Droplet-based microfluidics has proven a very effective tool for the miniaturization and automation of biological assays, for single cell analysis, DNA screening or drug screening [1]. In order to reliably function as microreactors, the droplets have to fulfill three major conditions: they must be stable against coalescence, biocompatible, and their components must remain encapsulated over time, three properties controlled by the surfactant molecules [2]. Fulfilling these constraints requires the design and synthesis of novel molecules as well as a detailed understanding of their properties in order to provide systems of practical interest, for example for industrial applications. The fine control of droplet actuation and of droplet size distribution, and the accessibility of short timescales (typically 1 ms) in microfluidic emulsification - difficult to achieve in bulk emulsification - makes it appealing as a new tool to quantitatively study the physics of surfactant-laden interfaces and emulsions [3]. The transport of molecules between droplets (an old problem linked to the aging of emulsions), is here revisited in microfluidic systems. Studies on solute exchange have already been performed in bulk emulsions, but they do not capture the microscopic details of the exchange at the single droplet or interface level. Such insights are now accessible by the microfluidic control of droplets, combining single droplet analysis and high-throughput measurement of large droplet population. Using microfluidic devices, we measure experimentally the relaxation of concentration differences in an emulsion (produced in microfluidics) initially containing droplets with two different concentrations of a fluorescent dye. We make the link between the microscopic exchange between two adjacent droplets and the macroscopic kinetics (at the scale of the emulsion) [4] and relate our results to classical models of transport through membranes demonstrating the key role of surfactant in the process. The transport - seen as a passive process - leads to equilibration of concentration and can be controlled -- for example by the use of additives. These additives can also be used to obtain selective transport for the development of novel systems usable to concentrate compounds of interest, leading to a versatile control of chemicals in emulsions. Notably, such transport processes are analogous to transport processes through cell membranes and controlling those could lead in the future to the creation of artificial cells or new biosensors for biotechnology applications, built from simple soft matter systems. [1] M.T.Guo, A.Rotem, J.A.Heyman and D.A.Weitz, Lab Chip, 2012, 12, 2146-2155. [2] J.-C. Baret, Lab Chip, 2012, 12, 422-433. [3] J.-C. Baret, F. Kleinschmidt, A. E. Harrak and A. D. Griffiths, Langmuir, 2009, 25, 6088-6093. [4] Y.Skhiri, P. Gruner, et al. Subm. (2012)

Single cell studies have gained increasing attention in recent years as efforts are being made to understand cellular functioning in complex processes (like cell division during embryonic development), and owing to realization of heterogeneity amongst population of a single cell type (for instance certain individual cancer cells being immune to chemotherapy). Therefore devices enabling powerful analytical methods like electrochemical detection, spectroscopy, optical detection, and separation techniques along with cell piercing and fluid transfer capabilities at the intra-cellular level are desired. Glass pipettes have conventionally been used for single cell operations, however their poor material properties and an intrusive conical geometry have led to limited precision and scope of successful experimentation, and resulted in increased research efforts to develop novel, non-intrusive cell probes. Carbon nanotubes (CNTs) are known for their superior physical properties and tunable chemical structure and possess a high aspect ratio, minimally invasive tubular geometry. Moreover, significant accomplishments have been made by researchers on chemical functionalization of CNTs, making the idea of multi-functionality of resulting probe tips realistic. Here we report a novel fluidic device fabricated by isolating and assembling a “single” CNT at the tip of a glass pipette using a fluid flow based technique. The nanotube-glass junction was sealed with epoxy, to allow fluid flow from the glass pipette to the nanotube, thereby enabling continuous fluid handling capability into cells. The nanotube was functionalized with paramagnetic iron oxide and gold nanoparticles facilitating simultaneous remote magnetic manipulation of the tip and surface enhanced Raman spectroscopy. Further, the glass pipette interior was coated with carbon facilitating electrophysiology. These probes were demonstrated to induce significantly less calcium response during cell piercing compared to glass pipettes. Thus, minimally intrusive "multifunctional cellular endoscopes” were developed (reported in Nature Nanotechnology 6, 57-64 (2011)). Meanwhile, we demonstrated an individual CNT to function as a nano-separation column for separating attoliter volumes of mixtures of different analytes (smallest volumes ever analyzed!). In this regard we demonstrated separation of two fluorescent dyes of significantly different molecular weights by flow through a 200 nm amorphous nanotube stuffed with smaller 20-30 nm multiwalled CNTs as stationary phase (reported in Scientific Reports (2012)). Moreover we performed the process of liquid-liquid extraction using 200 nm amorphous CNTs by selectively extracting a fluorescent dye from a dye mixture using a solvent having different solubility for the two dyes. These nanotubes can then be used as aforementioned "endoscope" tips to perform separation processes on single cells.

Recent biomedical research has sought to develop point-of-care diagnostic devices that have high sensitivity, provide rapid results, and are portable. While current detection methods are cumbersome. We propose the use of a potentiometric biosensor that incorporates the efficient and specific molecular imprinting (MI) method with a self-assembled monolayer (SAM) of thiol. This study sought to1) expand and verify the potential of disease detection and 2) characterize the detection interface for the final prototyping of a point-of-care diagnostic device. In preparation for future human studies, we first tested the biosensor in vitro using carcinoembryonic antigen, CEA, a biomarker associated with pancreatic cancer. In order to determine the level of sensitivity and the efficacy in low pH gastric environment, we tested the performance of the sensor as a function of imprinting and sensing pH. Computer simulations of the protein structure were performed in order to estimate the changes in morphology and determine the sensitivity of the biosensor to conformational changes in the proteins. Finally, to create a point-of-care diagnostic device, we designed and developed a commercial miniaturized biosensor that uses standard curves generated from the macro-scale biosensor and a microarray used to detect multiple biomarkers. At the same time, we also want to further apply our biosensor to other substances such as bacteria, virus. Since the size of the bacteria and virus is different from protein, to further apply the sensor to them, certain size of patterns are created for the detection of biosensor.

Nanofluidics devices consisting of nanochannels are used to stretch and visualize single DNA molecules. As a DNA molecule is forced in a nanochannel of height and width in the range of the DNA persistence length, the molecule stretches out. Single molecules can be isolated from the bulk and studied using high numerical aperture epifluorescence microscopy. This has been utilized to measure physical properties of DNA molecules such as their individual length. It has also recently been used to visualize sequence specific fluorescence barcodes obtained by labeling of nicking sites [1], the labeling of methylated sites [2], competitive staining [3] or partial melting [4]. Previous studies demonstrate barcoding for viral DNA or BACs in the length range of 50-200kbp, paving the way to applications within genomics. We have established a method for interfacing nanofluidic devices with samples such as a cell culture by extracting DNA from human metaphase chromosomes in lab-on-a-chip devices. This is implemented in two ways utilizing passive microfluidic trapping [5] or active optofluidic trapping [6] to select a single chromosome and extract the DNA by proteolysis. This method allows loading intact mega base pair long DNA strands onto a nanofluidic device. We will present our recent work toward visualizing mega base pair long DNA molecules and applying our method to genomics. References: [1] Das, S. K.; Austin, M. D.; Akana, M. C.; Deshpande, P.; Cao, H.; Xiao, M. Nucleic Acids Research 2010, 38, (18), e177 [2] Lim, S. F.; Karpusenko, A.; Sakon, J. J.; Hook, J. A.; Lamar, T. A.; Riehn, R. Biomicrofluidics 2011, 5, (3) [3] W. Reisner, N. B. Larsen, A. Silahtaroglu, A. Kristensen, N. Tommerup, J. O. Tegenfeldt, and H. Flyvbjerg, PNAS, 107, 30 (2010) 13294-1329 [4] Nyberg, L. K.; Persson, F.; Berg, J.; Bergström, J.; Fransson, E.; Olsson, L.; Persson, M.; Staring;lnacke, A.; Wigenius, J.; Tegenfeldt, J. O.; Westerlund, F. Biochemical and Biophysical Research Communications 2012, 417, (1), 404-408. [5] K. Rasmussen, R. Marie, J. M Lange, W. E Svendsen, A. Kristensen and K. U Mir, Lab Chip 11, 1431-1433 (2011) [6] Eriksen, J.; Thilsted, A. H.; Marie, R.; Luscher, C. J.; Nielsen, L. B.; Svendsen, W. E.; Szabo, P.; Kristensen, A. Biomicrofluidics 2011, 5, (3), 031

Most tissues are composed of repeating cellular functional structures, such as the lobule in the liver and kidney, islets in the pancreas. In the native microenvironment, the cells in these functional units are imbedded in a three-dimensional (3D) microenvironment composed of extracellular matrix (ECM) and neighboring cells. Approaches in tissue engineering attempt to recreate the native 3D architecture in vitro. Recently, the convergence of multiple fields including nano- and micro-scale technologies resulted in the emergence of bottom-up methods where cell-laden microgels can be used as building blocks for tissue engineering and regenerative medicine. Although various microgel fabrication and assembly methods have been developed based on modifying interfaces and using microfluidics, so far, two main challenges remain: (1) to fabricate microgels composed of multiple cell types spatially confined in 3D as functional units, and (2) to assemble microgels into large complex 3D constructs rapidly in an efficient way. Here, we present non-invasive field-based 3D assembly technologies based on magnetics, acoustics and bioprinting to create 3D complex tissue constructs and co-culture units in a high throughput manner with control over composition using multiple cell types. We demonstrate the utility of this method by fabricating four different types of co-culture units, where the quality of the units was optimized by the photomask design. Also, we developed a magnetic assembler that utilizes nanoparticles and microscale hydrogels as building blocks to create 3D complex multi-layer constructs via external magnetic fields using different concentrations of magnetic nanoparticles. These methods potentially enable a biologically relevant in vitro platform to investigate cell-cell interactions in a 3D microenvironment, holding a promising direction in various areas, spanning tissue engineering, regenerative medicine, pharmacological studies and high throughput applications.

We present a simple and fault-tolerant strategy for pathogen detection based on their specific capture onto patterned chips, with label-free optical imaging and 2D-FFT analysis for robust detection and readout. Pathogen capture is mediated by "immutable" ligands essential for cell-surface recognition or the sequestration of essential minerals. The ligands are presented as linear arrays by microcontact printing, or as dot-matrix arrays by inkjet printing. The patterned arrays are detectable upon pathogen capture using darkfield conditions, and encode peak frequencies that are easily monitored in Fourier space. The FFT readout produces signature peaks with remarkably low occupancy, and is highly tolerant to noise generated by nonspecific binding. This method has enabled us to detect select pathogens at limits below 1000 cfu/mL, and can be applied toward the design of array patterns for multiplex detection.

To truly realize the long-heralded potential of nanobiotechnology to advance human health, we must develop a way to manufacture such devices at low cost and in high volume. Moreover, we must beat the inherent resolution limitations and planar geometric constraints of traditional ‘top-down&’ manufacturing approaches. The human body spans a vast multitude of length scales with awesomely complex 3D geometries; both these attributes cannot be recapitulated with existing manufacturing approaches. This bottleneck is preventing the translation of critical technological innovations to move from the laboratory bench to the patient&’s bedside. To address these challenges, my lab has been pioneering a radically different approach to micro and nanofabrication. We enable high-resolution features directly in industry-standard plastics by leveraging the inherent heat-induced relaxation of pre-stressed thermoplastic sheets, commodity shrink-wrap film. By patterning at the large scale, which is easy and inexpensive, and subsequently shrinking down to 5% of the original, patterned sizes, we achieve intricate structures and devices with high fidelity. With this simple and scalable approach, we are able to circumvent the inherent resolution limit of traditional ‘top-down&’ fabrication defined by Rayleigh&’s criteria. Moreover, this allows us to pattern directly on flexible, lightweight, and conformal substrates. We have demonstrated that we can create fully functional and complete devices with robustly integrated nanostructures, printed electronics, and even optical components, all within minutes at a negligible cost, and importantly, with improved properties and performance characteristics. Integrated nanostructures self-assemble when the shrink film coated with a metallic thin film retracts; the stiffer metal layer cannot shrink and therefore buckles and cracks controllably into high-surface area nanostructures. This is a highly non-linear process sensitive to small changes in strain. Therefore, if well controlled, such folding of thin films is a rapid and effective way to generate deterministic 3D structures. Importantly, we have demonstrated these resulting nanostructures, which are robustly integrated into the plastic substrate, can be tuned in size and shape by controlling the thickness and composition of the deposited metal that can be optimized for a variety of applications. Using the toolbox that we have created with this approach, my lab has been investigating 2 main biomedical research thrusts, namely 1) high sensitivity / low cost molecular diagnostics and 2) differentiation of pluripotent human stem cells into functionally mature cardiomyocytes. Now, as we maturate our technologies, we are able to address these complex scientific problems in increasing intricacy and depth as well as to extend our technologies for other applications, including: chemical catalysts, embedded electronics, wound healing, environmental sensing, and energy applications.

Water-quality monitoring is an essential priority for global health. With microorganisms a primary cause for the occurrence of infectious diseases, the concentration of harmful pathogens should be routinely monitored to maintain microbiological quality control of drinking water. Currently testing is conducted in central labs by using plate-culture assay techniques which can take up to 24 hours to produce test result. In order to achieve more timely assessment of water quality, PARC is developing a compact and robust platform for rapid pathogen characterization in water. The presented approach is suitable for point-of-need testing and is able to provide test results in less than 20min. The enabling technique is termed “spatially modulated emission” and generates a time-dependent signal as a continuously fluorescing bio-particle traverses a predefined pattern for optical transmission. Correlating the detected signal with the known pattern achieves high discrimination of the particle signal from background noise. In conventional flow cytometry, the size of the excitation area is restricted approximately to the size of the particle. Our method allows a large excitation area to increase the total flux of fluorescence light that originates from a particle. Despite the large excitation area, the mask pattern enables a high spatial resolution which permits independent detection and characterization of near-coincident particles, with a separation (in the flow direction) that can approach the dimension of individual particles. In addition, the concept is intrinsically tolerant to background fluorescence originating from fluorescent components in solution or contaminants on the chip. We have demonstrated pre-concentration of Giardia and Cryptosporidium which substantially reduces the analyte volume (~1000 times) while retaining most of the pathogens (>90%). For pathogen detection we have assembled and tested a working prototype of a micro-fluidic-based flow cytometer which analyzes water samples with a throughput of 50ul/min. Measurements of the sensitivity and dynamic range were conducted with calibration particles and yielded a detection limit of ~500 MEPE, which clearly meets the requirements for a wide range of bio-particle-detection applications. Tests with water-borne pathogens clearly show that this instrument can be used to reliably identify and count specifically-tagged pathogens at meaningfully low concentrations. We will show results for Giardia, Cryptosporidium, and E. coli. Incubation studies with anti-body based reagents show that for Giardia incubation times as short as 2min and analyte-to reagent-ratios as low as 1:100 are sufficient for reliable detection. We will also show that the antibody-based reagents are highly stable, with little degradation over a period of months at 37C. *Acknowledgment: the research is funded by the U.S. Army Research Office, Contract # W911NF-10-1-0479. (PM Wallace G Buchholz.).

Self-assembly is a major component of bottom-up technologies in the development of nanoelectronics. It is, however, less common in the field of microfluidics. We shall show in this talk how self-assembly concepts and methods issued from the soft-matter and complex materials fields, can facilitate the implementation of complex microstructures, or offer new operational components in this fast evolving field. We shall, in particular, present new developments in the field of convective, capillary and magnetic self-assemby. We extended the capabilities of capillary assembly to organize multiplexed arrays of functionalized particles on surfaces. These particles were further used as traps for the selective capture of living cells, Capillary assembly is a powerful technique to pattern structured surfaces with high precision using micro or nano-object suspended in a liquid. A colloidal suspension is dragged along a surface topographically patterned with cavities or obstacles Particles are selectively trapped into the recessed or protruding structures while on the flat areas, particles are carried away by the capillary forces exerted by the meniscus and no deposition occurs. We extended this to the the multiplexed capture of different particles. We shall also present an application, in which these arrays are used as templates for reversibly creating in a microfluidic system arrays of posts bearing antibodies for the capture of circulating tumour cells (CTC). This system allows the creation of micropillars with an aspect ratio of several tens, impossible to create by microfabrication methods. As a bioseparator, it combines a high capture efficiency ( 90%) and the possibility to perform high resolution and high content imaging of the captured cells.

With more number of individuals becoming health conscious, the use of biosensors for medical diagnosis is growing rapidly. Many efforts are directed toward improving the performance of biosensors in terms of accuracy, sensitivity, reduced size, and increased portability. Consequently, nanotechnology emerges as an important role in biosensors field. Numerous ultrasensitive biosensors at nano-scale have been developed. Herein we present biosensors consist of individually addressable and dimension controllable conducting polymer single nanowires for ultrasensitive detection of proteins. Single conducting polymer nanowires (e.g. polyaniline, polyprrole) were electrochemical deposited in polymethylmethacrylate (PMMA) nano-channels patterned between two Au electrodes. A single polyaniline nanowire (PANI NW) based biosensor was established at first for the detection of immunoglobulin G (IgG) and myoglobin (Myo). The target proteins were detected by measuring the conductance change of functionalized PANI NWs. The detection limit was found to be 3 ng/mL for IgG and 1.4 ng/mL for Myo. To improve the sensitivity of this biosensor, instead of using antibodies, aptamer was then employed as the receptor. The use of aptamer generated an aptasensor that has excellent specificity and ultrasensitivity. The detection limit of the aptasensor for immunoglobulin E (IgE) can reach a few fM, which surpasses the requirements for the detection of most protein biomarkers. Due to the excellent stability and reversible conformational change of the aptamers, these aptasensors can be repeatedly regenerated and reused. To produce high throughput PANI NWs on large area with high uniformity, a novel NW fabrication method was developed. This approach combines nanofabrication techniques and a chemical synthesis method. E-beam lithography defined the dimension of the PANI NWs and allowed excellent shape uniformity. Chemical synthesis of PANI enabled and facilitated the mass production of PNAI NWs on large area. With an optimized surface modification method using 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimde (NHS) reagents, antibodies and aptamers can be immobilized onto the surface of these NWs. The fluorescence microscopy images of these PANI NWs modified by fluorescence-labeled antibodies showed that these NWs exhibited clear emission with uniform distribution over their surface. Remarkably, strong fluorescence can be observed from most of these NWs, suggesting the excellent reproducibility of this surface modification method. Hence, this new NW fabrication method has great potential for the development of nano-biosensors with excellent performances in point-of-care applications.

We implemented a disposable biochip that can be inserted onto a Printed Circuit board (PCB) which has reusable electrodes to perform contactless electrical impedance measurements. This significantly reduces the manufacturing costs (5 cents per consumable device in retail price), making it suitable for low resource settings, such as point-of-care testing in the developing countries. In this work, we demonstrated a novel and cost-effective approach to implement a disposable microfluidic flow-through impedance cytometer. Conventional methods for single cell impedance cytometry use microfabricated electrodes in direct contact with the buffer to measure changes of its electrical impedance when cells pass through the applied electric field. However, this approach requires expensive microfabrication of electrodes, and also, the fabricated electrodes cannot be reused without thorough and time-consuming cleaning process. Recently, contactless cell manipulation using Dielectrophoresis was demonstrated. Similar to this approach, we used a contactless measurement method to perform single cell impedance cytometry. Using cost-effective PCB technology, the copper electrodes (1 mm wide) are patterned on the substrate. The microfluidic channel (fabricated in PDMS using soft lithography) on a disposable micro glass coverslip of thickness 150 µm is placed on top of the electrodes. With this approach, the electrodes on the substrate are isolated from the buffer, by the thin glass coverslip. As the electrodes are no longer in direct contact with the fluid sample, the possibility of cross-contamination is eliminated which makes the electrodes reusable. Based on our analytical derivation and simulation results, for the frequency range of interest, the output noise stays constant in our system, while the measureable signal increases with frequency. In our system, signal at low frequency is degraded significantly by the small value of the glass coverslip&’s capacitance. Ideally, a large coverslip capacitance (higher dielectric material or thinner coverslip) is preferred. However, here we were limited by the commercially available coverslips. Therefore, in our experiment, we operated at sufficiently high frequency to capacitively couple the electrodes to the electrolyte in the channel, and thus, improve the signal beyond the noise level. The improvement of signal with frequency is limited by the amplifier&’s slew-rate limitation as the electrodes are shorting capacitively. To confirm our analysis and to demonstrate the functionality of our device we injected 2.8 µm-diameter beads in the channel. By analyzing the data for the passage of 60 beads (resulting in 60 distinct peaks), captured at 4 different frequency channels simultaneously we verified the increase in Signal-to-Noise ratio with frequency.

In this presentation, polymer-based modular microfluidics for building highly integrated systems for selecting biological cells from a variety of clinical samples, electrically enumerating the selected cells and molecular profiling them will be discussed. The specific example demonstrating the utility of these systems will be selecting circulating tumor cells (CTCs) from whole blood. CTCs are gaining popularity as potential biomarkers for a variety of epithelial-based cancers due to the ease of securing them (blood draw) and the wealth of information they can provide in terms of their numbers (response to therapy; disease recurrence; early detection) and the ability to guide therapy based on molecular signatures (personalized medicine). Highly functional diagnostic systems can be assembled using a modular design approach, in which task specific modules are interconnected to a fluidic motherboard. These modular systems are fabricated from a variety of polymeric materials, such as poly(methylmethacrylate), PMMA, polycarbonate (PC) or cyclic olefin copolymer (COC) using micro- and nanoreplication. The system consists of a cell selection module, an electrical detector for enumeration and a molecular analysis unit. For the cell selection module, monoclonal antibodies (mAb) must be covalently tethered to the wall of the selection bed using the appropriate chemistry following activation of the substrate walls. We will discuss the activation of polymer (COC and PMMA) walls using both UV and plasma activation processes, which produce a functional scaffold of carboxylic acids that can serve as anchoring points for the EDC/NHS coupling of the mAb to the channel wall. Effects of channel aspect ratio, material and irradiation dose on the density of carboxy functional groups will be discussed. We will also discuss the generation of nano-texturing using a 3D molding process of the channel walls to improve the adhesion strength of cells to the selection bed. Following release of the selected cells from the channel walls, enumeration is accomplished by single-cell impedance cytometry using thin metal films deposited onto polymer substrates. The cytometry can measure cell size under low frequency operation, but high frequency operation can provide cell composition information. The cells can be molecularly profiled by detecting sequence variations in their nucleic acids using a PCR coupled to an allele-specific ligation assay, which can detect single base mutations even from 1 cell. Readout of the molecular assay is accomplished using molecular beacons undergoing fluorescence resonance energy transfer with excitation via an embedded polymer waveguide. The use of the system for monitoring colorectal cancer patients for their CTCs and KRAS genotype will be discussed.

Microfluidic devices offer the ability to finely control physical and chemical conditions which is advantageous for microparticle synthesis. We have rinvented a new technique entitled Stop Flow Lithography (SFL) which couples microfluidics and projection lithography to create microparticles with chemical and geometric complexity. These particles can be both geometrically encoded and functionalized with biomolecules to create microparticles for use in multiplex assays. Importantly, the scaffold material for these microparticles is a hydrogel that offers many advantages for bioassays. In this talk I will discuss our progress in both particle synthesis, particle performance in sensing assays and development of a flow scanner for reading the particles. Specific application to miRNA sensing will be discussed.

The drive to improve the performance and reduce the cost of electronic, photonic and fluidic devices is starting to focus on the use of materials that are exotic for these applications but actually readily available in other fields. In this talk the use of paper in bio/medical chip applications will be reviewed. Paper is a very attractive material for many device applications: very low cost, available in almost any size, versatile surface finishes, portable and flexible. From an environmental point of view, paper is a renewable resource and is readily disposable (incineration, biodegradable). Applications of paper-based electronics currently being considered or investigated include biochips, sensors, communication circuits, batteries, smart packaging, displays. The potential advantages of paper-based devices are in many cases very compelling. Devices fabricated on paper for bio/medical applications frequently use the capillary properties of paper to operate without the need of external power sources, greatly simplifying the design and reducing the cost. Examples of paper-based biochips will be discussed.

In order to improve hemocompatibility and reduce thrombus formation on therapeutic devices used for blood pathogen separation and sensing applications, we applied a betaine polymer on the inner surface of a polydimethylsiloxane (PDMS) microfluidic construct through a wet chemistry process. The betaine monomer was directly polymerized on the microfluidic surfaces using a flow reactor. The entire inner surface of the device from tip-to-tip was modified, including the microfluidic device and silicone inlet and outlet tubes, while maintaining the microchannel geometry and bulk material properties. The resulting surfaces were characterized using optical and electronic microscopy, ATR-FTIR, contact angle, and XPS. Fibrinogen adsorption was evaluated using radio-labeled fibrinogen. Modified (n=16) and unmodified devices (n=11) were then challenged in a whole-blood flow loop test using freshly harvested bovine blood with radio-labeled platelets; heparin was added to the blood but at levels much lower than typically required for clinical applications. The pressure change of blood during the flow and the attached platelets after the flow test were measured. The modified device significantly improved the wettability by 73% by contact angle and reduced protein adsorption by 97 % when compared with the unmodified device. Visual evidence and pressure plots show extensive clotting in the control group and no clotting in the channels of the modified group during the 30-60 minute test. Additionally, few pressure changes in modified devices were found compared with the increased pressures observed in the unmodified control group. Previously, the carboxybetaine-modified surface plasmon resonance (SPR) substrates were additionally functionalized with antibodies and a strong specific binding was achieved with minimum non-specific adsorption, performed at a detection limit of 7.8 ng/mL in 100% human plasma. This work demonstrates the betaine modification is capable of improving blood compatibility of microfluidic devices designed for separation and sensing of pathogen and biomarkers in whole blood or plasma. This material is based upon work supported by DARPA and SSC Pacific under SPAWAR N66001-11-C-4187.

We present the development and manufacturing of microfluidic devices for a high definition immunoassay analyzer, enabled by the single molecule arrays (SimoaTM) technology. These devices, consisting of microfabricated polymer array assemblies, facilitate the isolation of individual micron-sized paramagnetic beads in arrays of femtoliter-sized wells and the detection of single enzyme-labeled proteins on these beads using sequential fluid flows. This approach allows for automated, low-cost and high-throughput precise measurements of clinically relevant biomarkers at unprecedentedly low concentrations over a broad dynamic range. In this presentation, we will give a brief introduction to the Simoa technology, as well as provide examples of measuring low abundance proteins and its potential in ultra-sensitive medical diagnosis. We will then describe the development of enclosed polymeric assemblies that perform the functions of loading and sealing of paramagnetic beads associated with single enzyme molecules in arrays of femtoliter-sized wells using only fluidic flow. Specifically, we will focus on various aspects of process development for a micro-replication method using cyclic olefin polymer (COP) to fabricate the femtoliter-sized well arrays and the fluidic structures to allow the delivery of fluids to the arrays. We will discuss results from characterization techniques that validated the integrity and the precision of the replication method of micro- and macro-structures, as well as critical optical properties. Finally, we will present results of a digital immunoassay for prostate specific antigen (PSA) performed using these devices.

Combining biomolecular function with integrated circuit technology could usher in a new era of biologically enabled electronics. A key challenge has been coupling different molecular functions to specific chip locations for communication with the circuit. We are developing directed assembly methods based on spatially confined electric fields to assemble different populations of DNA-coated nanowires to desired positions on a patterned Si wafer. This combination of off-chip synthesis and biofunctionalization with high-density, heterogeneous assembly and integration at the individual nanowire level points to new ways of incorporating biological functionality with silicon electronics. Off-chip functionalization however, requires that the molecular and/or biomolecular coatings on the nanowires be subjected to electric fields, non-aqueous solvents, photoresists, and resist removers. Evaluation of these treatments on DNA coatings will be discussed.

After the first demonstration of field effect-based biomolecular detection over a decade ago, there has been significant progress in BioFET sensors with improvements in detection limit, sensitivity, selectivity, biomolecule immobilization and integration with signal processing. The vast majority of FET biosensors utilize well-established Si MOSFET technology. In contrast, emerging nano-electronic approaches hold promise for drastic improvement of sensor performance. These include the use of nanoscale patterning, high-κ gate oxides, high mobility channel materials, and 3D gate architectures. Our proof-of-concept sensor platform expands on this work using nanoscale finFET devices. There are several reasons for using finFET geometry over the standard planar FET geometry. In this architecture, the gate and channel regions protrude from the device, similar to the dorsal “fin” of a shark. The fin works to increase the amount of sensing surface area per volume of the semiconductor channel, which is strongly believed to enhance sensitivity. Our work predicts that, unlike planar devices, the charge of a single molecule in the fin (i.e., the gate) could affect the entire cross-section of the channel, causing strong current blocking effects. While ultra-sensitive finFETs have been fabricated and are being studied for nanoelectronics applications, they have not yet been applied to biomolecular sensing. Further, there has been limited work on in situ growth of alternative nanostructures (nanowires, nanotubes, etc.) for electrical biosensing and “top-down” fabrication using CMOS-compatible processes. In contrast, our approach uses manufacturing processes to construct nanoscale finFET devices with distinct advantages for production and dissemination. We have previously shown that DNA probes can be directly immobilized to hafnium oxide gate dielectrics for FET-based detection of DNA targets. Here we will present the results of finFET biosensor fabrication and initial testing of device sensitivity for DNA detection. Briefly, finFETs were fabricated on 300 mm wafers by etching 28nm fins into SOI wafers. A gate stack of HfO2, TiN, and amorphous-Si was deposited and the gate area defined lithographically. SiN self-aligned spacers were formed and selective N and P implants were performed to dope the source and drain of N and P devices, respectively. A layer of SiN and SiO2 was applied to protect devices from aqueous solutions during testing. With subsequent processing steps these layers, and the TiN layer, were removed to reveal the gate region of devices (for DNA sensing). A final lithography step opened the oxide over the source and drain contact pads. This provided points for electrical contact, as well as a non-metalized gate for direct DNA deposition and detection on the gate oxide. To our knowledge this is the first example of finFET biosensors being fabricated on a production-scale 300 mm wafer platform, and the first DNA biosensor using the finFET geometry.

Electronic systems are believed as the best platform to perform biological sensing for fast point-of-care diagnostics or threat detection. One of the solutions is the lab-on-a-chip integrated circuit (IC) which is low cost and high reliability, offering the possibility for label-free detection. In recent years, similar integrated biosensors based on the conventional complementary metal oxide semiconductor (CMOS) technology have been reported. However, post-fabrication processes are essential for all classes of CMOS biochips, requiring biocompatible electrode deposition and circuit encapsulation. In this work, we present an amorphous silicon (a-Si) thin film transistor (TFT) array based solution, which greatly simplifies the fabrication procedures and even decrease the cost of the biosensor. The device contains several identical sensor pixels with amplifiers to boost the sensitivity. Ring oscillator and logic circuits are also integrated to achieve different measurement methodologies, including electro-analytical methods such as amperometric and cyclic voltammetric modes. The system also supports different operational modes. For example, depending on the required detection arrangement, a sample droplet could be placed on the sensing pads or the device could be immerged into the sample solution for real time in-situ measurement. The whole system is designed and fabricated using a low temperature amorphous silicon TFT process on glass substrate. No additional processing is required prior to biological measurement. A Cr/Au double layer is used for the biological-electronic interface. The success of the TFT-based system used in this work will open up new possibilities for flexible label-free or low-cost disposable biosensing.

A major technical challenge today in neuroscience centers on intracellular recording from multiple neurons with high spatio-temporal resolution in live intact brains. Currently electrophysiological recordings from neurons are achieved using patch or sharp glass pipettes for intracellular recordings, although these can be limited in-vivo due to required manipulators and chemical/physical disturbance of the cell to initiate measurement. Extracellular recording using planar devices can be implemented at multiple sites but have a low signal to noise ratio and must employ spike sorting to define single unit. To overcome the limitations of existing techniques, we have developed a type of probe with kinked three-dimensional (3D) silicon nanowire FET arrays, and show that it can readily record intracellular and extracellular signals from multiple neural sites within the brains of rats in-vivo. The overall probe is made from a silicon substrate shaped to resemble a pipette (with a tip diameter of ca. 200 µm). In the tip region of the probe, an array of multiple 3D kinked silicon nanowire FETs are fabricated with each FET connected to a source (S) and a drain (D) metal electrodes, and independently addressable. The nanowire FET conductance, which varies as a function intra or extracellular potential, is monitored during experiments and subsequently converted to potential using individually-determined device sensitivities. In this study we show that the 3D kinked nanowire FETs can record intracellular and extracellular signals from the somatosensory area of live anesthetized rats. Following probe insertion, both extra and intracellular signals were recorded from up to 8 devices. Details of the recording measurements, including baseline shifts during extra-to-intracellular recording, signal waveforms, and the response to pharmacological agents such as TEA and TTX will be described. In addition, subthreshold postsynaptic potentials (PSPs) were observed. Extracellular recordings have local field potential characteristics, although the signal amplitudes are substantially larger than observed with conventional recordings. In addition, attractive potential capabilities of this new nanotechnology probe will be presented, including (i) extremely small detection area down to hundreds of nanometers for a single FET, (ii) the ability for recording of intracellular voltage dynamics of neural networks in-vivo, (iii) the ability to record from small nerve structures (e.g. dendritic spines) and cell types (e.g. glial cells) difficult to achieve with conventional techniques.

The development of flexible biocompatible high-resolution neural probes is an important step in the correct understanding and treatment of neurological conditions such as Parkinson&’s disease, major depressive disorder, and spinal cord injury. Using fiber-inspired fabrication methods we have created flexible polymer-based hollow capillaries with composite conductive polymer-metal electrodes adjacent to the hollow cores. The diameters of the hollow cores can be tuned between 30-100 mu;m, which allows these devices to be used as flexible scaffolds for neuronal growth. To test our electronic scaffolds we have successfully cultured neurons inside their cores. These trapped neurons maintained viability for over two weeks and developed long processes. Furthermore, we have demonstrated the possibility of confined genetic modification by transfecting these neurons with fluorescent proteins. Finally, we have employed the embedded electrodes to detect action potentials from the trapped neurons. Using a similar processing approach, we have also fabricated polymer-based multielectrode arrays incorporating tin electrodes for in vivo neural recording. Our process allowed us to achieve electrode diameters as low as 2 mu;m with controlled pitch. As our fabrication method is highly scalable and adaptable to a variety of polymers and low-melting-temperature metals, it will allow us to reach up to hundreds of electrodes in arbitrary geometries. These polymer devices have flexural moduli approximately two orders of magnitude lower than those of the currently used silicon-based electrodes, making them promising candidates for neural recording implants that minimize tissue damage.

The use of reconstituted silk as a base material for optical and electronic applications has been recently gaining momentum. We will overview the various material formats obtainable and describe the high quality, micro- and nanostructured optical and optoelectronic elements largely or entirely composed of this organic, biocompatible and implantable protein. The interplay between the technological and biological worlds offered by this material platform allows for unusual opportunities at the biotic/abiotic interface and adds utility for biologically integrated and eco-resorbable device.

Graphene-based electric current detection of a DNA base has recently drawn significant attention to the next generation of rapid DNA sequencing because of graphene&’s spatial resolution (~0.34 nm) of a single base. However, direct identification among four DNA bases could be one of most challenges because of its broadly overlapped conductance values among bases. As discussed in recent literatures, the broad spectrum of current values depends on the rotation or translation of a base located between the planar graphene electrodes. One of the reasons, for example, was the variation of the coupling strengths between the channel and electrode associated with the size or orientation of the channel (base). Here, we theoretically investigated more specific quantum origin of the broad current ranges for a single base. Sharp dip appeared in transmission T(E) turned out to be originated from the destructive quantum interference (DQI) effect via out of phase between two quantum paths on a base located between the left and right graphene nanoribbon electrodes. Here, to enhance the transmission conductivities, we utilized the metallic properties of ketone-terminated zigzag-edges in our graphene nanoribbon electrodes. One of the conditions for the phase shift occurs whenever the oxygen is present in either of two paths and changes its potential barrier. The condition was sensitive to the rotational angles with respect to the axis of the electrode. To confirm our hyphothesis, we performed numerical evaluation based on DFT-NEGF method implemented in SIESTA/TRANSIESTA package. For the case of guanine (G), variation of conductance reached up to four orders of magnitudes by rotating the base. We also observed transmission dip for certain angles and energy, as well as for cytosine (C) and thymine (T). For the case of adenine (A), however, no transmission dip was observed. Thus, presence of oxygen in DNA base plays crucial role DQI. Further study of DQI would provide the methods for the distinctive identification of four different DNA bases.

Following the discovery of extraordinary optical transmission, metallic films perforated with subwavelength nanoholes have been the focus of intense research, in particular toward applications in sensing, imaging, and spectroscopy. Recent work also demonstrated the potential of the nanohole-based platform for “flow-through” sensing, which can improve the sample delivery and detection sensitivity for kinetic biosensing. Here we present our recent work on the high-throughput fabrication of large-area metallic nanohole arrays using nano-imprint lithography and template stripping. The resulting nanohole arrays, patterned over centimeter-sized areas, exhibit homogeneous optical properties with sharp (<10 nm in linewidth) and intense plasmon peaks. We describe techniques for surface modification, microfluidic integration, and multi-spectral imaging to utilize these nanostructured films for label-free biosensing in an aqueous environment. Also, we demonstrate a new mechanism to concentrate biomolecules on nanopore chips for improved detection sensitivity and also create ordered nanoscale arrays of beads and liposomes in a simple and facile manner.

Scanning Tunneling Microscope(STM) and Gating nanopores are the key Nano-Biodevices for third generation DNA sequencing technologies. These nanodevices will make sequencing kilobase length single-stranded genomic DNA or RNA or identifying individual small molecules using only electric currents and without fluorescent labels at low cost and unheard speeds. Nanopores approximately 1-5 nm in diameter are formed on a Si substrate, and nanogap electrodes with spacing equal to the diameter of the DNA molecules are fabricated in the Si3N4 membrane. This nanostructure is expected to detect molecules passing through the nanopore not by changes in the ionic current flowing parallel to the nanopore but by changes in the electric current flowing between the nanogap electrodes. The electric current passing between the nanoelectrodes comes from a tunneling current conducted via molecules. We have demonstrated identifying single-nucleotides using nano-fabricated mechanically controllable break junctions (Nano-MCBJ) by tunneling current across the nanogap electrodes. We found that the single peak current on current histograms determines the single-molecule conductivity of the order of dGMP >dAMP >dCMP >dTMP, and rGMP > rAMP > rCMP > rUMP. Recently, we have succeeded in sequencing micro-RNA by assembling contigs using a pair of nanogap electrodes. These results provide an essential scientific basis toward constructing Innovative Nano-Biodevices for the emerging DNA and RNA sequencing technology. References (1) T. Kawai et al, Nature Nanotechnology, 4, 518-522, (2009), (2) T. Kawai et al, Appl. Phys. Lett. 95, 123701-123703(2009). (3) T. Kawai et al, Nature Nanotechnology, 5, 286-290 (2010). (4) T. Kawai et al, Nature Communications, 1:138 (2010). (5) T.Kawai et al, Scientific Reports, 1, 46 (2011).

Electronic detection of biologically relevant species performed by means of disposable organic devices has the potential to revolutionize the current approach to strip testing. Presently, low-cost tests largely rely on paper based lateral flow strips capable of delivering a bare analogic output, inherently not processable. An organic electronic sensing device can produce a digital output allowing data processing for quantification. Such organic electronic devices can be produced, on a paper substrate, in principle at costs comparable to those for the manufacture of analogic strips. This is why electronic bio-detection is becoming a lively research field. Interesting developments involved the so-called electrochemically gated Organic Field-Effect Transistors (OFETs) [1, 2] or bottom gate OFETs with a different bi-layer architectures. [2 ] In fact, both the elicited device structures include a bi-layer architecture formed by a bioactive layer deposited or grafted on top of the OS film. A novel approach involving OFET devices comprising a Functional Biological Interlayer (FBI), was recently proposed. These devices comprise a supramolecular architecture with a bio-layer residing underneath the OS layer, right at the interface were the OFET two-dimensional transport occurs. [3 ]. This allows detection limits in the part per trillion concentration range to be achieved. In this presentation these novel developments will be reviewed and discussed. 1. L. Kergoat, et al., Organic Electronics 13, 1 (2012). 2. S. Cotrone et al. Organic Electronics, 13, 638-644 (2012). 2. L. Torsi et al., Nature Materials, 7, 412-417 (2008). 3. M.D. Angione et al. PNAS, 109, 6429-6434 (2012).

Nanowire sensors are emerging as one of the most outstanding platforms in various fields of the life sciences, including real-time, label-free in-vitro detection or cell monitoring of chemical and biological species. Although their potential applications in life science have been demonstrated successfully, the absence of effective surface modification routes still remains great challenges to achieve robust and multifunctional nanowire sensor platforms. To address these issues, we present a polymer-like nanosheath synthesized by nonthermal plasma technology that can provide robust and multifunctional platform for chemical and biological nanosensors. For ZnO nanowires which are well known as having chemical instability in aqueous solutions, we first demonstrate that ZnO nanowire field effect transistor (FET) with the nanosheath can be utilized as a pH sensor with long-time stability. Then, the importance of our approach will be discussed with comparison studies of conventional functionalization technologies. Based on the above considerations, we introduce various approaches to establish effective immobilization strategies of biomolecules on the nanosheath, including non-covalent and covalent attachments of proteins or aptamers. Using electrochemically top-gated FET, the electrical characterizations of nanosensors were performed systematically during each immobilization steps for the purpose of achieving the best performance of chemical and biological detections. Finally, we demonstrate that our nanosensor platform acts as pH/metal ion sensors, and a biosensor for the real-time, label-free detection of liver cancer markers.v

One of the best strategies to halt cancer&’s progress is the development of new diagnostic tools that allow one to detect the disease in an early stage. It would be desirable to develop simple and robust cancer detection systems without using unreliable biomarkers for a variety of tumor grades regardless of its origin in early stages. The development of non-invasive screening device for cancers with high specificity and selectivity enables more frequent monitoring of the early stage disease development, progress, recovery, and recurrence of cancers. Here we developed a new cancer detection platform incorporating electric cancer cell sensors on silicon chips. This sensing platform was designed to distinguish cells in different sizes and shapes by measuring their characteristic impedance signals on polysilicon microelectrodes. Due to the softness of cancer cells as compared to normal cells, cancer cells were observed to swell three times more than normal cells under hyposmotic pressure. By using this sensor chip and protocol, cancer cells can be distinguished from normal cells electronically without biomarkers; as strong hyposmotic stress is applied to cells, only cancer cells increase impedance signals due to the distinguished mechanical property. For example, we have examined six different cancer cell lines from prostate, kidney, ovarian, and breast, and all of these cancer cells were observed to expand their size about 35 - 50 % under osmotic pressure and their swellings could be detected sensitively and selectively by the robust impedance measurements of the sensor chip on the order of 10 cells/mL in less than 30 minutes even in contaminated samples. Recently, we improved the protocol to detect cancer cells in urine samples. As an important biomarker, mutant-p53 protein, encoded by mutated TP53 gene, is found to occur in more than 50% human cancers and mutant-p53 expressing tumors is more aggressive associating with poor-prognosis. Here, we studied mutant-p53 associate with metastases by using Impedimetric polysilicon sensor chip and and analyze the correlation between the aggressiveness of cancer cells and the cellular impedance values. Microscopic studies showed that the breast cancer cells with a high level of mutant-p53 can undergo a larger volume change under hyposmotic stress as compared to the cells without or with lower expression level of mutant-p53. When the volume change of each cell was exactly quantified and correlated with impedance values , we found that the knockdown of mutant-p53 in MDA-MB-231 and MDA-MB-468 breast cancer cells lowers impedance value, indicating that less aggressive cancer cells have lower impedance signal under hyposmotic pressure. In conclusion, the aggressive breast cancer cells could be distinguished from less aggressive ones by measuring impedance values of the samples, opening the possibility that circulating tumor cells, cancer stem cell, or metastatic cancer cells may be detected by this technology.

Early detection of harmful species in biofluids is paramount to enabling a drug regimen response to these species. Current virus detection methods, including enzyme linked immunosorbent assay (ELISA), are incapable of meeting this requirement due to the need for highly trained personnel and elongated period of time. Immunosensors function by electrically sensing highly specific antigen-antibody interactions that occur at the sensor&’s surface and are ideal for real-time analysis of biological samples. The primary objective of this work is to design and fabricate arrayable carbon nanotube thin film (CNT-TF) immunosensors capable of ultrasensitive, real-time, label-free and multiplexed virus detection. Chemical vapour deposition (CVD) will be utilized for CNT-TF synthesis, which will be followed by transfer/purification, integration into two-terminal devices, and coupled to microfluidic channels. MATLAB Monte-Carlo simulation will be used to predict the electrical behaviour of CNT-TF sensors and provide guidance in sensor design, including the optimization of CNT density and device aspect ratio, to achieve optimal reproducibility and sensitivity. Arrayed CNT-TF sensors will be designed and fabricated accordingly, and multiplexed whole-virus detection will be demonstrated. By combining computer simulation and experimental data, our work could provide scientific insights towards the mechanisms governing CNT based biosensors. Our platform could potentially overcome the reproducibility issue encountered by many 1D-nanostructure based sensing platforms and eventually lead to a real-world solution to cost-effective and rapid viral detection.

Cardiac electrophysiological signals that control contraction and release of cardiac muscles propagate rapidly over conducting paths of heart surfaces. Heart rhythm control starts from mapping and feedback therapy using cardiac electrophysiology. High precision mapping of rapidly changing electrical potentials through cardiomyocytes is key factor in clinical cardiology. Besides electrophysiology, other important physiological recordings to monitor and treat heart diseases, such as strain and temperature monitoring, require high performance electronics, which also require high quality electronic materials. However, high quality single crystal inorganic materials are intrinsically stiff and brittle, while biological systems are soft and curvilinear. This mechanical mismatch can be circumvented by processing thickness of inorganic materials to be hundreds of nanometer or even less. Deterministic assembly method using transfer printing technique integrates extremely flexible and ultrathin single crystal inorganic materials with substrates of mechanical properties of biosystem. By applying controlled pre-strains to designed ultrathin nanomaterials, stretchable systems with wavy and serpentine layout can also be developed. Neutral mechanical plane design further diminishes induced strain. This system based on mechanically optimized designs minimizes the influence of large external strain during deformations on vibrating heart surfaces. The concepts of flexible and stretchable bio-integrated devices are demonstrated through application examples including soft and conformable electrophysiological monitors integrated with epicardial and endocardial surface of heart. The value of this approach is also demonstrated in in-vivo and in-vitro experiments. The resulting bio-integrated electronic technologies realize important improvements in diagnostic and therapeutic surgical tools, including electrophysiological mapping systems and electrical stimulation therapies.

In our laboratory, we focus on a direct detection of ions or ionic molecules through ion-channels at cell membrane, because most of cell functions are closely related to transferring of charged conductors from cell to cell. In this study, we have clarified that a principle of semiconductor devices based on field effect realizes to detect ion charges in a direct, label-free, real-time and noninvasive manner for cell functional analysis. The principle of semiconductor-based biosensing devices is based on the potentiometric detection of charge density changes induced at a gate insulator/solution interface accompanied by specific bio-molecular recognition events. Ionic charges of ions or bio-molecules at the gate insulator electrostatically interact with electrons in silicon crystal across the thin gate insulator resulting in the threshold voltage change. Particularly, we are interested in ion transportations through membrane proteins such as ion-channels and ion-pumps at cell membrane and trying to detect ionic behaviors based on biological phenomena using a cell-coupled gate semiconductor (CGS). The semiconductor-based biosensing devices have good advantages of label-free, real-time and noninvasive method and we can make an arrayed device for a multi target analysis by use of the conventional semiconductor processes. In the point of detection of cell functions, we propose the device structure with three components such as target, signal transduction interface and detection device. Since we utilize the CGS, we are trying to design the signal transduction interface in order to detect ion charges specifically and selectively based on each cell function. In order to detect drug effect on cancer cells, we need to detect ion charges based on programmed cell death “apoptosis” using the CGS and develop the signal transduction interface to trap them. The previous work showed the possibility of potassium ions, chloride ions and water release in the early stage of apoptosis. Therefore, we have focused on potassium ion release based on apoptosis and succeeded in the real-time, direct and noninvasive monitoring of their flow. In particular, this result was accomplished by use of crown ether monolayer to trap selectively potassium ion as signal transduction interface of the CGS. Moreover, we have found the possibility of multi target detection for high throughput screening of drug effect using the CGS with some transfected cancer cells in this study. Using the CGS, furthermore, we have succeeded in a real-time and noninvasive monitoring of various cell functions, as follows.- Interaction between substrate and transporter at cell membrane for drug effect detection- Embryo activity based on in vitro fertilization (IVF) for assisted reproductive technology (ART)- Glucose response of pancreatic be-ta cells for insulin secretion- Differentiation of stem cells such as murine or human iPS cells - Autophagy for accommodation to starvation- Other cell functions

Breath analysis has the potential for early stage detection and monitoring of illnesses to drastically reduce the corresponding medical diagnostic costs and improve the quality of life of patients suffering from chronic illnesses [1]. In particular, the detection of acetone in the human breath is promising for non-invasive diagnosis and painless monitoring of diabetes (no finger pricking). Here, a portable acetone sensor consisting of flame-deposited and in-situ annealed, Si-doped epsilon-WO3 [2] nanostructured films was developed. The chamber volume was miniaturized while reaction-limited and diffusion-limited gas flow rates were identified and sensing temperatures were optimized resulting in a low detection limit of acetone (~ 20 ppb) with short response (10 - 15 s) and recovery times (35 - 70 s). Furthermore, the sensor signal (response) was robust against variations of the exhaled breath flow rate facilitating application of these sensors at realistic relative humidities (80 - 90%) as in the human breath. The acetone content in the breath of test persons was monitored continuously and compared to that of state-of-the-art proton transfer reaction mass spectrometry (PTR-MS) [3]. Such portable devices can accurately track breath acetone concentration to become an alternative to more elaborate breath analysis techniques [4]. [1] W. Q. Cao, Y. X. Duan, Clin. Chem. 2006, 52, 800-811. [2] M. Righettoni, A. Tricoli, S. E. Pratsinis, Anal. Chem. 2010, 82, 3581-3587. [3] K. Schwarz, W. Filipiak, A. Amann, J Breath Res. 2009, 3, 027002. [4] M. Righettoni, A. Tricoli, S. Gass, A. Schmid, A. Amann, S. E. Pratsinis, Anal. Chim. Acta, accepted.

For medical applications in neuroprostheses as well as for fundamental research on neuron communication, it is of utmost importance to develop a new generation of electronic devices which can effectively detect the electrical activity of nerve cells. Due to its maturity, most of the work with field effect transistors (FETs) has been done based on Si. However, the high electronic noise and relatively low stability associated to Si technology have motivated the search for more suitable materials. In this respect, the outstanding electronic and electrochemical performance of graphene holds great promise for bioelectronic applications. Here, we report on arrays of CVD-grown graphene FETs (G-FETs) for cell interfacing [1]. The high carrier mobility in graphene together with the large interfacial capacitance at the graphene/electrolyte interface leads to very high device sensitivities [2]. Furthermore, G-FETs exhibit very low electronic noise that allows detecting extracellular signals below 10 µV. The biocompatibility of graphene was investigated by culturing pure retinal ganglion cells from postnatal rats on bare and modified CVD-grown graphene layers. Retinal neurons on graphene surfaces show a healthy growth comparable to standard glass substrates. Finally, we will demonstrate the ability of graphene FETs to transduce with high resolution the electrical activity of individual electrogenic cells [3]. For instance, the generation and propagation of action potentials in cultures of cardiomyocyte-like HL-1 cells will be reported. Further, single cell-transistor coupling will be shown using Human Embryonic Kidney (HEK) cells and cortical neurons, in which the response of the graphene FETs to the electrical activity as well as the cell chemical activity will be discussed. This work highlights the potential of graphene to outperform state-of-the-art Si-based devices for biosensor and bioelectronic applications [4]. [1] Danker et al., Adv. Funct. Mater., 20 (2010) 3117 [2] Hess et al., Appl. Phys. Lett., 99 (2011) 033503 [3] Hess et al., Adv. Mater., 23 (2011) 5045 [4] C. Schmidt, Nature 483 (2012) S37

Single-nucleotide identification via transverse electron transport is a promising physical approach for high-speed and low-cost genome sequencing. Recently, we have developed single-molecule techniques for electrical detection of individual molecules trapped in an electrode gap (M. Tsutsui et al., Nano Lett. 8 (2008) 345-349; Nano Lett. 8 (2008) 3293-3297; Nano Lett. 9 (2009) 1659-1662; Nano Lett. 9 (2009) 2433-2439; J. Am. Chem. Soc. 131 (2009) 10552-10556; Nature Commun. 1, (2010) 138.), and reported that single nucleotides in a DNA can be identified by tunneling current measurements (M. Tsutsui et al., Nature Nanotechnol. 5, (2010) 286-290; J. Am. Chem. Soc. 133, (2011) 9124-9128.). Combining this capability, an electrode-embedded solid-state nanopore is considered as a promising detector structure offering prospects for $1000 genome, the device concept of which is based on identifying nucleobases in single-molecule DNA translocating through the pore by measuring the transverse current using the sensing electrodes. Despite the huge potential, fabrication of a molecular-scale nanoelectrode-nanopore has been a formidable task that requires atomic-level alignment of a few nanometer sized pore and an electrode gap. In my talk, I will present a self-alignment technique to reproducibly form a nucleotide-sized in-plane nanopore-nanoelectrode solid-state device compatible with silicon integrated circuit technology (M. Tsutsui et al., Sci. Rep. 1, (2011) 46.). I will also show results of transverse electron transport measurements of a DNA oligomer using the electrode-embedded in-plane nanopore sensor for electrical identification of the sequence. Unlike the conventional cross-plane configuration of nanopore detectors, the in-plane device geometry is amenable to integration of additional functionalities such as single-molecule tracking by fluorescence imaging, thereby suggesting the potential use of this synthetic electrode-in-nanopore architecture as a unique platform for the electrical DNA sequencing.

An electronic device capable of interfacing to the intracellular region of electrogenic cells has several important requirements such as small size, high signal-to-noise (S/N), and the ability to multiplex at both single cell and cell network levels. Here we report design, synthesis and fabrication of two novel intracellular probes based on nanowire field-effect transistors (NW FETs): (1) active nanotube transistor (ANTT), where the source/drain (S/D) is defined at one end of a silicon nanotube and electrically isolated from the surrounding medium, such that the intra/extracellular solution within the nanotube can gate the transistor and the variation of interior electrical potential is recorded as a change in device conductance; (2) branched intracellular nanotube field-effect transistor (BIT-FET), where the transistor remains outside of the cell but senses the intracellular potential of the solution inside a passive silicon oxide nanotube bridge. We show that both types of intracellular probes can sense intra/extracellular action potentials and can be readily multiplexed to record intracellular action potentials from a single cardiomyocyte as well as cellular networks.

Advantages associated with nanostructured materials such as the enhanced specific surface area, high aspect ratio, and size based confinement, the nanofiber arrays with localized and conductive carbon nanotube (CNT) inclusions have the potential to increase the sensitivity of the diagnostics platforms. We report a protein biosensor device consisting of CNT-polyvinyl alcohol (PVA) nanoweb (NW) layer and a printed circuit board (PCB) platform to detect C-reactive protein (CRP) at a femtogram level. An NW layer is deposited on the PCB microelectrode. Polydimethyl siloxane (PDMS) based microfluidic chamber is used for fluid encapsulation. (PVA-CNT) nanoweb was produced using a laboratory designed collector employed in traditional horizontal electrospinning process. PCB comprised of interdigitated finger pattern structures with gold as the exposed metal layer with thickness ranging from 50 to 100 nm, on top of a 3 micron thick nickel plating with 30 micron thick copper as the initial conducting path. SEM reveals that the average diameter of the NW is 170 nm. Optical micrograph shows the overall biosensor device with deposited NW. We focused on two specific experiments (a) detecting CRP in 0.15 M phosphate buffered saline (PBS) and (b) detecting CRP in commercially available human serum (hs). COMSOL is used to simulate the diffusion of CRP and anti-CRP in PBS based on the mobility of the protein molecules as a function of anticipated path and time through the NW. The percentage change in impedance ranged from 45% to 70% over a concentration range of 1 femtogram/mL to 0.1 microgram/mL. The change in the impedance in ionic buffer is comparable to that in human serum. The minimization of high background in the human serum buffer and the sensitivity enhancement of the device may be attributed to morphology of the NW that provides size matched confinement in the electrode for the target proteins.

Direct femtosecond laser writing is used to integrate optical waveguides that intersect the microfluidic channels in a commercial optofluidic chip [1]. With laser excitation, fluorescently labeled DNA molecules of different sizes are separated by capillary electrophoresis with high operating speed and low sample consumption (~ 600 pl). In the diagnostically relevant size range (~150-1000 base-pairs) the molecules are separated with high sizing accuracy (> 99%) [2]. An ultra-low limit of detection of 210 femtomolar is demonstrated. We introduce a principle of parallel optical processing. Different sets of exclusively color-labeled DNA fragments - otherwise rendered indistinguishable by spatio-temporal coincidence - are traced back to their origin by modulation-frequency-encoded multi-wavelength laser excitation, fluorescence detection with a single ultrasensitive, albeit color-blind photomultiplier, and Fourier analysis decoding. As a proof of principle, fragments obtained by multiplex ligation-dependent probe amplification from independent human genomic segments, associated with genetic predispositions to breast cancer and anemia, are simultaneously analyzed [3]. A principle of label-free optical sensing is demonstrated. Measurand-induced wavelength shifts in the transmission spectra of a resonant grating-based cavity are monitored. An interface layer containing receptors specific to the targeted analyte, the PepN enzyme, is applied. The analyte concentration adsorbed at the grating surface from a watery mixture of many compounds is determined with a resolution of ~4 pm ad-layer growth [4]. Improving optical sensitivity, we realize a distributed-feedback channel waveguide laser in Er- or Yb-doped Al2O3 on a silicon substrate. Single-longitudinal-mode operation with a Q-factor of 1.14×10^11 is achieved [5]. We demonstrate a dual-wavelength distributed-feedback laser based on the optical resonances induced by two local phase shifts. A stable microwave signal at ~15 GHz is created via heterodyne photo-detection of the two laser wavelengths [6]. By measuring changes in the frequency difference between the two laser modes as the evanescent field of the laser interacts with particles on the waveguide surface, we achieve real-time detection and accurate size measurement of single micro-particles with diameters down to 1 mu;m, which represents the typical size of many fungal and bacterial pathogens [7]. 1. R. Martínez Vázquez et al., Lab Chip 9, 91 (2009). 2. C. Dongre et al., Electrophoresis 31, 2584 (2010). 3. C. Dongre et. al., Lab Chip 11, 679 (2011). 4. S.V. Pham et al., "On-chip bulk-index concentration and direct, label-free protein sensing utilizing an optical grated-waveguide cavity", submitted. 5. E.H. Bernhardi et al., Opt. Lett. 35, 2394 (2010). 6. E.H. Bernhardi et al., Opt. Lett. 37, 181 (2012). 7. E.H. Bernhardi et al., "Intra-laser-cavity microparticle sensing with a dual-wavelength distributed-feedback laser", submitted.

Femto-molar DNA detection is important as this is the concentration needed for early-stage cancer and bacterial infection detection application [1]. Here, we present the first-ever nano-mechanical mass-sensing resonator with ordered vertical nanowire (NW) arrays on top of a Si/SiO2 bilayer thin membrane acting as a photonic crystal. The device has a very high surface area-to-volume ratio 10^8 m^-1, enabling DNA sensing of femto-molar concentration. Moreover, the nanowire array forms a photonic crystal that shows strong light trapping and absorption over broad-band optical wavelengths, enabling high-efficiency broad-band opto-thermal-mechanical remote device actuation and bio-sensing on chip, eliminating the need for any interconnect wires. This method represents a mass-based platform technology that can sense molecules at low concentrations. For mass-detection-based nano-mechanical bio-sensor, there are two important metrics [2]. The first metric is the minimum detectable mass, which requires a resonator to be as light as possible while maintaining high quality factor. The second metric is the minimum detectable mass per area, which requires maximizing sensor surface area, to bind as many detectable bio-molecules as possible. Previous nano-mechanical cantilevers have shown single-molecule level absolute mass sensitivity and single DNA molecule was detected [3]. However, single DNA-strand detection [3] requires high concentration of DNA solution (10^-9 mol/liter) to get even a single DNA-molecule binding, due to the small surface area of the device. Our NW array achieves the highest mass-per-area sensitivity 1.8x10^-12 kg/m2 amongst all the nano-mechanical bio-sensors ever reported, to the best of our knowledge. This high mass-per-area sensitivity sensor enables bio-molecule detection at ultra-low concentrations. Here we demonstrate 5x10^-16 mol/liter (0.5 femto-molar) concentration sensitivity, a value of one to six orders of magnitude better than other sensors [3-4]. Theoretical expression of the absolute mass resolution δm of a resonator can be derived as: δm = -2m δf/(αf), with initial resonant frequency f, spring constant k, effective mass m, resonance frequency shift δf and a numerical constant α that depends on the geometric localization of the added mass. Since the total mass of the bio-molecules bound to the surface will be proportional to its sensing area, then the concentration sensitivity (δC) of the sensor will be: δC = β δm/S, with constant β and total surface sensing area S. References: [1] F.Caruso et al., Anal. Chem. 69, pp.2043-2049 (1997). [2] T. P. Burg et al., Nature 446, pp.1066-1069 (2007). [3] B. Ilic et al., Nano Lett. 5, pp.925-929 (2005). [4] M. Li et al., Nat. Nanotechnol. 2, pp.114-120 (2007).

Plasmonics is a rapidly emerging field of nanophotonics that focuses on the ability of noble metal nanostructures to manipulate light at the nanoscale. For example, by using nanocorrugations etched in a metal film, light at optical frequencies can be coupled to surface plasmon polaritons (SPPs), which are electromagnetic waves that propagate along a metal-dielectric interface. SPPs are confined at the metal surface and are very sensitive to small changes in the refractive index of the dielectric (e.g. aqueous solutions with biochemical analytes). We have recently reported on a compact, high-throughput plasmonic sensor based on surface plasmon interferometry optimized for real-time monitoring of glucose in aqueous solutions [1]. The sensor consists of a spatially dense, planar array of plasmonic interferometers (>1,000/mm2), where each interferometer is composed of 100nm-wide, 10µm-long grooves flanking a 200nm-wide slit etched in a 300nm-thick silver film using focused ion-beam milling. The distances between each groove and the slit were varied between 0.25 to 10µm in steps of 25nm. The detection limit of the plasmonic sensor for glucose in aqueous solutions is 5.5mu;M with a sensitivity of 105,000%/RIU (refractive index units) at 590nm. In order to improve the sensor selectivity to glucose, we propose a novel molecular recognition scheme that couples plasmonic interferometry with dye chemistry, specifically using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). In this implementation, glucose oxidase is added in solution to rapidly convert D-glucose into D-gluconolactone and H2O2 in a 1:1 stoichiometry. The H2O2 reacts with horseradish peroxidase (HRP) to oxidize Amplex Red into resorufin, a dye molecule which is characterized by a strong optical absorption coefficient at ~571nm. The reaction is monitored in real-time by simply measuring changes in the light intensity transmitted through the slit of each interferometer. As a result of the increased concentration of resorufin, the solution&’s refractive index and the absorption coefficient will increase, resulting in, respectively (1) a spectral shift in the interference conditions (Δlambda;), and (2) an intensity decrease due to increased SPP absorption (ΔΙ). These two changes, (Δlambda; and ΔΙ) will provide information on the change in refractive index which can be directly correlated to the glucose concentration. In this work, we will describe the use of plasmonic interferometers for monitoring the kinetics of Amplex Red, glucose oxidase and glucose reaction. The effects of absorption using an oxidative dye on the spectral response of the plasmonic interferometer will be discussed. This approach will lead to better sensor specificity and sensitivity and allow the use of plasmonic interferometry to develop point-of-care diagnostic tools for biomedical sensing of glucose and other clinically relevant analytes. [1] V. Siu et al., Nano Lett. 2012, 12 (2), pp 602-609

By providing a comprehensive picture of the blood glucose profile over time and thereby enabling the patient to predict and minimize glucose excursions, continuous glucose monitoring (CGM) offers the potential to advance the treatment of diabetes and is widely recognized as the future of glucose self-monitoring. However, this potential has not yet been realized in clinical practice. Results of studies assessing the clinical impact of continuous glucose monitoring with the currently available glucose oxidase (GOx)-based sensors, while consistent with modest improvements in diabetes control and patient safety, have been unremarkable until recently — in part because of developmental needs of the current sensor technology. To overcome the limitations of GOx-based systems, Becton Dickinson (BD) (Franklin Lakes, NJ) is developing a sensor that detects glucose directly with fluorescently labeled glucose/galactose binding protein (GGBP). The conformational change that occurs when the fluorescently labeled GGBP reversibly binds glucose produces an optical signal that is the basis of a simple, direct, equilibrium-based method of estimating blood glucose values. The protein in the GGBP sensor has been genetically engineered at BD to bind glucose in the human physiological range and to produce a specific fluorescent response. The prototype investigational device, developed for subcutaneous (SC) or intradermal (ID) placement, is small and minimally invasive and requires only a minimal warm-up period. The robust performance of the GGBP-based fiber optic sensor in humans along with its resistance to interferents that commonly plague GOxbased systems indicates the potential of this optical technology for continuous glucose monitoring in humans.

In recent years the IoF has described in over 150 peer-reviewed publications the favorable interactions and outcomes of both plasmon supporting particles (Ag, Au, Cu, Zn, Ni, Cr) and substrates with electronically excited states. These favorable effects have included significantly enhanced fluorescence emission from singlet states, S1 and S2, as well as enhanced phosphorescence yields from triplet, T1, states (MEP). In addition, we have observed and described plasmon enhanced chemiluminescence intensities (MEC), as well as highly directional surface plasmon coupled Fluorescence. As a result of enhanced triplet yields, we have also observed both enhanced singlet oxygen and superoxide anion yields. These favorable influences on the photophysical properties of close proximity excited states to plasmon supporting substrates / particles has led to wealth of biochemical applications, such as the high sensitivity and ultra fast detection of proteins, DNA, RNA and ultra bright and photostable metal-enhanced fluorescence based particles for downstream cellular imaging applications. In addition, there are a lot downstream applications for MEP, such as in photodynamic therapy by surface plasmon controlled single oxygen generation. Current thinking, describes Metal-Enhanced Fluorescence as the near-field coupling of electronic excited states to surface plasmons (a surface mirror dipole), the particle subsequently radiating the photophysical characteristics of the coupled excited state quanta. In this lecture, we communicate our recent findings for metal-fluorophore interactions, our current thinking and progress towards developing a unified metal-fluorophore description and the subsequent development of ultra fast and sensitive sensing platforms for pathogen DNA.

Molecularly imprinted polymers (MIP) have been discovered by Wulff (1) and are since mainly developed as synthetic antibodies. Indeed, such polymeric matrices exhibit selective rebinding of the template used in their fabrication; this enables their use in sensing technologies. An important future challenge consists in performing a direct detection of analytes by coupling the recognition and the detection steps into one complete sensor (2,3). Thus, this kind of sensors can be used to real time monitor analytes in environmental or health applications. More than a linkage of MIP to an optical transducer, recent studies associated MIP hydrogels with opals (4,5). In this process, silica beads are deposited by evaporation on a substrate, forming an opal structure (hexagonal lattice) by simple self-assembly. A MIP pre-polymerisation mixture is then infiltrated by capillarity into the crystal and photopolymerized. Dissolution of silica beads and template extraction made it possible to obtain polymeric matrix as an inverse opal, which presents photonic crystal properties. Further template recognition by the matrix leads to a polymeric swelling which rhymes with a shift in the Bragg diffraction peak. Our work aims at combining MIP with opals in a steroid sensor by using a new deposition technique to build opals. The used online-coating device is a novel online-process enabling to deposit large areas of organized monolayers of nanoparticles on a substrate (6). The device was adapted to deposit successive layers, making opals with a controlled and uniform number of beads layers. Five layers opals were built with beads from 280 to1000nm on either glass or silicon substrate. Then, MIP pre-polymerisation mixture was infiltrated using spin-coating to give a thin and regular layer of polymer. Such a polymer repartition is permitted by a substrate functionnalisation, based on silanization, anchoring the first layer of beads. The few studies (4,5) published on opals and MIP focused only on spectrophotometric absorbance measurements to characterize the structures since a simple visible shift of the maximal absorbance wavelength proves the presence of the targeted molecule. In this work, more sophisticated optical characterizations of the crystal were investigated by performing diffractive measurements and by using goniometry. Finally, the novel synthesis of MIP inverse opal structure and its characterizations will be discussed. We will underline the potential industrialization of such built sensors. (1) G. Wulff, A. Sarhan, Angew Chem, 1972, 84, 364 (2) D. Kriz, O. Ramstrum, et al., Anal. Chem., 1995, 67, 2142-2144 (3) L. I. Andersson, C. F. Mandenius and K. Mosbach, Tetrahedron Lett. 29, 1988, 5437-5440 (4) Z. Wu, C. Lin, et al., Chem. Eur. J., 2008, 14, 11358-11368 (5) N. Griffete, H. Frederich, et al., Langmuir, 2012, 28, 1005-1012 (6) Z.Tebby, J. Gavillet, et al. Multi-Material Micro Manufacture, 2010 November, Oyonnax. Research Publishing, 2010

We describe stimulus-responsive DNA-functionalized gold nanoparticles that incorporate azobenzene-modified oligonucleotides. Beyond the classic self-assembly and sensing behaviors associated with oligonucleotide-modified nanoparticles, these particles also exhibit reversible photoswitching of their assembly. We show that perfectly complementary and partially mismatched strands exhibit clearly distinguishable photoinduced disaggregation properties, and we demonstrate that photon dose can thus be used in place of temperature or ionic strength to control hybridization stringency with the ability to discriminate single-base mismatches in sensing applications. We further study the influence of DNA complementarity on the photoswitching of gold nanoparticles by measuring the azobenzene photoisomerization quantum yield in different local environments. When inserted at different positions with respect to that of mismatched base, we find that the azobenzene demonstrates differently. We propose that this sequence dependent quantum yield underpins different photoinduced nanoparticle disaggregation rates, and underpins the ability to use photostringency to discriminate perfectly complementary and partially mismatched sequences using these materials.

Human breath contains a number of volatile organic compounds (VOCs) that are either disease markers (e.g., acetone or nitric oxide) or indications of consumption of substances such as alcohol. The presence of a large number of potential interferents, including water vapor, carbon dioxide, and many trace species demands that chemical sensors exhibit a high degree of selectivity, as well as high sensitivity and fast response. In many sensing devices a recognition chemistry is required to provide these properties. In this paper we describe the development of metal-organic framework (MOF) thin films for the detection of a wide range of VOCs. MOFs are crystalline nanoporous materials with pore sizes and chemical environments that can be tailored for selective and enhanced uptake of specific molecules. In particular, we will demonstrate selective detection of acetone and alcohols in the presence of water vapor and CO2, using novel multilayer MOF coatings integrated with surface acoustic wave sensors. In addition, we will show that strain-based detection of these molecules is feasible by depositing MOF coatings on microcantilevers. Our synthetic effort is guided by atomistic modeling, which shows that such selectivity is a direct consequence of the chemical environment of the MOF pore. Finally, we show that chemical “fingerprinting” is feasible by using a suite of MOF coatings. Overall, the results demonstrate that MOFs can be used to enable sensing with selectivity and sensitivity competitive with state-of-the-art devices.

Methods for probing mechanical responses of mammalian cells to electrical excitations can improve our understanding of cellular physiology and function. The electrical response of neuronal cells to applied voltages has been studied in great details, but less is known about their mechanical response to electrical excitations. Atomic force microscope (AFM) studies have shown that mammalian cells exhibit voltage-induced mechanical deflections at nanometer scales. However, AFM is complex, difficult to scale, and uses sharp nanotips which may be considered invasive. Piezoelectric PbZrxTi1-xO3 (PZT) nanoribbons/nanowires with piezoelectric charge constants up to 140 pm/V represent an alternative platform for sensing cellular deformations. Here we demonstrate that PZT nanoribbons can support healthy cellular growth and can sense tiny cellular deformations induced by electrical depolarization. Indeed, quantitative analyses show that cells deform ~1 nm upon ~100 mV membrane voltage excitation. The measured cellular forces agree with a theoretical model in which depolarization caused by an applied voltage induces a change in membrane tension, which results in the cell altering its radius so that the pressure remains constant across the membrane. We also transfer the arrays of PZT nanoribbons onto a flexible elastomer substrate over macroscopic scales to sense tissue deformation from a mimicked respiration process of a cow lung. The flexible PZT nanoribbon chip can generate 1 V peak-to-peak voltage via small strains when biointerfaced on a cow lung. The results suggest significant potential for using PZT nanoribbons as a powerful, scalable biomechanical sensing platform for monitoring mechanical signals from the single cell level up to multiple cells in tissues of biological systems

Ferromagnetic shape memory alloys (FSM) are an upcoming class of smart materials with strong magneto-mechanical coupling that has been attracting increasing interest during the past years. Exhibiting magnetization changes upon straining and, vice versa, reversible strains as high as 10% in response to an external magnetic field, they are perfectly suited for biomedical sensing or actuation - which particularly holds true as they can be operated at constant (body) temperature at frequencies ranging from quasi-static to some kHz. In contrast to the "prototype" Ni-Mn-Ga alloy, where the FSM effect was initially discovered, Fe-Pd FSM alloys reveal excellent biocompatibility, which makes them hot candidates for in vitro and in vivo use. As straining is physically mediated by reorientation of twin variants within the martensite phase, a sufficiently high twin boundary mobility is prerequisite on the materials side. This requires single crystallinity with low defect density. After reviewing these fundamentals we demonstrate that freestanding single crystalline Fe-Pd membranes, that are grown as single crystals on MgO substrates by molecular beam epitaxy (MBE) and subsequently lift-off their substrate, fulfill all requirements of a biomedical FSM sensor and actuator [1]: By appropriate choice of deposition and annealing conditions, they reside within the face-centered-tetragonal (fct) martensite phase at body temperature, which is precondition for the FSM effect. Employing density functional theory (DFT) calculations we demonstrate the underlying physical principles of phase stabilization, including constraints by open surfaces and magnetic microstructure as well as influence of order/disorder and lattice defects [2]. Biocompatibility as well as coupling to bone and tissue is addressed in extensive studies ranging from simulated body fluid (SFB) to cell test. They demonstrate that Fe-Pd FSM membrans are perfectly suited as biomedical strain sensors or actuators. [1] T. Edler and S.G. Mayr, Adv. Mat. 22, 4969 (2010) [2] Y. Ma, A. Setzer, J.W. Gerlach, F. Frost, P. Esquinazi and S.G. Mayr, Adv. Func. Mat. 22, 2529 (2012) [3] Y. Ma, M. Zink and S.G. Mayr, Appl. Phys. Lett. 96, 213703 (2010)

Artificial noses have attracted a great deal of attention in the past decade for a wide range of applications, including foodstuff monitoring, security, environmental monitoring and disease diagnostics. By use of combinatorial responses coming from a variety of sensing units, artificial noses provide a platform for the analysis of samples with complex composition without the necessity to identify individual components in a mixture. The Photonic Nose (P-Nose) is a simple and cost effective concept for the analysis of both liquid and vapor phase samples. It is based on arrays of specially designed photonic crystal sensors, each displaying a bright and intense reflection of a specific band of color. When exposed to a chemical environment, each sensing unit will expand to different degrees, with each of these expanded states leading to distinct and vibrant color states. The combinatorial response of an array of P-Nose units can be analyzed by use of simple digital cameras for high-precision analysis. Furthermore, P-Nose devices can instead be optimized for single-unit, high optical contrast mode, in which case these can form the basis of highly effective visual indicators. In this presentation, the theory and operation of the P-Nose platform will be described, and some device concepts will be expanded upon.

Direct interfacing of nanosensors onto the human body could revolutionize areas ranging from health quality monitoring to adaptive threat detection.[1] Due to its exceptional electrical properties, nanosensors based on graphene have been shown to exhibit extremely sensitive analyte detection. Further, the high interfacial adhesion exhibited by graphene renders it ideal for interfacing onto curvilinear and rugged surfaces. Here we introduce a transformational approach to directly interfacing graphene nanosensors onto biomaterials. Specifically, we demonstrate that graphene can be printed onto water-soluble silk. This in turn permits intimate biotransfer of graphene nanosensors onto biomaterials including tooth enamel and skin, alike the attachment of a temporary tattoo. The result is a fully biointerfaced sensing platform, which can be tuned to detect specific target analytes. For example, via bifunctional self-assembly of antimicrobial peptides[2] onto graphene, we demonstrate bioselective detection of bacteria at single-cell levels. The incorporation of a resonant circuit consisting of interdigitated electrodes and an inductive coil eliminates the need for onboard power and external connections. Combining these elements yields two-tiered interfacing of peptide-graphene nanosensors with biomaterials. In particular, we demonstrate integration onto a tooth for remote monitoring of breath and detection of bacteria in saliva at single cell levels.[3] The key functionalities of this hybrid sensing element are thus derived from a synergistic integration of the individual materials properties and components. Overall, this strategy of hierarchically interfacing biomolecules with nanosensors and biomaterials, represents a versatile approach for ubiquitous detection of biochemical targets. References [1]. D.H. Kim, N. Lu, R. Ma, Y.S. Kim, R.H. Kim, S. Wang, J. Wu, S.M. Won, H. Tao, A. Islam, K.J. Yu, T.I. Kim, R. Chowdhury, M. Ying, L. Xu, M. Li, H.J. Chung, H. Keum, M. McCormick, P. Liu, Y.W. Zhang, F.G. Omenetto, Y. Huang, T. Coleman, J.A. Rogers, Science 333, 838 (2011). [2]. M.S. Mannoor, S. Zhang, A.J. Link, M.C. McAlpine, Proc. Nat. Acad. Sci. U.S.A. 107, 19207 (2010). [3]. M.S. Mannoor, H. Tao, J.D. Clayton, A. Sengupta, D.L. Kaplan, R.R. Naik, N. Verma, F.G. Omenetto, M.C. McAlpine, Nat. Commun. 3, 1767 (2012).

Second harmonic generation resulting from nonlinear interaction of ultra short pulses of light with nanoparticles is a quite promising tool for biological imaging applications, regarding the capability of detecting single particles without harming the specimen or the detection probe [1]. Recent research clearly demonstrated the superiority of exploiting nonlinear optical properties of nanoparticles over fluorescent chemical molecules or emergent quantum dots [2]. Nanoparticles for second harmonic generation could be produced via chemical synthesis from salts of metals [3], where size and shape could be tightly controlled but the available materials are restricted. Nanoparticles could also be generated from bulk material via numerous methods, among which laser ablation [4] is quite effective in producing nanoparticles of almost all solids, while size and shape mostly vary to a great extent as a trade off. This study investigates the nanoparticle characteristics related to efficiency in nonlinear optical imaging of biological samples by comparing synthesized and laser generated nanoparticles. Nanoparticles of various materials with different geometries and sizes were prepared via chemical synthesis and ultra short high power laser ablation techniques. The biological imaging capabilities of these nanoparticles were investigated using a custom built multi photon microscope optimized for nanoparticles based on nonlinear imaging. Mammalian cells were incubated with different concentrations of various types of nanoparticles, and several methods of surface chemistry were utilized for biocompatibility and targeting. The results demonstrate that nonlinear optical properties of nanoparticles could be tuned precisely according to the requirements of the experimental setup, depending on the aim of nanoparticles use. It could be expected that with the advance of fabrication techniques towards large scale production, nanoparticles will supersede conventional fluorescent molecules in near future. [1] L. Tong, J. X. Cheng, "Label-free imaging through nonlinear optical signals," Materials Today, vol. 14, 264-273, (2011). [2] P. Pantazis, J. Maloney, D. Wu, and S. E. Fraser, "Second harmonic generating (SHG) nanoprobes for in vivo imaging," PNAS, vol. 107, 14535-14540, (2010). [3] X. H. Ji, X. N. Song, J. Li, Y. B. Bai, W. S. Yang, and X. G. Peng, "Size control of gold nanocrystals in citrate reduction: The third role of citrate," JACS, vol. 129, 13939-13948, (2007). [4] M. Ullmann, S. K. Friedlander, and A. Schmidt-Ott, "Nanoparticle formation by laser ablation," Journal of Nanoparticle Research, vol. 4, 499-509, (2002).

Action potentials have a central role in neurons, cardiomyocytes, and many other cells with ion channels. Traditionally, patch clamp pipettes and microelectrode arrays are used to record action potentials. However, patch clamping suffers drawback of invasiveness and low throughput whereas microelectrode arrays recording gives low signal. Nanoelectronic devices hold unique advantages to solve these problems in cell electrophysiology measurement. Here we present vertically aligned Pt nanopillar arrays for both extracellular and intracellular recording of rat cardiomyocytes. The small footprint of our nanopillar arrays provide high spatial resolution recording. We discover that the tight cell membrane-electrode interface allows large extracellular recording signal despites the small electrode area. After local electroporation of cell membrane around the nanopillars, we perform parallel intracellular recording of action potentials from multiple cells in the same culture. Because this method is minimally invasive, we are able to record action potentials from single cells over a span of three days. We are also able to monitor the maturation of cultures at single-cell level. Furthermore, we study changes in the intracellular action potentials induced by drugs, demonstrating the capability of our nanoelectrodes for large scale drug screening. (Nat. Nanotechnology 7, 185-190 (2012))

Nowadays, due to the increasing need for new therapeutics that target different mechanisms of action and methods for earlier diagnosis, new methods and tools are required to increase understanding of disease processes and progression [1, 2]. Especially, the clinical utility of immunoreactions to discriminate health and disease states requires the capability to measure extremely low concentration proteins, which is also important to understand cellular processes and to search for new therapies [3, 4]. In the meanwhile, the developments of novel sensors using nanomaterials provide promising approaches to offer high performance in resolution and detection limits. The nanomaterials such as nanoparticle [5], carbon nanotube (CNT) [6], graphene [7], and silicon nanowire [8], provide effective approaches for novel biosensors with better performance. But the detection limits and response time still have much space to improve. A variety of methods demonstrate neither ultra-low detection limits nor very large detection ranges, and many of them are incredible expensive and complex to realize. We are exploring unconventional methods that have the characteristics that they provide accurate information that is useful in diagnosis, and do so at costs that are very competitive. In special, combination of “bottom-up” layer-by-layer (LbL) nano self-assembly and “top-down” micromanufacturing techniques are to fabricate MEMS and flexible electronics for biomedical applications. With nano self-assembly and surface micromachining, highly flexible cantilever platform for micro sensing and actuation was investigated successfully. With respect to flexible electronics, self-assembled graphene- and carbon nanotube-based field-effect transistors with embedded self-assembled films as dielectric and active layers were fabricated and characterized successfully. These self-assembled field-effect transistors were primarily investigated for high-performance bio-sensing applications. [1] L. A. Tessler, J. G. Reifenberger, R. D. Mitra, “Protein Quantification in Complex Mixtures by Solid Phase Single-Molecule Counting”, Anal. Chem. 81, 7141, (2009). [2] J. Todd, B. Freese, A. Lu, D. Held, J. Morey, R. Livingston, P. Goix, “Ultrasensitive Flow-based Immunoassays Using Single-Molecule Counting”, Clinical Chemistry 53, 1990, (2007). [3] G. Zheng, F. Patolsky, Y. Cui, W. U. Wang, C. M. Lieber, “Multiplexed Electrical Detection of Cancer Markers with Nanowire Sensor Arrays”, Nat. Biotechnol. 23, 1294, (2005).

Self-assembly of plasmonic nanoparticles involves the spontaneous organization of individual building blocks to form nanometer-micrometer sized superstructures. Hitherto, molecular cross linker mediated self-assembly of plasmonic nanoparticles is limited to 1D linear and branched chains with polydisperse architecture and nanoparticle units. However, in order to successfully exploit the plasmonic nanoparticles in various technological applications, precise control over self-assembly is required. Here, we demonstrate the formation of flower-like nanoclusters and its chains with precise control of surface chemistry and zeta-potential of two different sizes of shape-controlled gold nanoparticles. The central big particle (core) is modified with a bi-functional molecular cross linker, p-aminothiophenol (p-ATP), and the zeta-potential of surrounding small nanoparticles (satellites) are adjusted by lowering the pH of the solution to about 1.5. At this pH, corss-conjuation of planets and satellites is triggered resulting in isolated, uniform and organized plasmonic clusters. We demonstrate the versatility of this method with a broad range of gold nanoparticle shapes and sizes. Furthermore, single nanocluster surface enhanced Raman spectroscopy (SERS) measurements reveal their large SERS enhancement and establish the feasibility of deploying them as Raman probes. This technique is affable, highly efficient, cost-effective, and controllable to design fastidious 2D and 3D plasmonic nanoclusters which can be useful to address several fundamental questions in plasmonics.

Nanoscale drug delivery systems have generated significant interest because of their ability to distribute within biological systems with kinetics that approach the molecular level, while allowing the addition of cell specific targeting molecules and/or multiple therapeutic agents. Iron based nanomaterials are particularly unique due to their inherent magnetic properties and surface, whose physical and chemical properties can be easily modified using suitable silano-organic compounds. Despite these advantages, the biological efficacy of an iron based nano-carrier system depends upon its bio-distribution and pharmacokinetic properties in vivo. With this in mind, water dispersed, iron oxide nanoparticles (~10 nm) with either a carboxylic acid (-COOH) or an amine (-NH3) functional group were synthesized. The in vivo bio-distribution was determined by detecting 14C labels (t1/2=5730 years) that were covalently linked to the surface of the nanoparticles. The nanoparticles were radiolabeled by incorporating 14C into the organic functional groups, with the radioactivity controllably varied from 0.1 nCi/mg up to 0.1 mu;Ci/mg. The radio-labeling approach used in these studies was significant, as the probes had the same chemical properties as the non-labeled probes that they were intended to mimic. This approach provides comparable data sets. 14C labeled nanoparticles in tissue samples were detected using Accelerator Mass Spectroscopy (AMS), an ultrasensitive (10-18 moles) quantitative spectrometric technique with small sample requirements. Furthermore, the use of a magnetic core as the nano-carrier for the radiolabels allows for dual detection schemes, which increases the accuracy of the bio-distribution data. The nanoparticles synthesized in this work have implications for use in different biological applications and the approach described is broadly applicable to the synthesis of nanoscale materials with multiple core and surface functionalities.

The spatial organization of cell surface receptors has significant implications for signaling and regulation processes since it influences the interactions and interplay of the receptors. To obtain information about the spatial organization of cell surface receptors robust assays are required that provide detailed insight into their spatial distribution on nanometer length scales. In this presentation we will introduce an effective Au nanoparticle (NP) labeling approach for specific cell surface receptors. We will demonstrate through combination of scattering spectroscopy, electron microscopy, and generalized multiple particle Mie theory (GMT) simulations that the density- and morphology-dependent spectral response of NP immunolabels bound to the epidermal growth factor receptors ErbB1 and ErbB2 encodes quantitative information of both the cell surface expression and spatial clustering of the two receptors in different unliganded in vitro cancer cell lines (SKBR3, MCF7, A431). The systematic characterization of the collective spectral responses of NPs targeted at ErbB1 and ErbB2 at various NP concentrations indicates differences in the large-scale organization of ErbB1 and ErbB2. Validation experiments in the scanning electron microscope (SEM) confirm that NPs targeted at ErbB1 on A431 are more strongly clustered than NPs bound to ErbB2 on SKBR3 or MCF7 at overall comparable NP surface densities. This finding is consistent with the existence of larger receptor clusters for ErbB1 than for ErbB2 in the plasma membranes of the respective cells. The large optical cross-sections of the NP labels in combination with the ability to resolve the receptor self-organization on nanometer to tens of nanometer length scales provides new opportunities for quantitative optical biomarker profiling.

Since the first demonstration of surface plasmon resonance as a label-free optical biosensor, there has always been a desire to pursue high sensitivity, high resolution, robust, and inexpensive detection approaches based upon the properties of optical resonators to extend the limits of detection of label-free assays to lower concentrations and to increase the signal-to-noise ratio for observation of lower concentrations or smaller molecules. Here, utilizing a tunable photonic crystal (PC) resonant reflector as the wavelength selective element of an external cavity laser (ECL) cavity, we have demonstrated a novel single-mode continuous-wave narrow bandwidth emission and widely tunable external cavity laser biosensor, representing a fundamentally different approach to the problem of achieving high Q-factor resonance and simultaneously high-sensitivity for label-free resonant optical biosensors. The PC is fabricated inexpensively from plastic materials using nanoreplica molding, and its resonant wavelength is tuned by adsorption of biomolecules on its surface. Gain for the lasing process is provided by a semiconductor optical amplifier (SOA), resulting in a robust and simple detection instrument that operates by normally noncontact illumination of the PC and direct back-reflection into the amplifier. Through the process of stimulated emission, the laser biosensor generates its own resonant emission as narrow as 0.03 pm (lambda;0=850 nm; Δlambda;=0.03 pm), corresponding to a Q-factor (lambda;0/Δlambda;) of 2.8×107. By exposing the PC surfaces to dimethyl sulfoxide (DMSO) solutions at varying concentrations, sensor&’s bulk refractive sensitivity of 212 nm/refractive index unit is determined. The widely adopted metric for the intrinsic resolving power of resonator sensors is the figure of merit (FOM), defined as FOM = Sb×Q. Our obtained FOM is 5.9×109, representing the state-of-the-art resolving capability. We demonstrate single-mode, biomolecule-induced tuning of the continuous-wave laser wavelength with the ability to generate wavelength shifts as large as 13 nm, and the ability to discriminate wavelength shifts as small as <1 pm. For the first time, we have demonstrated the use of such a system for the dynamic detection of an adsorbed monolayer of a protein polymer, the real-time detection of the incorporation of two distinct small molecules (biotin and estradiol) by immobilized large proteins (streptavidin and estradiol receptor), and finally, the selective detection of DNA using an immobilized capture-strand of DNA. Because this approach includes a mechanism for optical gain, the device represents a departure from previous optical biosensors using passive resonators. It enables simple optical coupling and a sensor format that is amenable to high-throughput multiplexed analysis for a broad array of applications in life science research, pharmaceutical screening, diagnostics, and environmental monitoring.