on electrospinning of three different 2-Nitrobenzyl-chitosan compositions namely 1:1, 1:0.5, and 1:0.25 and evaluating chitosan matrices for scaffold properties and cell compatibility. The mechanism of photolysis to obtain neat chitosan from 2-Nitrobenzyl-chitosan followed by UV exposure was confirmed by FTIR analysis. The morphology of the electrospun fibers, pore structure, and fiber diameter was examined using scanning electron microscopy. Biocompatibility was evaluated by measuring cell proliferation and metabolic activity of MC3T3 cells seeded onto the nanofiber scaffolds

We have developed an all-inorganic, enzyme-mimicking electrocatalyst composed of a submicron magnetite (Fe3O4) bead decorated with Ag-Pd alloy nanocages. The Fe3O4@Ag-Pd hybrid particles can be used for highly efficient electrocatalytic oxidization of organic dyes instead of peroxidase enzymes even in the absence of H2O2. The Fe3O4 is capable of catalyzing the electrochemical reduction of the substrates due to its enzyme-mimicking properties, while the Ag-Pd nanocages dramatically amplifies the resulting catalytic currents due to their hollow interiors and porous walls. The as-prepared Fe3O4@Ag-Pd hybrid particles show greatly improved stability in electrocatalytic activity than peroxidase over a broad range of pH and temperature. Based on this finding, we have developed a novel electrochemical cytosensor, which involves the aptamer-functionalized nitrogen-doped graphene nanocomposites electrode interface and the cell-targeting aptamer-functionalized Fe3O4@Ag-Pd nanoprobes, toward ultrasensitive detection of circular tumor cells (CTC). A superstructured sandwich-type sensor geometry is adopted for electrochemical cytosensing, with the cells of interest sandwiched between the nanoprobes and the electrode interface. Such electrochemical sensing strategy allows for ultrasensitive CTC cytosensing with detection limit as low as ~ 10 cells mL-1. This nanotechnology-enabled platform provides a powerful tool for the study of cancer progression and metastasis and holds great potential in early diagnosis of cancer.

Much recent biomedical research has been focused on advancing prosthetic implants to give the patient increased mobility, reduced pain and faster rehabilitation. One of the most important aspects in the successful implementation of biomaterials is the characterisation of the external coating. Hydroxyapatite (HA) is the most attractive and widely used material as a bioactive surface layer for orthopedic implant applications due to its potential for inducing osteoconduction and osseointegration. However, the thermal treatment of HA before and after deposition creates many microstructural defects, due to the recrystallization, such as porosity, cracks, non-uniform grain size and grain size distribution which causes distinctive variations in its mechanical strength at different locations on the implant. This, coupled with extremely rough implant surfaces, makes it very difficult to extract the properties of the material and determine how it is exactly adhered to the substrate.
We use novel nanoindentation techniques to determine the mechanical properties of the material and to map the stiffness of the inhomogeneous material at different depths. The data extracted through indentation are parameters that are strongly dependant on tip geometry, material geometry and elastic and inelastic tip behaviour. The geometry of biomedical implants as well as the material thickness makes characterisation of materials very difficult. We use a Berkovich tip to extract local data which allows us to statically analyse the material properties and use cross section analysis to map the material.
By adapting this technology, we manufacture cantilevers of HA, which are attached to the rough substrate and use the nanoindenter to determine the interfacial adhesion of the material. This is done by propagating an interfacial crack and debonding the material from the substrate. The main advantage of this technique is the control we have over the process; we can move the indenter tip at speeds as low as nm/s and get a precise data from the debonding process.

Counterfeit drugs cause a large number of injury, disability and death in the past decades, and are responsible for profit loss of drug manufactures. Incorporating markers in drug formulation has been proposed as a major step forward. Simple methods via color and shape recognitions, composition/chemical analysis, printing and packaging technologies have been used for authentication purposes, unfortunately, these approaches are too vulnerable to counterfeiting. More sophisticated approaches such as graphical encoding achieved by state-of-the-art lithographic techniques or embedded images in hologhaphic images have been developed. However, code information stored by these taggants may be lost once the taggants are damaged. Additionally, high cost production makes these taggants less attractive to pharmaceutical companies. Phase change materials have received significant interest in many thermal applications. During the melting process, a large amount of heat is absorbed and used to transform a solid to liquid without rising temperature, characteristic melting peak of a PCM can be observed in a linear thermal scan by differential scanning calorimetry (DSC). Many organic PCMs such as poly-waxes or fatty acids are well known for their biocompability, high latent heat of fusion and especially, most of them are on the approval lists of the Food and Drug Administration (FDA). In this work, we present a new platform of covert taggant based on phase change particles for anti-counterfeiting medicine. We have synthesized and encoded acetaminophen with four different organic PCMs: stearic acid (SA), 12-hydroxydodecanoic acid (12-HDA), polywax 1000 and polywax 3000. The physical appearance of the encoded drug is indistinguishable compared to that of before mixing. In addition, cytotoxicity study confirms that these particles do not have side effects on the viability of cells, indicating the biocompatibility of the organic PCM particles. Authenticating of genuine drugs can be achieved by matching melting peaks of encoded particles with the standard values. Decoding requires very small amount of material (5-10mg) with the taggant loading as small as 1 wt.% and the process takes less than 5 minutes, but can be shorten if higher ramp rate is used. We believe the low cost of production, ease in encoding, simplicity in decoding of our thermal taggant platform make it become a powerful solution to the on-dose authentication of drugs that is urgently needed for pharmaceuticals.

We present a novel detection scheme for biological analytes based on antibody-functionalized silicon nanowires utilizing a miniature Wheatstone bridge.¹ Silicon nanowire (SiNW) field-effect transistors² are promising candidates for point-of-care diagnostics and personalized medicine. They have been used to detect glucose,³ biomarkers for various cancers,⁴ as well as myocardial infarction and viruses.
Due to their high surface-to-volume ratio, SiNW devices are susceptible to surface effects, such as the binding of molecules and related induced changes in the charge density in the biofunctionalized layer on the nanowire surface. Associated with their small size and large response is an amplification of noise due to changes in the fluid dynamics, temperature effects, electrical noise and chemical changes of the gate oxide. In order to reach their full sensing potential, the signal-to-noise ratio (SNR) needs to be increased. For that purpose, we devised a lithographically fabricated Wheatstone bridge with 4 sets of wires as arms. The sensing set of wires and the reference set of wires are exposed to the fluid and the other two sets of wires are tunable by top gates to enable balancing of the bridge. In this case, the output voltage is zero and hence, the signal can be greatly amplified.
We showed that the SNR can be enhanced in this setup with respect to conventional single wire setups by filtering out background, signal drift, and effects of thermal fluctuations and the dynamics of fluid flow. We performed a detailed analysis for the sensing of pH with increased SNR by a factor of 5 and overall gain in signal stability with the balanced bridge. We also showed the detection of TROY, which is a novel melanoma biomarker that is suspected to be shedded into blood by circulating tumor cells and thereby can serve as a surrogate marker for the presence of these cells. Our goal is to measure abnormally elevated levels of TROY in serum of melanoma patients before metastasis becomes clinically detectable, and subsequently incurable. Using the nanowire Wheatstone bridge, the detection limit was enhanced by an order of magnitude with respect to a conventional differential conductance measurement. We thank Larry House, Battelle Memorial Institute, and the National Science Foundation for support.
¹C. Maedler, S. Erramilli, L. J. House, M. K. Hong and P. Mohanty, Appl. Phys. Lett.102, 043112 (2013).
²Y. Chen, X. Wang, M. K. Hong, S. Erramilli, P. Mohanty and C. Rosenberg, Appl. Phys. Lett.91, 243511 (2007).
³X. Wang, Y. Chen, K. A. Gibney, S. Erramilli, and P. Mohanty, Appl. Phys. Lett.92, 013903 (2008).
⁴Y. Chen, X. Wang, M. K. Hong, C. L. Rosenberg, B. M. Reinhard, S. Erramilli and P. Mohanty, Appl. Phys. Lett.97, 233702 (2010).

Surface charge is known to influence cell adhesion and proliferation. For example, poled hydroxyapatite (HA) has been shown to accelerate attachment and proliferation of osteoblast cells. However, the process by which HA is fabricated can lead to variations in Ca/P ratios, surface roughness, and surface potentials, all of which will impact the results obtained. Here, we use a model template with natural permanent polarization to study the effect of surface charge on osteoblast proliferation. Lithium niobate (LN) is a ferroelectric material which we show has the potential for clinical relevancy. LN has the advantage of controllable polarization via the application of an electrical bias and through surface modification via chemical patterning, i.e., proton exchange. LN is also optically transparent, which is ideal for confocal fluorescence microscopy and is polished optically flat (typical surface roughness of 2 nm but can be further tailored using various chemical etching techniques).
The effect of LN surface charge on cells proliferation and mineralization was studied by culturing with an osteoblast precursor cell line (MC3T3). Firstly, we have shown that LN is biocompatible in vitro and the cells proliferate past 11 days. Secondly, we show that even without processing, charged (z-cut) LN templates enhanced attachment and proliferation of osteoblasts compared to electrostatically neutral (x-cut) LN samples and control glass slides. By processing the material with proton exchange and reactive ion etching we can create nanostructures on the surface which can aid the preferential alignment of osteoblasts. We also show that by exploiting the photovoltaic nature of LN to photoreduce silver and gold nanoparticles on the surface we can combine cell fluorescence and surface enhanced Raman for potential cell sensing.

Transient electronics represents an emerging class of technology whose key characteristic is that it physically disappears, in whole or in part, in a controlled fashion after it has served its targeted function.[1] Devices with this property provide capabilities that complement those of traditional integrated circuits, enabling applications for implantable medical sensors that can degrade inside the body after service to avoid second surgery. Here, the constituent materials must be carefully formulated and integrated together to accommodate a desired transient process, such as dissolution in biofluids.
Silicon and silicon oxides which undergo hydrolysis in basic aqueous conditions, are attractive choices for the semiconductor and dielectric materials respectively. Conductive material, is another essential component for water-soluble electronic devices. Biodegradable metals thin films (e.g., Mg, etc) fabricated from physical vapor deposition are therefore studied to assess the potential for use of these metals in transient electronics. Reactive dissolution and its effects on electrical conduction, morphological change and chemical transformation in these thin films in de-ionized (DI) water and simulated body fluids (Hanks&’ solution pH 5-8) are systematically investigated. The results indicate that the electrical dissolution rates in thin films can be much different that traditionally reported corrosion rates in corresponding bulk materials. Silicon metal oxide field effect transistors (MOSFETs) built with these metals through micro-fabrication process demonstrate feasibility for use in transient electronics. Degradation at device level of integrated circuit with transient metals transfer printed on biodegradable substrate is also demonstrated in simulated body fluids.
Reference
[1] Hwang, S.-W., H. Tao, D.-H. Kim, H. Cheng, J.-K. Song, E. Rill, M.A. Brenckle, B. Panilaitis, S.M. Won, Y.-S. Kim, Y.M. Song, K.J. Yu, A. Ameen, R. Li, Y. Su, M. Yang, D.L. Kaplan, M.R. Zakin, M.J. Slepian, Y. Huang, F.G. Omenetto, and J.A. Rogers. Science, 2012. 337(6102): p. 1640-1644.

Silk-based porous scaffolds have been used extensively in tissue engineering because of their excellent biocompatibility, tunable biodegradability and robust mechanical properties. Although many silk-based scaffolds have been prepared through freeze-drying, a challenge remains to effectively control the porous structures during this process. In the present study silk fibroin with different nanostructures were self-assembled in aqueous solution by repeat drying-dissolving process and then used to improve the porous structure formation in lyophilization process. Viscosity, secondary structures and water interactions were also studied to elucidate their influence on the formation and control of porous structures. Following nanofiber formation in aqueous solution, silk scaffolds with improved porous structure were directly formed after lyophilization and then stabilized with water or methanol annealing treatments. Compared to silk scaffolds derived from fresh solution, the nanofibrous scaffolds showed significantly improved cell compatibility in vitro. Therefore, this nanoscale control of silk offers feasible way to regulate the matrix features including porous structure and nanostructure, which are important in regulating cell and tissue outcomes in tissue engineering and regeneration, and then achieve silk-based scaffolds with improved properties.

Thermoplastic polymer blends were prepared from polytrimethylene terephthalate [PTT] and polyamide 6,10 [PA6,10] by melt processing in a Brabender mixer to assess the morphology and the degree of immiscibility developed between the domains. PTT and PA6,10 were selected as a pair of engineering polymers with complementary properties and as polymers that could be prepared substantially from bio-based precursors. We estimate that blends near the 50/50 composition will possess a bio-based content of nearly 50%. In addition the numerous functional groups of significant dipole character in both of these materials present excellent opportunities for utilization in biomechanical structures. The broader objective of our overall studies is focused on developing engineering blends with good stiffness, strength, and dimensional stability while simultaneously simplifying processing.
In our current work, the blend homogeneity was evaluated by preparing blends in a designed experimental matrix with the experimental factors of mixing time and temperature in the range of t=0-25 minutes and T=240-260 C. Differential scanning calorimetry was conducted to define the melting and cold crystallization behaviors, but more importantly to assess the degree of immiscibility by identifying the glass transition temperatures of the component polymers as well as any additional phases formed during processing. Subsequent to processing at the indicated times and temperatures, samples were cold-fractured to reveal morphology, domain characteristics, and the nature of the blend interfaces. Optical microscopy and SEM were used to capture images and a standard image analysis program was used to characterize the morphology both in terms co-continuity and domain size as the mixing parameters varied. Principal results of this work show that domain size decreases strongly with mixing time and temperature such that domain size, originally about 2 micrometers at the lower end of the experimental range, is reduced smoothly and monotonically over the experimental range to about 100 nanometers at the highest temperatures and longest mixing times. Image analysis allowed for the detection of immiscibility and the occurrence of phase inversion. Further differentiation of the polymer blend structure was determined using energy-dispersive spectroscopy [EDS] to identify the nitrogen-rich amide functional groups in the PA6,10. In addition to morphology assessment using SEM, integrated torque/mixing time curves were evaluated to compute dispersive energy imparted to the blends during processing.
Preliminary mechanical testing results are presented for the first time in this paper. Although we ultimately seek comprehensive mechanical characterization, the initial elastic modulus data show strong linkages between the phases and the composite moduli are interesting and important for the engineering of bio-inspired materials.

Nanofluidic channels are important for a host of biological and chemical applications, including manipulating and sensing DNA molecules. Although fluidic channels with nano-scale dimensions have been demonstrated, the development of a wafer-scale nanochannel process, compatible with CMOS devices, has been challenging. Here, we report a 200 mm wafer-scale sub-20 nm nanochannel fabrication method that enables stretching, translocation, and real-time fluorescence imaging of single DNA molecules, compatible with CMOS circuits and targeting lab-on-a-chip applications. Given a well-established Si patterning technology, sub-20 nm Si patterns are converted into nanochannel structures through the highly-selective isotropic XeF2 etching process. An integration scheme was developed to yield a fluidic chip comprising macro-scale fluidic ports, micro-scale fluidic feed-channels, and nano-scale channels for DNA imaging, by combining different scale lithography methods, including mid-UV (MUV), deep-UV (DUV), and e-beam lithography (EBL). In addition, gradient nanopillars were located within the channels to stretch DNA molecules, which are subsequently delivered into the nanochannels and imaged by fluorescence microscope. Our method offers a manufacturable approach to producing biochips for a wide spectrum of bio-applications, such as DNA sequencing, drug delivery, and molecular detection.

For ultrafast transdermal drug delivery (TDD), we propose the spiral-shaped microneedle patch inspired by a unique structure of venomous snake&’s fang to minimize infection by transcutaneous pierced wound after administrating the patch on skin.
Previously introduced microneedle systems deliver the drug through the encapsulation or coating of polymer microneedle; those systems supply the drug across skin by diffusion. However, the microneedles relying on diffusion for TDD cannot provide fast drug release and short-term opening on skin which is required to prevent infection of diseases. Therefore, the creation of microneedle which can release drug in the fast (within a few second) and simple manner has been a great interest.
To address this issue, we propose a snake-fang-inspired microneedle through which drugs are delivered into target skin tissue within a second by capillary force. The envenomation mechanism of venomous snakes provides critical clues to address this challenge. In the majority of venomous snakes, and many other reptiles, venom is conveyed from the animal&’s gland to the prey&’s tissue through an open groove on the surface of the teeth and not through a tubular fang. There are two key aspects of the unique structural characteristic of the grooved delivery system: first, the hydrodynamics of venom as it interacts with the groove geometry and second, the efficiency of the tooth-groove-venom complex as the tooth penetrates the prey&’s tissue. The surface tension of the venom is the driving force underlying the envenomation dynamics.

In this research, we mimic these unique structural properties of envenomation. The newly designed microneedles rapidly delivered the liquid type drug into target skin tissue due to the minimization of the drug&’s surface-tension energy. To verify our snake-fang-inspired microneedles, we conducted vaccination with inactivated influenza virus and ovalbumin to mice in which microneedle was inserted for 3 seconds. After vaccination, immunoglobulin level was highly increased compared to syringe injection manner. Furthermore, the pierced wound on skin after administration was found to be promptly closed, minimizing any infection.
We expect that our suggested ultrafast drug delivery system will greatly contribute in the field of vaccination, drug research and practical applications.

The scaffold with appropiate mechanical, biodegrable and biocompatibility properties plays a key role in bone tissue engineering and regeneration. In this study, a facile and green starch-gel foaming method was developed for fabricating biodegradable ceramic composite scaffolds with desirable properties at low temperatures. The method utilizes the gelatinization and retrogradation properties of modified starch, which allows starch-hydroxyapatite suspension to transform into porous scaffolds via foaming and drying processes at temperatures lower than 90°C. Before drying, the scaffold can be machined or cast into various shapes. Porosity control of the scaffold was achieved by altering the solid contents in the starch-hydroxyapatite suspension and the highest apparent porosity of 64% was obtained. The interconnectivity of the pores in the scaffold was confirmed by micro-CT measurment with 3-D reconstruction of the pore structures. Uniaxial compression tests revealed that the yielding strengths of the dried porous scaffolds were between 2~9 MPa depending on the porosity, which are close to or better than that of cancellous bones. The biocompatibility of the scaffolds for hosting tissue cells was evaluated by osteoblast (bone forming cell) viability tests up to three days and the results indicates good biocompatibiliy and osteocondutivity. The results demonstrate that the starch-gel foaming method is a green and facile approach for fabricating high strength and biocompatible bone substitution scaffold at low temperatures.

The combination of force and flexibility enables controlled, soft movements in organisms. In sharp contrast, presently used human-made machines are solid and mostly based on stiff driveshafts or cog wheels. Magnetic elastomers were realized through controlled, reliable dispersion of nano- and micron metal particles in polymer matrices and have attracted significant interest as soft actuators for controlled movement or conveying and are particularly attractive candidates for magnetic pump applications [1-3].
The human body contains a number of highly efficient, non-classical, non-rotatory pumps. In this project, we were inspired by the skeletal-muscle pumps that aid in the blood circulation by venous contraction and designed a new, flexible and highly magnetic, active tube. Venous contractions cause a sufficiently large pressure change in our body to create a vacuum that then draws blood (1st half of duty cycle) which is pumped towards the heart during the contraction (2nd part of a duty cycle). Clinically well accepted medical grade silicon (polydimethylsiloxane, PDMS) was used as a soft and flexible polymer matrix for the present study. We then demonstrate the successful incorporation of up to 75 wt% metal particles (carbonyl metal iron) in silicone, resulting in stable, flexible and homogeneous magnetic composites [4] of considerable bulk magnetization. In order to demonstrate the application of iron doped silicone tubes, a magnetic peristaltic pump similar to the skeletal-muscle pump was designed and operated in various continuous modes (type of contractions with up to 4 locations of contractions in a single tube; frequency of compaction/release).
Our study demonstrates a reliable use of soft and flexible magnetic silicones for peristaltic pumps with very low shear stress and without any moving parts, suggesting their use for pumping of sensitive fluids, such as human blood or large scale cell cultures in biotechnological production.
Reference:
1) P. M. Xulu, G. Filipcsei, M. Zrinyi, Macromolecules 2000, 33, 1716.
2) R. Fuhrer, E.K. Athanassiou, N.A. Luechinger, W.J. Stark, Small, 2009, 5(3), 383
3) R. T. Olsson, M. Samir, G. Salazar-Alvarez, L. Belova, V. Strom, L. A. Berglund, O. Ikkala, J. Nogues, U. W. Gedde, Nat. Nanotechnol. 2010, 5, 584.
4) R. Fuhrer, C. M. Schumacher, M. Zeltner and W.J. Stark, Adv. Funct. Mater. 2013, doi: 10.1002/adfm.201203572

Uniformly-sized, nanostructured peptide microparticles were generated by exploiting the ability of enzymes to serve both as catalysts, to control self-assembly within monodisperse, surfactant-stabilized water-in-oil microdroplets, and as destabilizers of emulsion interfaces, to enable facile transfer of the produced microparticles to water. This approach combines the advantages of biocatalytic self-assembly with the compartmentalization properties enabled by droplet microfluidics. Firstly, using microfluidic techniques, precursors of self-assembling peptide derivatives and enzymes were mixed in the microdroplets which upon catalytic conversion underwent molecular self-assembly into peptide crystals or gel particles, depending on the chemical nature of the precursors. In addition, due to their amphiphilic nature, enzymes adsorbed at the water-surfactant-oil interface of the droplets, inducing the transfer of peptide microparticles from the oil to the aqueous phase. Ultimately, through washing steps, enzymes could be removed from the microparticles which resulted in uniformely-sized particles composed of either gel phase or crystalline, nanostructured aromatic peptide amphiphiles.

Freeze casting is a novel fabrication method for the production of highly porous hybrid materials with a fine hierarchical architecture based on the directional solidification of a polymeric solution or ceramic particle slurry. The process involves freezing of a water-based colloidal system under conditions that promote the formation of lamellar or columnar ice. It allows the fabrication of porous bulk materials (several cm^3) or entire components, including those with a complex shape, and therefore has become an ideal route for the manufacturing of biomedical devices. Freeze casting is a two-step process. First, a water-based polymeric solution or ceramic particle slurry is directionally solidified and “ice-templated” when phases separate into ice and a vitrified polymer or ceramic-polymer composite. In the second step, the frozen material is lyophilized where the ice crystals are removed by sublimation, leaving behind the vitrified polymer or ceramic-polymer composites dried further through the removal of bound water. Both steps combined determine the structure and properties of the overall freeze-cast scaffold and its cell wall properties.
Of great importance in freeze-cast scaffolds are the scaffolds&’ mechanical properties which are determined by those of the cell wall material and the cell wall structure; all of those also affect cell adhesion and growth. We, therefore, report the cell wall properties during freeze casting using various materials characterization methods. After initial qualitative observations of component self-assembly in the cell walls of freeze-cast scaffolds though optical and scanning electron microscopy, we applied synchrotron X-ray techniques (small angle X-ray scattering, wide angle X-ray scattering and full-field transmission X-ray microscopy) to investigate this phenomenon systematically both in-situ during directional solidification and afterwards on freeze-cast and lyophilized scaffolds. In-situ freezing of polymer and ceramic particles was performed with a Hele-Shaw cell-like setup for directional solidification. Diffraction data from SAXS and WAXS measurements revealed that the scaffolds exhibited three structural layers, namely random layer at the bottom, partially-organized layer from 2 mm above the cooling source, and a well-aligned layer from 6 mm above the cooling source. A FIB cut in the well-aligned layer illustrated the ability of the ice-templating process with which a nacre-like wall structure is obtained. 3D morphology information from X-ray nano-tomography experiments showed, in combination with electron microscopy, the overall porosity and nanoporosity in the cell wall of the well-aligned layer.

Mercury has been known to be highly toxic metal and can pose serious dangers to the environment and human health. Therefore, monitoring the mercury concentration is an extremely important issue to prevent such toxic metal from endangering human life. In this paper, we demonstrate that the principle of triboelectric nanogenerator (TENG) can be used as a sensing system for the detection of mercury ions (Hg2+). Our first step is to improve the performance of the TENG through the assembly of Au nanoparticles (NPs) onto the metal plate. These assembled Au NPs not only act as steady gaps between the two plates at strain free condition, but also enable the function of enlarging the contact area of the two plates, which will increase the electric output of the TENG. Through further modification of 3-mercaptopropionic acid molecules on the assembled Au NPs, the high-output nanogenerator can become a highly sensitive and selective nanosensor to determine Hg2+ ions because of the different triboelectric polarity of Au NPs and Hg2+ ions. On the basis of this unique structure, the output voltage and current of the TENG reached 105 V and 63 µA with an effective dimension of 1 cm x 1 cm. Under optimum conditions, this TENG is selective for the detection of Hg2+ ions, with a detection limit of 30 nM and linear range from 100 nM to 5 µM. The commercial LED lamp was tested as the indicator to replace the expensive electrometer and showed the potential to simplify the detection system. With the advantages of simplicity, low cost, high sensitivity and high selectivity, this self-powered and stand-alone triboelectric nanosensor demonstrates its abundant potential to be a next-generation environmental sensor for the eco-minded future.
References:
1. Lin, Z.-H.; Zhu, G.; Zhou, Y. S.; Yang, Y.; Bai, P.; Chen, J.; Wang, Z. L. Angew.
Chem. Int. Ed. 2013, 52, 5065-5069.
2. Zhu, G.; Lin, Z.-H.; Jing, Q.; Bai, P.; Pan, C.; Yang, Y.; Zhou, Y.; Wang, Z. L. Nano
Lett. 2013, 13, 847-853
3. Lin, Z.-H.; Xie, Y.; Yang, Y.; Wang, S.; Zhu, G.; Wang, Z. L. ACS Nano 2013, 7,
4554-4560.
4. Fan, F.-R.; Lin, L.; Zhu, G.; Wu, W.; Zhang, R.; Wang, Z. L. Nano
Lett. 2012, 12, 3109-3114.

Fungal keratitis is a leading cause of ocular morbidity throughout the world, and it is also a major eye disease that leads to blindness in Asia. Fungal keratitis is mainly caused by yeast-like fungi (such as C. albicans) and filamentous fungi. Current therapies against fungal keratitis are often ineffective due to several reasons. (i) Fungal keratitis infection often exists as a biofilm, which is particularly difficult to clear because of its encasement in a protective and impermeable extracellular matrix. Much higher doses of antimicrobials are needed for biofilm clearance as compared to planktonic microbials. (ii) Shortage of broad-spectrum efficient antifungi drugs. Current clinical drugs for fungal keratitis include azole compounds (such as fluconazole) and polyenes (such as amphotericin B). The fungistatic nature of azole compounds, which function by enzyme inhibition, requires a prolonged course of application. Polyenes, which function via disrupting the permeability of ions through the cell membrane, are rather expensive. Due to their poor penetration property, solubility and stability, they are limited in application. These limitations, together with the development of drug resistance, have led to the low efficacy and unsatisfactory outcome associated with the current therapies for fungal keratitis.
We have developed amphiphilic main-chain imidazolium polymer (PIM-45) and oligomer (IBN-1) materials that can efficiently inhibit the growth of fungi with low minimal inhibition concentration values, and successfully clear the fungal biofilm. In order to test keratitis treatment effects, fungi keratitis disease model was induced on mice. In vivo keratitis treatment indicates that topical solutions of these polyimidazolium salts are safe and as effective as that of amphotericin B, the most commonly used agent for the treatment of C.albicans keratitis. Compared to the costly and unstable amphotericin B and fluconazole, PIM-45 and IBN-1 are easy to prepare, inexpensive and stable. They can be stored in phosphate-buffered saline solutions with long shelf life for routine topical use.

In this study, we proposed and demonstrated a novel analysis of bio-sensing system on allergy test using a semiconductor-based field effect transistor (FET). Allergy is one of the closest diseases at the present day. Almost half of the population are affected with some type of allergy in Japan, and the number of patients are increasing throughout the world. There are still a lot of questions on its mechanism that remain unexplained and strong demands for approach to treatment. However, its precise diagnosis requires not only a lot of time but also collection of large amounts of blood from patients. Therefore, easy-to-use evaluation approaches are expected in clinical practice and drug discovery. Diagnostic test kit lacks precision because it assesses only the presence of antibody using enzyme. This study focused on type I reaction, the immediate allergic reaction such as food allergy and pollen allergy. We detected the cellular activities based on antigen-antibody reaction at mast cells in an easy, quantitative, economic and label-free way by monitoring its change in respiration and chemical release using FET. Mast cell that we used in this study was Rat basophilic leukemia (RBL), which has been commonly used in allergy studies, and IgE antibody had been added before antigen was introduced. RBL was adhered to the gate sensing surface of the cell-based FET. The principles of cell-based FETs was based on the detection of charge density changes such as pH variation at the interface between the cell membrane and the gate surface. The gate surface potential of IgE-RBL-based FET decreased continuously and specifically after introduction of antigen, the potential reached its bottom after 10 to 20 min. This result indicates that the electrical activity of RBL was successfully monitored on the basis of pH changes, i.e., decrease in the concentration of hydrogen ions, at the cell/gate interface using the IgE-RBL-based FET. Generically, histamine secretion from RBL following the antigen-antibody reaction is the key factor to cause allergy action. β-hexosaminidase, which is widely used for evaluating histamine was fluorescently labeled in medium after the reaction. Thus, we assume that the pH variation based on hydrogen ion accumulation at the cell/gate interface was mainly induced by basophilic histamine secretion following antigen addition. From now on, we are focusing on the cell/gate interface for the further study and considering the highly-responsive method by additional procedures using an extended-gate FET whose surface is designed with gold to make it responsive to a specific molecular. The platform based on the field-effect devices is suitable for application in a real-time, and label-free detection system for allergy test.

The synthesis of gold nanocages via galvanization has drawn attention from multiple areas. Due to their tunable localized surface plasmon resonance (LSPR), high surface-to-volume ratio and hollowed structure, their biomedical potential has been studied in areas like drug delivery, bio-imaging and photothermal blast of tumors. Meanwhile, the hollow inside a nanocage can serve as a protective protein immobilizer. Combined with their other properties, gold-nanocage protein immobilizers can have great potential in a variety of biomedical applications ranging from biomedicine delivery and sensors to functioning surfaces that resist microbial fouling. However, due to the difficulties in the delivery of proteins into the nanocage hollow, their applicability for protein delivery has not yet been thoroughly investigated,.
In this research, based on our previous studies on gold nanoparticle-protein nanobioconjugates, we developed a methodology that not only is able to deliver enzymes into nanocages, but also is able to characterize the behavior of the immobilized enzymes. Precise morphological controls and surface chemical modifications were incorporated into the synthetic procedure to obtain protein uptake surfaces with appropriate topography and surface chemistry. Lysozyme was used in our study to probe the efficacy of nanocage&’s protein internalization. Rigorous approaches were adopted to characterize the synthesized nanocages in order to investigate their geometry in detail. In subsequent protein experiments, non-hollowed gold nanocube particles, which are very similar to the nanocages in size and surface modification, were used as a reference. Lysozyme adsorption isotherms were obtained for both types of nanoparticles to measure their relative affinities to the proteins. The nanobioconjuagtes&’ activities were then assayed with two substrates that have very different sizes, so that the activity of the lysozyme that immobilized inside nanocages could be deconvoluted. Moreover, a monolithic protease was used to digest the immobilized lysozyme to test the efficacy of nanocages to protect the encapsulated proteins.
According to our study, lysozyme was not only able to be delivered and immobilized at the inside of the nanocage, but also able to maintain a significant part of its native activity, as well as stay active after the digestion of monolithic protease. Moreover, compared with lysozyme that adsorbed at the external surfaces, the lysozyme inside nanocages were apparently more active. This phenomenon is believed due to the concave surfaces inside nanocages that altered the surface-protein interactions. This study demonstrated the potential of gold nanocages in protein delivery and also render them to be a promising platform to study nanostructure-biomolecule interactions at concave and confined surfaces.
This work is supported by the US National Science Foundation under Grant No. DMR-0642573.

Custom-made biomedical implants made of bioresorbable materials are in great demand in reconstructive surgery. These implants should overcome common drawbacks of currently commercially available products such as brittleness, incompressibility and difficulty in shaping. In this context, electrospinning allows the production of shapeable and porous materials. Their porosity plays an important role when adaptation to the different requirements in tissue engineering is intended.
Various techniques are available to tailor the porosity of the electrospun fibrous implants. For example, we increased the porosity from 79% to 95% by low-temperature electrospinning using dry ice that renders the material cotton wool-like [1]. If scaffold design needs reduction of porosity, dry heat treatment is suited to obtain the desired features regarding structure and morphology (porosity: 40%) [2]. Herein, the influence of these modifications on the mechanical properties of the electrospun scaffolds was investigated.
We further complemented the polymeric cotton wool with amorphous, flame spray-derived bioceramics such as calcium phosphate (a-CaP) nanoparticles [3]. The latter present advantages in terms of size, accessible composition and reactivity and excel in biocompatibility, bioactivity and osteoconductivity.
A highly porous and fibrous composite material made of a-CaP and poly(lactide-co-glycolide) (PLGA) was developed and resulted in a flexible and compressible cotton wool-like filling material [4]. Bioactivity was proven by the formation of nano-sized hydroxyapatite and suggests application as substitute in non-load bearing bone defects. Additionally, a shapeable anisotropic electrospun bilayer made of a-CaP/collagen/PLGA with compositional and structural similarities to natural bone tissue on one side and pure PLGA fibers on the other side was fabricated [5].
Next to homogenous vascularization [6], animal studies in rabbit and sheep confirmed the biocompatibility of the bone cotton wool with enhanced porosity (95%) that results from the low-temperature electrospinning [7] [8]. Specifically, the bone regeneration in vivo was investigated in detail (> 40% area fraction of new formed bone in defect, negative reference: < 30%, positive reference: < 35%). The findings suggest possible usage of the bone cotton wool as alternative to granulates.
[1] Simonet, M et al., Polym Eng Sci (2007)
[2] Hild, N et al., Macromol Mater Eng (online)
[3] Loher, S et al., Chem Mater (2005)
[4] Schneider, OD et al., J Biomed Mater Res B (2008)
[5] Hild, N et al., Nanoscale (2011)
[6] Buschmann, J et al., Injury (2012)
[7] Schneider, OD et al., Acta Biomater (2008)
[8] Schneider, OD et al., Open Orthop J (2011)

Modern trans-catheter devices (TCDs), such as vascular stents and embolic coils, are generally fabricated using metal alloys. While these materials are durable and radio-opaque, their bulk mechanical, imaging, and surface characteristics are static, resulting in biocompatibility and device performance issues. We aim to develop a highly-tunable composite material for TCD design, which combines the characteristics of shape-memory polymers (SMPs) and gold nanoparticles (GNPs). With adequate thermal energy, SMPs can repeatedly recover from strains of several hundred percent, a useful property for catheter-based storage and delivery. GNPs are expected to confer x-ray and ultrasound visibility, significantly extend the spectrum of attainable mechanical properties, and allow for precise control of thermal transitions with visible light through surface plasmon resonance-enhanced energy absorption. Here, we present the initial results of our effort to disperse GNPs in acrylate SMPs through targeted surface-functionalization and study the resulting composite material properties. GNPs were synthesized chemically via the reduction and capping of hydrogen tetrachloroaurate(III) trihydrate (HAuCl4-3H2O) with oleylamine at elevated temperature, followed by ligand exchange with dodecanethiol and 2,5,8,11-Tetraoxatridecane-13-thiol. X-ray photoelectron spectroscopy of GNP films was used to confirm surface thiolation. Surface-stabilized GNPs were then suspended by sonication in a mixture of two acrylate monomers: 80 wt% tert-Butyl acrylate and 20 wt% poly(ethylene glycol) dimethacrylate (Mn 550). This mixture was polymerized in the presence of photo- or thermal initiators until > 99% vinyl group conversion was achieved, as determined by fourier transform infrared spectroscopy. UV-Vis spectroscopy and transmission electron microscopy revealed well-dispersed clusters of ~15 nm GNPs. The resulting composite materials demonstrated shape recovery characteristics similar to the original SMPs - no concentration-dependent effect was seen on glass transition temperature or shape recovery sharpness during dynamic mechanical analysis. Moreover, the glassy-rubbery transition was successfully induced by 500 mW/cm2 532 nm light in a spatially-controlled manner. Uniaxial thin-film tensile testing showed that, at low concentrations (< 5 mg/ml), GNPs reduce the Young&’s modulus of the composite, but recover and eventually increase modulus at higher concentrations. To improve the quality of the composite, future work will introduce heterogeneity in molecular weight to the GNP surface brush. Transport phenomena, including electrical and thermal conduction, will also be assessed.

The use of chitosan, a biomaterial derived from sea-shells, in nanofibrous form would offer a powerful platform to exploit its inherently beneficial properties in biomedical therapeutics. However, nanofiber formation of chitosan is difficult unless used with toxic solvents or in combination with inert blends. Our approach entails blending chitosan with a small molecule sugar, cyclodextrin, to facilitate chitosan nanofiber formation. In this case the cyclodextrin with its complexation guest-host properties could serve to incorporate useful moieties (e.g. drug molecules) in its core, thus serving as a functional blend while enhance chitosan electrospinning. In this study, we examine the role of each component and the possibility of synergistic effects in nanofiber formation. Multiple fiber morphologies are produced from a range of material concentrations, and key solution and operating parameters are identified, including polymer entanglement and molecular interactions.

In drug therapy, most therapeutic drugs are of low molecular weight and could freely diffuse in the biological milieu depending on the administration route applied. The main reason for the development of polymeric drug carriers is to obtain desired effects such as sustained therapy, local and controlled release, prolonged activity and reduction of side-effects. Alternatively, polymeric carriers can be made bioerodible in order to be eliminated by natural ways after a certain time of therapy. Core-shell fibres from coaxial spinneret or emulsion electrospinning are good candidates for the development of such devices; however difficulties remain especially in controlling the release over a sustained period. Here, we present a novel technique combining coaxial and emulsion electrospinning to produce micro-structured core-shell fibres. The design of drug micro reservoirs of variable size within the bulk of the fibre combined with a tailored diffusive barrier allows modulating the release kinetic of these novel carriers. A nearly constant and linear release of the model drug Levetiracetam (MW~170 g/mol) from PLGA emulsion-coaxial electrospun fibres is observed over 20 days. This device is aimed to be implanted into the brain for the treatment of epilepsy and is an example of the new capabilities and the promising potential that emulsion-coaxial electrospinning can provide towards the development of future drug carriers.

Nanoscale chemical and biological sensors are emerging as one of the most outstanding platforms for specific identification of biomolecules in the life sciences. Especially, field effect transistors (FETs) using semiconductor nanowires have been demonstrated to be fashinating platforms for the real-time and label-free detection of biological and chemial species. In this sensing platform, it is one of the most important issues that the electrostatic potential changes caused by chemical or biological binding events on the surface of FET occur within the solution Debye length for highly sensitive detection. At this point, aptamer with smaller size than the Debye length is one of the promising materials for the effective surface immobilization. In this work, we demonstrated aptamer-immobilized ZnO/PAC(polymer-like amorphous carbon) core-shell nanowire FET sensors for the sensitive and real-time detections of metal ions and cancer marker. Based on the Cu aptamer-immobilized nanowire FET sensor, the sensitive and real-time detections of the Cu metal ions in the solution were performed sucessefully in this work. Furthermore, the theoretical and experimental studies for effects of the solution Debye length on the surface of FET sensor will be discussed in detail through comparison of both antibody-antigen and aptamer-antigen bindings of alpha-fetoprotein as liver cancer marker.

Microfluidics, the ability to manipulate fluids that are geometrically constrained to sub-millimetre spaces has revolutionized lab-on-a-chip technology. Microfluidics enables such analytical and bioanalytical tasks as sample preparation and extraction, molecular separations and waste processing to be carried out on nanolitre and picolitre volumes by confining the liquids to micro-channels. Many LOC devices are used in a wide array of biomedical applications including rapid pathogen detection, clinical diagnosis, forensic science, flow cytometry, blood chemistry and DNA analysis. One challenge facing this area is the introduction and integration of easily-controllable and miniaturised electrochemical sensing formats. The use of electrochemistry offers immense benefit because of the ease with which electrodes can be implemented in microfluidic chips without any loss of analytical sensitivity and the positive benefits of the use of electrodes in fluidic structures. We are interested in exploring electroanalytical tasks that could be achieved by the integration of conducting polymers into microfluidic channels.
The fabrication of microfluidic chips housing thin layer electrochemical cells for fabricating and controlling functional polymeric structures will be presented. Conducting polymers can be both chemically and electrochemically grown within microchannels as flow-through, high surface area structures using porogens or sacrificial colloidal crystal templates to induce porosity. Subsequently, they can be used for a range of electroanalytical applications. Work is currently underway in our laboratory using these new microfluidic chips incorporating conducting polymer-based monoliths as flow-through stationary phases for lab-on-a-chip (LOC) chromatographic applications, drug delivery and flow-through electrochemical biosensors.

Long-term brain activity mapping and stable brain machine interfaces require seamless integration of electronic probes with brain tissue. Micro-fabricated probes have been widely used in both basic neuroscience and clinical neural prosthetics, although these structures have important limitations, including chronic deterioration of the implant-tissue interface elicited by the mismatch between implants and brain tissue. Semiconducting nanowire based macroporous nanoelectronic networks[1,2], can overcome these limitations since they have (i) extremely low bending stiffness similar to that of brain tissue, (ii) three-dimensional (3D) macroporous structure that allows interpenetration of the implant and tissue, (iii) only biologically-relevant microscale-to-nanoscale feature sizes, and (iv) inert and bio-compatible properties. Here, we demonstrate the design and implantation of a novel 3D macroporous nanoelectronic neural probe. These probes are free-standing in solution, and are designed with built-in strains to control both global and local device structures, which allow the probe-tissue interface to be optimized. The soft 3D macroporous nanoelectronic neural probes were inserted in a ‘frozen&’ state into the brains of anesthetized rats with minimal surgical damage. Multiplexed recordings from both the motor cortex and somatosensory areas will be described. In addition, histology analysis and long-term stability of the probe-tissue interface will also be discussed. Opportunities of these novel probes for brain activity mapping and as chronic implants for next generation brain-machine interfaces will be highlighted.
[1] B. Tian, J. Liu, T. Dvir, L. Jin, J.H. Tsui, Q. Qing, Z. Suo, R. Langer, D.S. Kohane and C.M. Lieber, “Macroporous nanowire nanoelectronic scaffolds for synthetic tissues,” Nature Mater. 11, 986-994 (2012)
[2] J. Liu, C. Xie, X. Dai, L. Jin, W. Zhou and C.M. Lieber, “Multifunctional three-dimensional macroporous nanoelectronic networks for smart materials,” Proc. Natl. Acad. Sci. USA 110, 6694-6699 (2013)

Nanowire field-effective-transistors (FETs) have been exploited as highly-sensitive subcellular resolution detectors for recording from electrogentic cells, and as functional elements in recently reported bio-compatible 3D macroporous nanoelectronic scaffolds.[1,2] This latter direction represents an unprecedented opportunity for seamlessly interfacing electronic and biological systems in 3D, thereby opening up many directions in basic and applied research. Here we demonstrate the capability to take full advantage of 3D macroporous nanoelectronics networks for long-term biomedical studies, including chronic neural implants, by fully-passivating the critical ultra-small nanoelectronic devices in these networks. Conformal atomic layer deposition (ALD) of ultrathin Al2O3 shells on nanowire surfaces were found to significantly improve the long-term chemical stability of nanowire FET devices, while maintaining device performance. Time-dependent studies of the the morphology, electronic conductance and electronic transconductance of nanowire FET devices with and without protective coating characterized at 37 oC will be described, where the results show clearly the excellent long-term stability and performance of core/shell nanowire/Al2O3 devices. Fabrication of highly-stable 3D macroporous nanoelectronic networks as well as their applications in long-term recording in cyborg tissues and as tissue implants will be discussed.
[1] B. Tian, J. Liu, T. Dvir, L. Jin, J.H. Tsui, Q. Qing, Z. Suo, R. Langer, D.S. Kohane and C.M. Lieber, “Macroporous nanowire nanoelectronic scaffolds for synthetic tissues,” Nature Mater. 11, 986-994 (2012)
[2] J. Liu, C. Xie, X. Dai, L. Jin, W. Zhou and C.M. Lieber, “Multifunctional three-dimensional macroporous nanoelectronic networks for smart materials,” Proc. Natl. Acad. Sci. USA 110, 6694-6699 (2013)

Biomimetic modification of materials has attracted intense scientific research effort [1]. We have developed an efficient method for preparing highly water repellent and self cleaning three-dimensional (3D) structures by direct laser texturing of silicon and subsequent coating with hydrophobic or hydrophilic layers respectively [2]. The laser patterned surfaces exhibit controlled dual-scale roughness at both the micro- and the nano- scales, which mimics the hierarchical morphology of surfaces found in nature. The superhydrophobic and superhydrophilic properties of the artificial surfaces are compared to various natural surfaces, and remarkable similarities were observed. The implementation of laser engineered hierarchically structured surfaces for the development of biomimetic scaffolds for neural tissue engineering was investigated. We demonstrate that it is possible to tune primary neuronal cell adhesion differentiation and growth on such surfaces, in the absence of chemotropic growth factors, which is desirable for biomedical applications. At the same time the compatibility of Si with current integrated circuit technology may allow development of novel 3D neurobiosensor devices onto the laser textured surfaces. As a proof of concept, we demonstrate the fabrication of laser textured Si multielectrode arrays that could potentially be exploited for recording conductivity in neurophysiology studies.
[1] ‘Biomimetic Artificial Nanostructured Surfaces&’ V. Zorba and E. Stratakis (2007) in Nanotechnologies for the Life Sciences, Wiley#8208;VCH Verlag GmbH & Co. KGaA
[2] “Biomimetic artificial surfaces quantitatively reproduce the water repellency of a Lotus leaf”, Zorba et al., Advanced Materials 20:4049, (2008).

Localized surface plasmon resonance (LSPR)-based biosensors promise highly sensitive, cost-effective, and point-of-care diagnostic tools. However, similar to conventional immunoassays, current plasmonic biosensors use natural antibodies, which are expensive and exhibit poor environmental and temporal stability. Macromolecular imprinting aims to overcome storage, stability and variability problems of natural antibodies by synthesizing specific artificial antibodies with high stability, reusability and cost-efficiency. Urinary neutrophil gelatinase-associated lipocalin (NGAL) is a biomarker of acute and chronic kidney injury; however, the current measurement of NGAL requires natural antibodies. We used organo-siloxane monomers to provide amine, hydroxyl and methyl functional groups mimicking a natural antibody and when polymerized mold a reversible recognition cavity specific for NGAL, detected as a shift in LSPR wavelength. These artificial antibodies, cast on gold nanocages (about 60nm/side) reliably detect 25 ng/ml of NGAL and each sensor pad may be recycled at least 5 times. The LSPR signal from NGAL was stable over a pH range from 5.0 to 8.5 and specific gravity from 1.005 to 1.030. Other human urinary proteins with lipocalin-like domains such as FABP1, FABP3 or orosomucoid; and myoglobin or hemopexin (1-10 ug/ml); and hemoglobin or serum albumin (up to 500 ug/ml) interfered less than 20% with the LSPR signal of NGAL. Thus, molecular imprinting of nanocages with surface synthetic antibodies opens up a novel class of plasmonic nanostructures with sensitive and specific biorecognition capability. The technical and conceptual development here open up new possibilities in biomedical applications of plasmonic nanostructures particularly in label-free and antibody-free diagnostic tools relevant to disease diagnosis, toxicology testing, and biotechnology.

We have explored different aspects of galvanostatic electrochemical deposition (ECD) of Pt interconnects for applications in flexible implantable bio-medical devices [1-3]. The goal was to replace physical vapor deposition methods (PVD) with ECD in cases when this is cost effective, i.e. when only a small fraction of the substrate needs to be coated with Pt [1-4]. We have also considered implementation of Pt plating on large, wafer scale substrates, in a fashion similar to fabrication of copper (Cu) interconnects for needs of microelectronics industry, since this could further reduce the production costs. Initial studies were done on coupons diced from 200 mm silicon (Si) wafers coated with PVD TiN. Optimal conditions were chosen for through-mask plating on 2 inch patterned Si or glass wafers with TiN plating base sputtered on flexible, medical grade, releasable polyimide layers. The use of Si or glass substrates enabled easier manipulation and transport of the samples, but in the end, flexible polyimide layers with Pt interconnects were released from the carriers and served as stand-alone entities. Physical and chemical properties of deposited Pt lines were determined using characterization techniques such as scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion-mass-spectroscopy (TOF-SIMS), X-ray diffraction (XRD), atomic force microscopy (AFM), and 4-point probes, and, as needed, post-plating processing steps were developed so that ECD Pt characteristics closely matched those of PVD Pt. Based on these experiments, we examine and discuss the potential for further up-scaling with special emphasis on high throughput manufacturing of Pt interconnects with good adhesion to TiN/flexible substrates, low impurity content and resistivity, and acceptable roughness and uniformity.
References:
[1] M. Op de Beeck, J. O&’Callaghan, K. Qian, B. M. Morcos, A. Radisic, K. Malachowski, M. F. Amira, and C. V. Hoof, in Proc. of the 45th Intern. Symp. on Microelectronics, San Diego, CA, USA, 45 (2012) 215-224.
[2] M. Op de Beeck, K. Qian, P. Fiorini, K. Malachowski, and C. V. Hoof, in Proc. of the 44th Intern. Symp. on Microelectronics, Long Beach, CA, USA, 44 (2011) 152-160.
[3] A. Cowley and B. Woodward, Platinum Metals Review 55 (2011) 98-107.
[4] E. Slavcheva, G. Ganske, G. Topalov, W. Mokwa, and U. Schnakenberg, Appl. Surf. Sci. 255 (2009) 6479-6486.

Although regenerative medicine approaches using stem cell therapies are well established for soft tissue, they have not been widely implemented in dental medicine. The tooth is a complex structure where a high level of organization must occur within a confined region and with minimal perfusion. One of the major obstacles to modeling differentiation in vitro, in the absence of chemical inducers, has been a lack of substrates that mimic the organization of the in vivo matrix. Entangled elastomer surface confinement has allowed us to produce substrates whose modulus varies in a continuous and differential manner. These polymers support dental pulp stem cell (DPSC) growth and allow us to simultaneously probe the mechanical properties of substrates and cells. Using this principal to produce multi-scaled, topographically flat, mechanically heterogeneous patterned surfaces made by spin casting polybutadiene, elastomer polymer, on imprinted silicon wafers, we have also been able to evaluate how DPSC respond to complex gradients, and organize focal adhesions and their associated signals. We found that the length scale of the mechanical gradient is a critical determinant and that micro-scale patterns suppressed biomineralization as measured by SEM/EDX, even when chemical inducers (dexamethasone) were present, whereas nanoscale patterns increased hydroxyapatite deposition.

For recent years, droplet-based microfluidic systems have been widely investigated for material synthesis, genetic assays, and biomedical applications. For these applications, droplets are used as isolated chamber for reactions or mixing. The advantage of droplet-based microfluidics comes from this isolation against containment and the small amount of reagents. In addition, the separation between reagents and carrier fluid enables independent fluidic control using the inert continuous phase. While multi-phase droplet generation on chips has been actively studied, robust techniques for manipulation of droplets on chips have not been well established. To process complex tasks inside droplets, the microfluidic devices need to perform several functions on individual droplets; these include target encapsulation, droplet merging, droplet flow timing, reagent injection, droplet splitting, droplet trimming, and droplet sorting. Injecting reagents into an existing droplet is particularly important for multistep analysis or synthesis, since different reagents are to be injected in a pre-determined order at different times. Droplet merging may as well provide a way to mix multiple reagents at designated times. However, merging or injection can be significantly hindered with stable emulsion droplets, because the surfactants prevent droplet surfaces from allowing aqueous reagents to break into the droplets. Here, we introduce the multifunctional microfluidic droplet processor, a robust device to manipulate droplets on chips. It can generate multi-sized emulsion droplets at various programmed rates or on demand, only encapsulating desired reagents in the droplets; it can control droplet flow rates individually for multiple droplets to coalesce at a designated spot using electromicrofluidics; it can fine-tune the size of droplets by trimming the existing droplets after initial formation and coalescence; it can also provide ways to sort droplets based on fluorescence and size.

Enzymatic lithography is a new approach to create arrays of micro-structures and has promising applications in biotechnology1. With this method, enzymes can be selectively patterned to create specific micro or nano topologies. In previous studies, it was necessary to immobilize enzymes on the printing tip to prevent the loss of image quality resulting from enzyme mobility along the surface2. Since the enzyme must be held in contact with the polymer surface to catalyze its degradation, long incubation times were required in these studies to image films only a few nanometers thick. The goal of our study is to develop a printing process where the enzyme need not be immobilized on the printing tips, but transferred completely to the surface, while maintaining good image quality.
The enzyme, Candida antartica lipase B (CALB), is known to catalyze the decomposition of poly (ε-caprolactone) (PCL) films in humid environments at 37oC. A Polymer Pen Lithography3 (PPL) technique was used to deposit an aqueous solution of CALB onto a spin-cast PCL film. The PPL printer used in this study employs an array (typically 10 x 10) of polydimethylsiloxane (PDMS) printing tips mounted to a multi-axis robot such that multiple patterns, one from each tip, are created in parallel. By programming the printing sequence, any arbitrary pattern of enzyme ink deposits can be generated, one pattern for each tip in the array. The tips are coated with enzyme solution before each printing cycle by dipping into an inkwell. The PDMS tips are then brought into contact with the PCL surface using a controlled force and time to transfer the enzyme. After printing, the film was incubated at 37oC and 87% RH. CALB selectively catalyzes the degradation of the PCL film during incubation, forming etched features through the film thickness with straight sidewalls and no lateral spreading.
Interactions between the enzyme and the PCL film restricts lateral diffusion of the enzyme and improves resolution compared to previous printing methods. These etched micro-wells may be used as containers in functional material synthesis, tissue engineering, drug discovery and drug testing. The design of the printer and the effect of printing parameters and incubation conditions on lithographic resolution will be presented. In addition the effect of PCL spherulite size will be discussed.
REFERENCES:
1. Hyun, J.; Kim, J.; Craig, S.L.; Chilkoti, A. J. Am. Chem. Soc. 2004, 126, 4770-4771.
2. Guyomard-Lack, A.; Delorme, N., Moreau, C., et al. Langmuir 2011, 27(12), 7629-7634
3. Huo, F.; Zheng, Z.; Zheng, G.; Giam, L. R.; Zhang, H.; Mirkin, C. A. Science 2008, 321, 1658-1660.

In hospitals and clinics worldwide, medical device surfaces have become a rapidly growing source of nosocomial infections. Contamination can occur from the presence of just a small number of microorganisms due to surgical procedure, improper sterilization, and more commonly the simple migration of bacteria from the skin into the body after an operation. Almost immediately after adhering to a device surface, bacteria can begin to form a biofilm: a robust, sticky matrix that provides protection against the host immune system and antibiotics. This makes the infection especially difficult to treat, and often necessitates device removal. Adding to the severity of this problem is the spread of bacterial genetic tolerance to antibiotics, in part demonstrated by the recent and significant increase in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA).
Nanomaterials are beginning to be used for a wide variety of biomedical applications due to their unique surface properties which have the ability to control initial protein adsorption and subsequent cell behavior. This “nanoroughness” gives nanomaterials a greater functional surface area than conventional materials, which do not have significant features on the nanoscale. In addition, it is theorized that nanoparticles may also have general mechanisms of toxicity towards bacteria that do not cause problems for mammalian cells.
The objective of the present in vitro study was to develop a nanocomposite material by embedding conventional polyvinyl chloride (PVC) with zinc oxide nanoparticles through a simple and inexpensive procedure. The effect of different nanoparticle sizes and %wts were investigated, and the surface roughness and features of the ZnO/PVC nanocomposites were visualized and compared using SEM and AFM. Results demonstrated that this technique significantly decreased S. aureus density and biofilm formation without the incorporation of antibiotics or other pharmaceuticals, as well as increased the adhesion of human osteoblast cells. Thus, this material could have much promise for use in the manufacture of common implanted medical devices.

In the last years, several nanodevices have been proposed as a result of the widespread interest focused on diagnostic and therapy by nanoparticles. Specifically for cancer therapy, the traditional treatments as chemotherapy and radiotherapy are yet extensively employed. Therefore, improving these traditional techniques is potentially important and the nanosystem based on lanthanum orthophosphate (LaPO4) arises like an interesting approach. The 4f15d1->4f2 emission of Pr3+ as doping on phosphate was studied in previous work dedicated to lighting sources; the broadband ultraviolet emission occurs in the so-called ‘‘UVC&’&’ range and could be used in UVC light emission for germicidal purposes [a,b]. The target of UV therapy is the nucleic acid of pathogenic microorganism or tumors cells. The high energy light could induce the dimerization process between adjacent thymine residues in the same DNA strand, and it has blamed for malfunctions of replication process leading to cell death. The phosphors have been prepared by spray pyrolysis (SP) followed by thermal annealing, a very efficient way to produce good quality UVC emitting phosphor. Among the systems studied, the LaPO4:Pr3+ was excited more efficiently by X-ray source than by plasma excitation. Further, the powder obtained has a broad size distribution as characteristic from SP methodology. However, the particles were achieved as hollow spheres composed of nanocrystals (50-100 nm) and these ones may be separated by outer action. In this purpose, organic-inorganic nano-composites have been built by the interaction of an amino acid (asparagine = ASN) with LaPO4:Pr3+ particles and this combination could bring reactive amine groups on the NPs surface became possible a future bio-compatibility. All systems were characterized by different methods, as transmission electron microscopy (TEM), X-Ray Excited Optical Luminescence (XEOL), Raman and FTIR spectroscopies. The proposed composite may improve the radiotherapy since the nanoparticle could be linked at the tumor cells and be excited together. Thus, the UVC emission will be localized under the neoplastic cells and could enhance the therapy action.
[a] - J.M.A. Caiut, S. Lechevallier, J. Dexpert-Ghys, B. Caillier, Ph. Guillot, Journal of Luminescence 131 (2011) 628-632.
[b] - C. Muja, B. Colin, B. Caillier, J.M.A. Caiut, J. Dexpert-Ghys, Ph. Guillot, XXXI International Conference on Phenomena in Ionized Gases (ICPIG), July 14-19, 2013, Granada, Spain.

Breath NO2 detection of low ppm or ppb levels is essential for early diagnosis of respiratory disorders like asthma. A highly selective and sensitive sensing material is thus required for use in a breath analyzer device for asthma montoring. Nanoparticles of γ-WO3, synthesized at 350C, are highly sensitive and selective to oxidizing gases [1] like NOx, Nanowires of metal oxides have been shown to exhibit enhanced sensing properties [2]. WO3 polymorphs in nanowire structure are synthesized and there sensing behavior towards different gases like NOx, NH3, Isoprene, is analyzed. . The sensing behavior is elaborated and their usefulness in a hand held breath analyzer as a diagnostic tool is discussed.
[1] P.I Gouma and K. Kalyanasundaram, "A Selective Nanosensing Probe for Nitric Oxide", Appl. Phys. Lett. 93, 244102, 2008.
[2] P. Gouma, K. Kalyanasundaram, and A. Bishop, "Electrospun Single Crystal MoO3 Nanowires for Bio-Chem sensing probes", Journal of Materials Research, Nanowires and Nanotubes special issue, 21(11), pp. 2904-2910, 2006

Bioinspired self-assembling hybrid materials with hierarchical order were synthesized using bottom up approaches. Synthetic polymers or their conjugates with biomineralization proteins/peptides were used as templates for the bioinspired synthesis of magnetite and zirconia nanocomposites. Amphiphilic triblock and pentablock copolymer templates that self-assemble at the nanoscale and at the macroscale based on changes in temperature and/or pH were used as templates. Uniform superparamagnetic magnetite nanocrystals with a size of about 30 nm, were only synthesized in the presence of Mms6 protein, and not by its mutants, indicating that the number and placement of charged groups in the protein were critical for its function. Synthesis of other iron-containing magnetic materials in the presence of this protein is being studied. Zirconia nanocomposites were synthesized using lysozyme conjugated to block copolymer templates and natural self-assembling macromolecule agarose. Removal of the templates left behind a porous network of zirconia with a much higher surface area and thermal stability than those made without any template. Based on these studies, we developed a robust and modular method with control over the formation of an inorganic phase in the nanocomposite structure, and are able to design tailored functional organic templates for room-temperature bioinspired synthesis.

With the rise of ageing population, the need to restore the function of degenerative bone greatly drives the market for bone grafts. Hydroxyapatite (HA) is chemically similar to natural bone mineral and has been widely used in bone graft applications. However, its slow osseointegration process and lack of antibacterial property could lead to implant-related infection, resulting in implant failure. Studies on ionic substitution of apatite have gained attention in recent years with greater understanding of the composition of bone mineral being a multi-substituted apatite. An integrated approach is proposed by co-substituting silicon and silver into hydroxyapatite (Ag,Si-HA) to modify its surface for bi-functional properties. Incorporation of silicon can enhance the biomineralisation of hydroxyapatite and introduction of silver can create antibacterial property. Ag,Si-HA containing 0.5 wt.% of Ag and 0.7 wt.% of Si was prepared by a wet precipitation method. A phase-pure apatite with a nanorod morphology of dimensions 60 nm in length and 10 nm in width was synthesized. Surface Ag⁺ ions of Ag,Si-HA were demonstrated to prevent the replication of adherent Staphylococcus aureus bacteria for up to 7 days. Furthermore, mechanism of the antibacterial action by the incorporated Ag⁺ ions was also ascertained in this study. Biocompatibility tests revealed that human adipose-derived mesenchymal stem cells proliferated well on Ag,Si-HA with culturing time. Enhanced cell attachment in turn permits greater bone differentiation as evidenced in greater production of collagen type I and osteocalcin on Ag,Si-HA as compared to HA from day 14 onwards. Overall, co-substitution of Ag and Si could complement the benefits of each substituent by endowing HA with antibacterial property, and concurrently promoting its biological performance. Their synergistic effects can serve unmet medical needs and solve the problem of implant-related infection. This work also enhances the understanding of substituted apatite with multiple ions for bifunctional properties.

Implants for total joint replacements and grafts to fill large bone defects can repair tissue, restore mobility, and alleviate morbidity. Occasionally, these devices fail by post-surgical loosening or stress shielding due to a modulus mismatch between the bone and implant. To attenuate these incidences, endogenous progenitor cells can be recruited to (i) promote the direct integration of the implant with the host bone tissue and (ii) repair critical size defects. The use of modular nanostructured coatings containing multiple biological growth factors and inorganic bone minerals, can mimic aspects of the natural bone wound healing cascade and direct cell behavior.
Implant surfaces were coated using a layer-by-layer (LbL) approach, with individual layers of materials with nanoscale control over their deposition. Angiogenic vascular endothelial growth factor (VEGF) and osteoinductive bone morphogenetic protein (BMP-2) were incorporated in gradient degradable coatings, such that VEGF released rapidly during the early acute phase of wound healing, along with slower releasing BMP-2 for longer term osteogenic effects. In vivo, coated polycaprolactone/β-tricalcium phosphate scaffolds were implanted in a rat quadriceps muscle pocket model. Dual growth factor from the scaffold surface resulted in superior bone formation compared to single growth factor release.
In a complementary study, hydroxyapatite (HAP) and BMP-2 LbL coatings were used for the biological fixation of surrogate bone implants to the host tissue. The films contained a permanent osteoconductive HAP base layer and a degradable BMP-2 releasing top layer.The mode and rate of growth factor release significantly influenced bone regeneration. In combination, HAP and slow releasing BMP-2 accelerated progenitor cell response and implant integration with the host bone. Coated implants bonded to the host bone, with a time-dependent increase in the pull-out force that was sustained over the long term.
To repair bone tissue in large defects, we developed degradable poly (lactic-co-glycolic acid) (PLGA) membranes that served as temporary scaffolds. Nanogram quantities of BMP-2 and platelet derived growth factor (PDGF-BB) coated on the scaffolds accelerated repair and rapidly close critical size rat calvarial defects within 4 weeks after implant. Regenerated bone had similar mechanical properties to the native calvaria.
Taken together, these results demonstrate that surface modification on next-generation biologically integrated implants can be used to induce tissue regeneration. Precisely assembling materials using this technique in modular nanoscale assemblies can result in a synergistic response which accelerates bone regeneration and enables implants to last without failure through their natural lifetime, and scaffolds to introduce rapid bone healing.
Acknowledgements: NIH (R01 AG029601), NCI (P30 CA014051), U.S. Army Research Office (under contract W911NF-07-D-0004) and Pfizer Inc.

Nanopores fabricated in thin solid-state films can detect single charged biopolymers, such as DNA, via resistive sensing. These charged biopolymers are electrophoretically drawn through a nanopore, where their presence causes a transient reduction in pore conductance. Although these biosensors show great promise for a range of biomedical applications (e.g., DNA sequencing), the challenge remains to tailor analyte translocation dynamics independent from pore chemistry or geometry. To date, the primary means of controlling translocation have been changing sensing buffer composition and chemically functionalizing the nanopore surface, approaches which also alter the electrical sensing properties of the nanopore. We hypothesized that a permeable mesh occupying the volume immediately outside the nanopore would interact with analytes as they move through the nanopore, affording control over translocation dynamics, while leaving the nanopore unaltered and available for further functionalization. Here we have selected an electrospun mesh (NFM), composed of a network of fibers (100-1000 nm diameter) formed from poly(ε-caprolactone) (PCL) doped with poly(glycerol-co-ε-caprolactone) (PGC), to modify nanopore (NP) chips. The PGC is functionalized with stearic acid to confer hydrophobicity; thus the copolymer ratio PCL:PGC in the electrospun NFM determines its overall hydrophobicity. Fiber size and mesh density is controlled by adjusting the electrospinning parameters. We demonstrate fabrication of nanopore-nanofiber mesh (NP-NFM) sensors using a range of hydrophobic NFMs, and show that the electrical properties of the nanopore are unaltered by the presence of the mesh. Importantly, DNA translocation speed is slowed by the presence of a hydrophobic NFM, increasing resolution along the length of the DNA compared to a bare nanopore by a factor of > 30. Because an NFM applied to a nanopore a) is simple to fabricate via electrospinning; b) affects translocation in a manner orthogonal to nanopore electrical properties; and c) interacts with analytes in the volume outside the nanopore, NP-NFMs can be diversified to include a wide range of mesh modifications which would allow further control over translocation dynamics.

We explore the morphology and tribo-mechanical properties of polyacrylate catheter coatings variably UV-crosslinked (1,4- butanediol diacrylate) and following tribological history (mimicking shear stresses during surgery). A rich surface morphology is revealed over micro- to nano-scales, strongly dependent on both the extent of crosslinking and tribo-history. Submicron defect structures resulting from the coating deposition process include shallow (nm's) circular depressions hundreds of nanometers across and much deeper/narrower pinholes; from the UV treatment we find deep but larger in diameter "craters" plus meandering "fissures" running between the deep holes and craters. The deepest defects exhibit modified behavior, as seen in multiple AFM modes (friction, adhesion, phase) sensitive to physicochemical properties. Mapped nanoindentation measurements interrogate both elastic and dissipative property changes as a function of UV treatment and tribo-history. AFM-based nanowear discerns substantial variations in coating behavior correlating with processing and tribo-history. Both nanomechanical and nanotribological responses are further explored under aqueous immersion. We include some discussion of the optimal UV crosslinking levels to achieve sought durability and lubricity.
Broadly, this study exemplifies (1) technologically important phenomenology over scales ranging from tens of microns to tens of nanometers; (2) connections of nanoscale behavior to macroscale properties; and (3) the complementarity that multiple AFM methodologies [1] afford in assessing the performance of thin film technologies for surface modification of biomedical materials.
[1] G. Haugstad, Atomic Force Microscopy: Understanding Basic Modes and Advanced Applications (Wiley, 2012)

Poor dispersibility and agglomeration of unmodified ceria nanoparticles in biological relevant media have limited their promising catalytic antioxidant applications in the field of biomedical research. In this work we present a simple, solution-based synthetic process to prepare silica supported ceria nanoparticles. The ceria nanoparticles of size ~2-3nm with mixed valence states (+3/+4) are bonded to the surface of silica via silanol-hydroxyl functional groups. The ceria attached silica hybrid nanostructure are resistant to agglomeration in broad pH range and cell culture media conditions as measured by particle size and suspension stability measurements. We found that the bio-catalytic SOD activity of silica supported ceria was structurally stable and not limited by adverse pH and chemical constituents of culture media. The silica supported ceria hybrid nanostructure developed in this work show promising properties for bio-catalytic applications.

Advanced biomedical devices such as the PillCam have brought the concept of non-invasive diagnostics and therapeutics one step closer to reality. In addition to imaging devices, Gultepe et al, have recently demonstrated the successful in vivo biopsy of the gastrointestinal (GI) organs such as the bile duct using sub-millimeter sized untethered metallic microgrippers (1). In order to broadly implement this concept of using untethered tools for surgery, there is a need to construct devices that are biocompatible and possibly biodegradable so that any tools left behind are completely cleared away from the body. Here, we describe polymeric thermoresponsive six-membered grippers composed of poly propylene fumarate (PPF) and poly N-isopropylacrylamide-acrylic acid (PNIPAM-AAc). We utilized a soft lithographic photopatterning approach to design 2D thin film PPF-PNIPAM-AAc bilayer structures composed of rigid PPF panels atop a continuous PNIPAM-AAc hydrogel layer. These gripper structures activate an anisotropic swelling response between the top and bottom layers when triggered by an external thermal stimulus so that spontaneous 3D folding occurs. We discuss implementation of a modified Flory-Huggins model to describe the reversible thermal actuating PPF-PNIPAM-AAc polymeric gripper mechanism. We highlight possible applications of these grippers in soft-robotics and surgery by incorporating ferromagnetic iron oxide so that they can be guided from afar.
References: E. Gultepe, J. S. Randhawa, S. Kadam, S. Yamanaka, F. M. Selaru, E. J. Shin, A. N. Kalloo, D. H. Gracias, Biopsy with thermally-responsive untethered microtools, Advanced Materials 25, 4, 514-519 (2013)

Poly(ε-caprolactone) (PCL) is a promising material for tissue engineering applications, however, pure PCL scaffolds exhibit undesirable mechanical properties. This study addresses these issues by incorporating surface modified Al₂O₃ nanoparticles to reinforce PCL membranes. Surface modified Al₂O₃-reinforced PCL composites were manufactured with an electrospinning technique to investigate the effect of interfacial adhesion on the mechanical properties of PCL-based scaffold materials. It was shown that even at low additive content; the mechanical properties of the composites can be improved significantly. The strength of the electrospun composite membranes was affected significantly by inorganic filler/polymer matrix adhesion. However, the tensile modulus was not sensitive to particle surface modification.

Electrospun nanofibrous mats for pH-dependent anti-bacterial activity were fabricated with two Eudragit® polymers with opposite charges in aims to control enterobacterial infections. Eudragit® has been widely employed as a material for biomedical devices because of their biocompatibility and pH sensitivity. Among those, Eudragit® EPO (EEPO) having dimethylamino group and Eudragit® L100 (EL100) having carboxylate dissolves in less than pH 5.0 and above 6.0. Inter-polymer complex (IPEC) by electrostatic interactions between oppositely-charged polymers has been often employed to fabricate drug carriers in aims to control drug release profiles. Upon mixing of two polymers, however, they forms insoluble aggregates by electrostatic interactions, thus could not be simultaneously employed for drug carriers. We newly employed electrospinning techniques to fabricate nanofibers (NF) with co-axial electrospinning to resolve this issue. In this study, positively-charged EEPO and negatively charged EL100 was co-electrospun with tetracycline (Tet) and subsequently complexed at the tip of co-axial needle during the electrospinning process. The composition of the respective polymer was conveniently adjusted by changing the flow rate ratios of EL100 (outlet) and EEPO (inlet). Morphology and mass loss profile of the NF according to different IPEC composition in the NF were characterized. Scanning electron microscopy (SEM) revealed that the NFs with different compositions of Eudragit polymers showed the similar morphology. In pH 2.0 buffer, NF with higher EEPO contents was dissolved rapidly. Tet loaded NF was prepared by electrospinning of mixture of EEPO and Tet. Release property of Tet in pH 2.0 and pH 6.0 were evaluated by UV-vis spectrophotometer. Higher EEPO composition in NF accelerated release of Tet in pH 2.0 and pH 6.0. In order to figure out anti-bacterial activity of Tet loaded Eudragit® NF, the release fraction of Tet was treated to Escherichia coli (E.coli) culture medium and the turbidity changes were monitored. The release fractions from high EEPO composition NF in pH 2.0 and pH 6.0 successfully inhibited the growth of E.coli. In conclusion, the oppositely-charged EL100 and EEPO formed an inter-polymer complex during electrospinning and Tet was efficiency encapsulated within NF.

Surface enhanced Raman scattering (SERS) is a sensitive technique that allows rapid identification and composition analysis of extremely small amounts of chemical substances and biological species (up to 1 molecule) adsorbed on the nanorough metallic substrates. It has been found that localization of surface plasmons on the metallic roughness makes the most significant contribution in the Raman signal enhancement. A number of advances in processing metallic nanostructures for use in biomedical sensing by SERS have already been achieved including fabrication of solid substrates consisted of single metal or bimetallic nanoparticles (NPs) as well as nanovoids (NVs). Unfortunately, developed materials have not found wide practical application as it requires careful systematic study of their fabrication process and properties. In the present work we proposed fabrication of SERS-active substrates by electroless/electrochemical deposition of metals on porous silicon (PS) template. PS was formed by anodization of Si wafers in HF-based solutions. Managing types of initial Si substrate and anodizing regimes provided fabrication of arrays of pore channels with controlled dimensions. Therefore, PS played a role of the template which determined the shapes and sizes of deposited metallic nanostructures. Use of PS with pore diameters about 20-100 nm and thickness more than 5000 nm led to deposition of metallic NPs on the external surface of PS avoiding pore walls. It was explained by diffusion limits for reagents which take place in pores of nanosized diameters and high aspect ratio. From the other hand pores of 500-1200 nm diameter and 900-2600 nm length had no limitations for penetration of reagents. As a result conformal metallic film covered internal pore surface and final structure presented an array of metallized NVs.
SERS activity of metallized PS substrates was tested using water-soluble cationic Cu(II)-tetrakis(4-N-methylpyridyl) porphyrin (CuTMpyP4) as an analyte compound which was chosen due to its well-studied Raman spectrum and relative photostability. Tetrapyrroles are known as natural macroheterocyclic pigments that play an important role in the metabolism of living organisms. Moreover, tetrapyrrole compounds can be used as a photosensitizer in photodynamic therapy of tumors.
SERS-activity of metallized PS was demonstrated. In order to exclude the contribution of PS into enhancement we made measurements for the analyte&’s molecules adsorbed on the fresh PS which showed no SERS signal. The influence of metallized PS structure on the SERS signal intensity was deeply studied. As structure was found to be directly dependent on the deposition conditions, we were able to determine the optimal regimes for fabrication of the most active substrates. The minimal concentration of analyte detected with use of metallized PS was about 1e-12 M.
The present research was supported by the Belarusian Foundation of Fundamental Research under the Grant T13M-161.

Despite the large success of silicon-based MicroElectroMechanical Systems (MEMS), substituting rigid inorganic materials by soft organic materials offers basic economical and functional benefits. Indeed the wide panel of functional properties given by polymers (electrical, mechanical, chemical, biological) allows the elaboration of low-cost, tailor-made MEMS devices according to the targeted application. In this context original organic MEMS can be designed and used as mass sensors for the specific, selective and label-free molecular recognition of analytes relevant to biomedical applications. However the field of organic MEMS remains emerging, therefore new and original ways have to be opened, ranging from the materials view to their patterning, as well as the strategies to improve the performances of such mass sensors.
Our recent developments allow us to propose a complete valued chain for the microscale processing of organic materials for the fabrication of MEMS sensors in an all-organic approach. First a shadow-masking process is applied, consisting in the combination of spray-coating through organic microstencils. Several organic materials can be patterned with this approach (either photosensitive or thermosensitive) since deposition and patterning occur simultaneously. Four generations of flexible microstencils are currently developed. The first one is based on the use of SU-8 epoxy photoresist while the others are developed using PolyDiMethylSiloxane (PDMS) or silicone derivative materials, taking benefits from their elastomeric behavior for a simple and reliable manipulation.
The shadow-masking process is applied for the fabrication of free-standing microcantilevers using methacrylate polymers, characterized by a low and tuned elasticity allowing large deformation capabilities. For this, ethylene glycol chains of different lengths enable the control of anchor points of methacrylate monomers. Cantilevers with Young modulus ranging from 1 to 5 GPa are obtained. This process is also used for the fabrication of monolayered MEMS mass sensors using Molecularly Imprinted Polymers (MIPs, tailor-made biomimetic polymers) as constitutive material capable of binding specifically and selectively low molecular weight analytes. The monolayered format of the mass sensing devices combined with the efficiency of imprinted cavities in the volume of the MIP film give a limit of detection of 100nM towards S-propranolol, a beta-antagonist drug. Such results confirm the potential of organic MEMS as efficient label-free sensing platform for low molecular weight analytes, a challenge when using MEMS as mass sensors.
Last, but not least, a composite material based on carbon nanotubes dispersed in SU-8 is evaluated as piezoresistive strain gauge. Around the percolation threshold, gauge factors around 100 have been measured while the integrated transduction of the dynamic behavior of free-standing bridges has been recently demonstrated.

As an emerging manufacturing technique, nanoimprint lithography (NIL) can fabricate micro and nanoscale features of microfluidic devices at very high accuracy and reliability. The process parameters such as pressure, temperature, and material properties play critical roles in the NIL process. Leakage between channels or significant variation in channel width can occur in micro-fluidic devices if mold deformation exceeds design tolerances. In this work, the process of thermal nanoimprint lithography (TNIL) is studied computationally and the developed model can accurately predict the nano- and micro-pattern geometry and quality from TNIL processes based on complex mold-resist interaction.
In high-temperature TNIL process, a polymer melt such as polymethyl-methacrylate (PMMA) is heated beyond the melting temperature so that it behaves predominantly as a fluid during the imprint process, and is appropriately solved with computational fluid dynamics (CFD). The effects of surface tension and shear thinning become significant at or above the melting point, whereas the polymer melt between the glass transition and melting temperatures can be modeled as a viscoelastic solid, solved with finite element analysis.
The mold used in TNIL deforms significantly even at very low imprinting pressure when it is made of soft-rubbery elastomer such as polydimethylsiloxane (PDMS), and it is of interest to solve for subsequent mold deformation and interaction with resist flow field. In the current work, fluid-structure interaction (FSI) technology is leveraged to solve for significant mold deformation and its effect on the polymer melt flow field during TNIL process. The simulation result is compared to experimental results. The FSI simulation result is also compared to the equivalent case with a rigid mold in place of flexible material, which shows results of differing mold materials.

After coronary or peripheral artery bypass grafting surgery, restenosis or occlusion of blood vessel often occurs due to the damage in endothelial cells or abnormal growth of smooth muscle cells in vascular tissues at the sites of anastomosis. Later, this can lead to serious complications or fatality. Although devices including drug eluting stents and perivascular wraps have been developed, the efficiency of such drug delivery methods to target tissue, tunica media, is still limited or unclear. Recently, we have developed cuff-shaped microneedle (MN) devices that can be attached on the exterior surface of blood vessels for more efficient delivery of drug molecules to the inner vascular layers. The MN array of the device is inserted into the vascular tissue and the drugs are slowly released from the MN tips based on biodegradation to prevent neointimal hyperplasia after bypass grafting. MN cuff devices were fabricated by thermal drawing of MN arrays and post-annealed for cuff shape. 90/10PGLA was used as material for MN arrays and paclitaxel (PTX) as a model drug mixed in 50/50PGLA was coated on the surface of the MN for slow release. From 2 week in vivo study using rabbit abdominal aorta model, MN cuff devices delivered about 200 times higher amount drug than plain cuff devices (without MN) coated with the identical drug formulation. In another set of in vivo study (n=4) for 4 weeks, use of MN cuffs resulted in 44% reduction of neointima formation after balloon injury compared to the cases without MN cuffs.

Biomaterials-associated infection is now a leading complication of tissue-contacting biomedical devices. Bacteria can colonize implant surfaces, develop into antibiotic-resistant biofilms, and chronically infect the surrounding tissue, thus inhibiting the healing process. Biomaterials-associated infection occurs because implant surfaces generally cannot differentiate between desirable tissue cells and undesirable bacteria. Surfaces that are adhesive to tissue cells are typically adhesive to bacteria. We are studying surfaces whose cell adhesiveness is laterally modulated at cellular and sub-cellular length scales using electrostatically deposited poly(ethylene glycol) [PEG] based microgels. In contrast to surfaces that are rendered entirely non-adhesive by a continuous coating of PEG, surfaces with microgels spaced at approximately 1-2 micron distances preferentially inhibit the adhesion of staphylococci but enable osteoblast adhesion, spreading, and proliferation. We create such surfaces using the electrostatic deposition of sub-monolayers of anionic PEG-based microgels. Their negative charge also enables post-deposition electrostatic loading of the microgels by a number of small-molecule cationic antimicrobials. These provide an active bacterial killing mechanism in addition to the passive anti-adhesion property. Significantly, electrostatic self-assembly is a non-line-of sight deposition method, and the surfaces of complex shapes can be easily modified. In addition to results using 2-D substrates such as silicon and glass, we use microgels to modulate the fiber surfaces within topographically complex 3-D nanofiber tissue scaffolds. Not only is staphylococcal colonization substantially inhibited on all of these, we also find that osteoblast response is either maintained or enhanced, an effect we attribute to the rich interplay of the modulated surfaces with the transduction processes associated with cell adhesion, spreading, motility, and proliferation.

In-situ synthesis of superparamagnetic Fe3O4 nanoparticles within poly (acrylic acid) (PAA) nanogels could be a highly integrated nano-platform which can be used as biomedical materials for MR imaging, anti-cancer targeting,and drug delivery. This nano-platform is consisted of the internal layer of Fe3O4 nanoparticles and the external layer of PAA nanogels. PAA nanogels display pH-stimuli responsitivity and good biocompatibility, and supplies the highly reactive carboxyl group which has potential for loading cancer drug and conjugating targeted ligands; on the other side, the internal layer of Fe3O4 nanoparticles performs superparamagnetic behavior and biodegradability which is important in MR imaging, protein isolation and cancer magnetic targeting hyperthermia (MTH).
In this work, the green method for synthesizing surfactant-free PAA nanogels in aqueous media was developed by using hydroxypropylcellulose (HPC) as a template. The results showed that all the PAA nanogels demonstrated a narrow size distribution and were very stable in aqueous media. Furthermore, in-situ synthesis of Fe3O4 nanoparticles into PAA nanogels was carried out via electrostatic interaction. The results obtained by TEM and XRD confirmed the iron oxides dispersed within the nanogels network and its crystal structure. The interesting TEM results showed that the PAA- Fe3O4 nanoparticles displayed a nanoworm-like morphology. The superparamagnetic behaviorof the resulting nanogels was characterized by vibrating sample magnetometer (VSM). The obtained saturation magnetization was as high as 39.521 emu/g. The cytotoxicity was evaluated by Alarma Blue Assay and Live/Dead fluorescence observation, and the cell proliferation was only slightly inhibited by the PAA- Fe3O4 nanoparticles. This result showed that the cytotoxicity of the PAA-Fe3O4 nanoparticles was very low.

A facile technique was developed for a long-term increase in silicone elastomer surface hydrophilicity, eliminating the need for post-cure surface treatment (e.g. oxygen plasma or surface grafting). Well-defined silicones (1-4 kDa) with a central vinyl functionality and discrete PEG2, PEG3 and tetrahydrofurfuryl (THF) pendant endgroups were synthesized, characterized and used as comonomers in addition-cure, platinum catalyzed silicone RTV formulations. The central vinyl functionality reacts into the elastomer network during copolymerization of the RTV leading to no increase in undesirable extractables in the silicone elastomer. The modified silicone elastomers were optically clear and maintained the mechanical performance characteristic of this class of material with up to 20 wt.% comonomer in the RTV formulation. The silicone comonomer provides intrinsic control of surface hydrophilicity as the surface active pendant endgroups can orient on the bulk silicone elastomer surface. Deionized water contact angle measurements on the modified silicone elastomers decreased with higher loadings of comonomer. The elastomer surface shifted from hydrophobic (contact angle ~120oC) to hydrophilic (contact angle < 900C) at ~5 wt.% comonomer loadings for prolonged time frames (> 5 months). The PEG3 surface active pendants yielded a greater reduction in contact angle over the comparable PEG2 and THF contact angles. Hydrolytic stability of the elastomer modifications at various pH increased with the surface active pendants PEG3< PEG2

Reconstitution of ion channels in artificial bilayer lipid membranes (BLMs) provides an excellent system for drug screening under chemically controlled conditions. However, the mechanical instability of BLMs prevents them from being widely used. Recently, we succeeded in the formation of mechanically stable BLMs by preparing membranes in microfabricated silicon chips (1). The key feature that stabilized the BLMs is probably the tapered shape of the pore edge, which allows reducing the stress on the bilayer at the pore edge. In this presentation, we report on an application of this stable BLM device to measure ion currents of biological channels, including gramicidin and human ether-a-go-go-related gene (hERG) channel, which has been found to be related to serious arrhythmic side effects following drug treatment (2).
The basis of the device was a silicon wafer covered with a silicon nitride (Si3N4) layer. Micropores were fabricated in the Si3N4 layer using wet chemical etching. The surface of the device was further coated with thermal oxide and Teflon-AF (3). After the device was treated with a silane-coupling reagent, artificial BLMs were prepared in the micopores by the monolayer folding method. Owing to the use of SiO2/Teflon dielectric layer, the current noise level of the BLM was as small as 1-2 pA in peak-to-peak after low-pass filtered at 1 kHz. Owing to the low noise level, clear single-channel events were observed for both gramicidin and hERG channels. Average hERG single-channel conductance was 11 ± 1.2 pS, which is similar to a reported value (12 pS) by the patch-clamp method. The membrane lifetime was 43-65 h with incorporated gramicidin or hERG channels. The BLM containing hERG channel was tolerant to repetitive (17 times) solution exchanges. Compatibility of the BLMs to solution exchanges can be useful for increasing the experimental throughput from single BLMs. The next step is to extend this stable BLM device to a multisite array format. Since we previously reported on the simultaneous and automated formation of stable BLMs (4), in future studies, we plan to improve the throughput by combining the channel-containing BLMs with the array format. The realization of an ion channel array will open a variety of applications, including high-throughput drug screening procedures that can be a complement to the patch-clamp method.
References
(1) A. Hirano-Iwata, K. Aoto, A. Oshima, T. Taira, R. Yamaguchi, Y. Kimura, M. Niwano, Langmuir, 26, 1949-1952 (2010).
(2) A. Oshima, A. Hirano-Iwata, H. Mozumi, Y. Ishinari, Y. Kimura, and M. Niwano, Anal. Chem., 85, 4363-4369 (2013).
(3) A. Oshima, A. Hirano-Iwata, T. Nasu, Y. Kimura, and M. Niwano, Micro and Nanosystems, 4, 2-7 (2012).
(4) A. Hirano-Iwata, T. Nasu, A. Oshima, Y. Kimura, and M. Niwano, Appl. Phys. Lett., 101, 023702 (2012).

Magnetic hyperthermia for cancer operates on the principle that magnetic nanoparticles introduced into a tumor produce heat when subjected to an alternating magnetic field of suitable frequency and amplitude. A key issue with magnetic nanoparticles is that they must have a high specific absorption rate (SAR) so that not only is the dose of nanoparticles required for hyperthermia treatment minimized, but also so that low values of the product of magnetic field strength and frequency are used, thereby minimizing eddy current heating of tissue. Recently, we developed a new type of magnetic nanoparticle (NP) with high specific absorption rate. The NPs are made of gamma-Fe2O3 with saccharide chains implanted in their crystalline structure. The saccharides that we have used are mono- (glucose), bi- (sucrose) or poly (starch, dextran and others) as well as their derivatives (hydroxyethyl, carboxymethyl and others). Unlike NPs commonly used for magnetic hyperthermia that have relatively large single crystals (12-30 nm) and/or irregular shape, our NPs are built out of small (2-4 nm) single crystals, gathered in 20-40 nm aggregates with a hydrodynamic diameter of 110-120 nm. The NPs form stable (>12 months) colloidal solutions in water, show very low saturation magnetization (2-7 emu/g) and no hysteresis, but produce significantly more heat than commercially-available NPs at 300-400 Oe and 135 kHz. Even more valuable is the fact that they produce enough heat for therapeutic treatment at magnetic field strengths as low as 100-200 Oe while commercially-available NPs do not.