Low-dimensional nanomaterials are improving the detection of molecular binding phenomena. The resulting optical technologies allow for the quantification of disease biomarkers and other bioanalytes. The fluorescence of one such material, single-walled carbon nanotubes, may facilitate quantification of analytes within biological environments. Semiconducting single-walled carbon nanotubes emit bandgap photoluminescence in the near-IR spectral region and exhibit unique photostability. To build optical probes using these materials, we developed a new spectroscopic imaging technique in order to observe the near-infrared photoluminescence of carbon nanotubes and measure their spectral changes spatially. The imaging method allows the emission of individual nanotube chiralities to be observed and quantified within live cells and tissues.

Superb electromechanical properties of graphene, where large elastic deformation is achievable without significant perturbation of electrical properties, provide a substantial promise for flexible electronics, advanced nanoelectromechanical and bioelectronic devices. We report three-dimensional (3D) field-effect transistor biosensors built from the monolithic integration of crumpled graphene and graphite. First, we present monolithic synthesis of graphene-graphite for all-carbon bioelectronic transistor arrays. Second, we develop a rapid and scalable method of texturing 2-dimensional (2D) graphene by using soft-matter transformation of shape-memory polymers into 3D bioelectronic sensors. We demonstrate that the thermally-induced transformation of graphene on a polymeric substrate creates 3D textured graphene. Quantitative analysis shows that both the wavelength and height of textured graphene are a few micrometers at an applied strain of 300% and that the 3-dimensionality of graphene (i.e., wavelength and height of texturing) can be controlled by the processing parameters. We further characterize the electrical and mechanical properties of 3D graphene, and demonstrate the robust electromechanical properties of 3D textured graphene. Finally, we explore biosensor device applications by constructing an array of field-effect biosensors. We believe our approach to forming textured graphene by soft-matter transformation offers a unique avenue for creating advanced and 3D bioelectronic devices, and furthermore, these unique capabilities could be exploited in chemical and biological detection and conformal interface with biological systems in the future.

Graphene, one atom-thick carbon hexagonal sheet, holds great promise in nanoelectronics. Development of methods for producing higher-quality graphene in larger quantities is essential for realizing their applications. Current research on liquid-phase exfoliation (LPE), chemically separating graphite into graphene, has increased to develop graphene paper and nanocomposites. This study has examined the efficiency of LPE with three surfactants, 1-Pyrenesulfonic acid sodium salt [Py-1SO₃] and 1-Pyrene methylamine (ionic) [Py-NH₂], and Pluronic ®P-123 (non-ionic). After continual centrifugation / ultrasonication, the graphene filtrates are investigated with Raman spectroscopy, UV-Vis spectroscopy, and atomic force microscopy to determine the characteristic electronic bands of graphene, the quality and thickness. These findings lead to developing more defined procedures for higher yields of single-, bi-, and oligo-layered graphene(s) in addition to graphene paper. Moreover, due to high electron mobility and its unique surface properties such as irreversible protein adsorption, they may serve as ‘smart&’ bio-platforms for accommodating proteins and facilitating protein electron transfer. Therefore, we attempt to demonstrate that graphene and its&’ variants including GO (graphene oxide) and rGO (redued graphene oxide) support efficiently the redox centers of several heme-containing metalloproteins (cyt c, myoglobin and horseradish peroxidase) to the electrode. Importantly proteins retain their secondary structure thus biological activity upon forming mixtures with graphene and variants investigated using a range of electrochemical modes. The observed features are indicative of promising applications of graphene-based systems/protein complexes in the development of biosensors and biofuel cells.

Glucose oxidase (GOx) is a glycoprotein that catalyzes the conversion of β-D-glucose to δ#8209;gluconolactone and hydrogen peroxide in the presence of oxygen. In 1962, Clark and Lyons (1) demonstrated that the presence of glucose in blood plasma could be detected by the change in the potential of the electrode adjacent to GOx during catalysis of glucose. The oxidation overpotential of the electrode reaction can be reduced by modifying the electrode with graphene or other 2D material to facilitate a direct electron transport to the GOx active site being a redox center and containing FAD (flavin adenine dinucleotide). Graphene, because of its properties as being strong, flexible, transparent and highly conductive, could be relatively advantageous for sensing compared to other carbon nanomaterials (2). Furthermore, nowadays, graphene can be fabricated in many shapes and sizes, including recently introduced growth in lithographically patterned hexagonal boron nitride atomic layers (3), which makes it highly suitable for encapsulation (4).
Using all-atom molecular dynamics simulations in water environment, it was possible to demonstrate spontaneous and tight encapsulation of GOx dimer by graphene 7 nm x 7 nm flakes linked together by linkers of different width and forming a flower-like shape. The partially overlapping graphene flakes compacted the structure of GOx dimer, bringing the monomers much closer to one another. We found that the most complete wrapping of the enzyme by the flower-like graphene was achieved when the broadest linkers between graphene flakes were employed. Encapsulation can be an efficient way to obtain a large contact surface. However, an exceptionally tight binding of the flower-like graphene can also influence the positions of amino acids in the enzyme binding site resulting in less efficient catalytic reaction. Furthermore, such extensive encapsulation could block the access of the substrate to the active site of the enzyme. Contrary, a partial encapsulation by graphene caused only small distortions of GOx structure while the contact surface with graphene was still high. We are continuing the above studies on single graphene layer adsorbed on SiO₂ and decorated by GOx.
References
1. Clark LC, Jr. & Lyons C (1962) Electrode systems for continuous monitoring in cardiovascular surgery. Ann. N. Y. Acad. Sci. 102:29-45.
2. Yang W, et al. (2010) Carbon Nanomaterials in Biosensors: Should You Use Nanotubes or Graphene? Angew. Chem. Int. Ed. 49:2114 - 2138.
3. Liu Z, et al. (2013) In-plane heterostructures of graphene and hexagonal boron nitride with controlled domain sizes. Nat. Nanotechnol. 8(2):119-124.
4. Chen Y, et al. (2012) Aerosol synthesis of cargo-filled graphene nanosacks. Nano Lett. 12(4):1996-2002.

Doped graphene nanostructures have emerged as important materials for a range of applications. However, most doped carbon materials are highly inhomogeneous, precluding a systematic study of structure-activity relationships to optimize their performance. We have prepared well-defined N-doped graphene nanostructures with precisely known structures and functionalities using a synthetic approach with solution chemistry [1]. In particular, N-doped graphene structures with precisely controlled sizes (monodisperse) where the nitrogen occurs as phenazine moieties have been synthesized. Characterization of the materials has been carried out with infrared, Raman, and XPS studies. Associated electronic structure studies have been carried out using density functional theory to understand the spectroscopic properties measured experimentally. Our synthesized N-doped graphene quantum dots show catalytic activity for the electrochemical oxygen reduction reaction, and our studies reveal that the reaction occurs predominantly via a four electron pathway [2]. Density functional theory has been used to calculate the intermediates and transition states of the oxygen reduction reactions to derive the associated reaction mechanism including detailed energetics. The spectroscopic properties of N-doped graphene structures along with our proposed mechanistic picture for the catalytic activity will be presented.
[1] Q. Li, S. Zhang, L. Dai, and L.-S. Li, “Nitrogen-doped colloidal graphene quantum dots and their size-dependent electrocatalytic activity for the oxygen reduction reaction”, J. Am. Chem. Soc. 134, 18932-18935 (2012).
[2] Q. Li, B. W. Noffke, Y. Wang, B. Menezes, D. G. Peters, K. Raghavachari, and L.-S. Li, “Electrocatalytic oxygen activation by carbanion intermediates of nitrogen-doped graphitic carbon”, J. Am. Chem. Soc., 136, 3358-3361 (2014).

Boron Nitride Nanomaterials for Prostate Cancer Therapy
Xia Li, Qunhong Weng, Jun Zhang, Yoshio Bando and Dmitri Golberg
World Premier International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), Namiki 1-1, Tsukuba, Ibaraki 305-0044, Japan. E-mail: [email protected], [email protected]
Boron nitride (BN) nanomaterials¹, as a structural analogue of carbon (C) ones, have recently started to attract increasing attentions in biomedical field. However, the exploration of BN nanomaterials in cancer therapy has been rarely reported and some problems limited their application, such as high hydrophobicity, easy aggregation, poor degradability, difficulties in targeting and manipulation, etc. Here, we report on our latest advances in regards of BN nanomaterial syntheses, surface modifications, and their potential use in prostate cancer therapy: (i) BN nanotubes@mesoporous silica core@shell structures with adjustable surface zeta potential and improved dispersibility were fabricated to enhance intracellular drug delivery into LNCap prostate cancer cells for chemotherapy²; (ii) BN nanotubes@NaGdF₄:Eu core@shell structures with multimodal luminescent-magnetic properties were fabricated giving the opportunity to trace, target and thus to manipulate BN nanotubes in vitro³; (iii) Highly water-soluble, porous, and biocompatible BN nanomaterials were synthesized, which can effectively load anticancer drugs up to 300% and exhibit much higher potency for reducing the viability of LNCap cells than free drugs⁴; (iv) Hollow BN nanospheres with controlled boron release and good biodegradability were explored, and these exhibited unique chemopreventative effects for prostate cancer in vivo, compared with other conventional nanomaterials ⁵.
1 . Golberg, D.; Bando, Y.; Huang, Y.; Terao, T.; Mitome, M.; Tang, C. C.; Zhi, C. Y., Boron nitride nanotubes and nanosheets. ACS Nano 2010, 4, 2979-2993.
2 . Li, X.; Zhi, C. Y.; Hanagata, N.; Yamaguchi, M.; Bando, Y.; Golberg, D., Boron nitride nanotubes functionalized with mesoporous silica for intracellular delivery of chemotherapy drugs. Chem Commun 2013, 49, 7337-7339.
3 . Li, X.; Hanagata, N.; Wang, X. B.; Yamaguchi, M.; Yi, W.; Bando, Y.; Golberg, D., Multimodal luminescent-magnetic boron nitride nanotubes@NaGdF4:Eu structures for cancer therapy. Chem Commun 2014, 50, 4371-4374.
4 . Weng, Q.; Wang, B.; Wang, X.; Hanagata, N.; Li, X.; Liu, D.; Wang, X.; Jiang, X.; Bando, Y.; Golberg, D., Highly water-soluble, porous, and biocompatible boron nitrides for anticancer drug delivery. ACS Nano 2014, DOI: 10.1021/nn5014808
5 . Li, X.; Wang, X. P.; Zhang, J.; Hanagata, N.; Weng, Q.; Bando, Y.; Golberg, D., Hollow boron nitride nanomaterials with controlled B release as next generation chemopreventative agent for prostate cancer. 2014,unpublished work.

Pyrolyzed Photoresist Films (PPF) have electrochemical properties similar to glassy carbon electrodes with the unique feature that they can be lithographically defined to create microstructures and microfeatures. Porous carbon electrodes were fabricated by interferometric lithography (IL) to generate 3-D periodic structures in PPF that contain five patterned layers with microporous hexagonal lattices (~ 800 nm in diameter). Because IL is a maskless approach porous carbon structures are able to be produced with defect-free 3-D lattices and sub-wavelength periodicity uniformly over large volumetric areas in excess of 2 cm a side. Despite a high degree of interconnectivity, the relatively large pore sizes preserve hemispherical diffusion inside the structures which exhibit diffusion profiles similar to microelectrodes. We demonstrate that these porous carbon structures can be used as a highly adaptable electrode material with increased mass transport properties for the deposition of metal nanoparticles and conducting polymers with applications in such areas as fuel cells, ultracapacitors, and sensors. We also discuss the conversion of these structures into 3D graphene.

The non-covalent interaction of biomolecules with materials interfaces and/or nanoparticles is of great interest due to potential applications in biosensing and nano-medicine.¹ The interaction of peptides with graphene has shown particular promise in this area due to graphene&’s unique chemical and physical properties.^2,3 However, to fully exploit the peptide-graphene interaction a deeper understanding of the interfacial adsorption occurring at the molecular level is required. Molecular dynamics (MD) simulations, with the ability to predict and reveal interactions at the atomic level, can play a contributing role in elucidating the structure/property relationships of such systems.⁴
Using a recently developed polarizable force-field specifically designed to model the interaction of biomolecules with graphitic nanostructures⁵ in combination with an advanced sampling technique^6,7 the interaction of peptides with graphene interfaces is investigated. Two peptides known to have a strong affinity to the basal plane of graphene, P1 (HSSYWYAFNNKT)) and GrBP5 (IMVTGSSDYSSY), have been modeled.^2,3 We have then explored the effect of sequence on the adsorption behavior by modeling analogues of these sequences where one or more residues has been mutated. Our findings provide valuable information regarding how peptide sequence influences the behavior of peptides at the aqueous graphene interface, moving us closer to the ultimate goal of de novo design peptides for bio-sensing and/or nano-medicine.
[1] Grey, J.J., The interaction of proteins with solid surfaces, Curr. Opin. Struct. Biol., 2004, 14, 110-115.
[2] Kim, S.N., et al., Preferential Binding of Peptides to Graphene Edges and Planes, J. Am. Chem. Soc., 2011, 133, 14480-14483.
[3] So, C.R., et al., Controlling Self-Assembly of Engineered Peptides on Graphite by Rational Mutation,ACS Nano, 2012, 5, 1648-1656.
[4] Tang, Z., et al., Biomolecular recognition principles for bionanocombinatorics: an integrated approach to elucidate enthalpic and entropic factors, ACS Nano, 2013, 7, 9632-9646.
[5] Hughes, Z.E., Tomásio, S.M. and Walsh, T.R., Efficient simulations of the aqueous bio-interface of graphitic nanostructures with a polarisable model, Nanoscale, 2014, 6, 5438-5448.
[6] Terakawa, T., et al., On easy implementation of a vairent of the replica exchange with solute tempering in GROMACS, J. Comput. Chem., 2010, 32, 1228-1234.
[7] Wright, L.B. and Walsh, T.R., Efficient conformational sampling of peptides adsorbed onto inorganic surfaves: insights from a quartz binding peptide, Phys. Chem. Chem. Phys.,2013, 15, 4715-4726.

Nanowire nanomaterials enable unique opportunities at the interface between physical and life sciences. The interface between nanoelectronic devices and biological systems makes possible communication between these two diverse systems such functional biological and electronic elements are on similar size scales. Here, we describe the design, realization and application of novel nanoelectronic scaffolds for engineered tissue and in-vivo studies. First, we introduce the fabrication of ultra-flexible macroporous nanoelectronic network with 3D structure, porosity larger than 99%, hundreds of addressable nanodevices and feature sizes ranging from 10 micrometers to 10 nanometers. Second, we discuss how these nanoelectronic networks or scaffolds can be easily merged with organic gels, polymers and biomaterials without altering their physical/chemical properties. Third, we introduce the concept of cyborg tissues that are prepared by culturing different cells within 3D macroporous nanoelectronic scaffolds. Examples of the unique capabilities enabled by the embedded nanoelectronic scaffold, including spatially-dependent neuronal activation by focal drug addition and real-time monitoring of microenvironment pH, will be discussed. Last, we will describe a new method by which freestanding macroporous nanoelectronic networks can be delivered by syringe injection and subsequently ‘unfold&’ to their originally-designed geometric configuration. The electronics can be injected into in-vivo systems to facilitate minimally invasive integration with neural networks. Multiplexed recording of brain activity from the addressable nanodevices within the nanoelectronic scaffolds will be described, as well as, detailed histology studies demonstrating unique neurophilic nature of the these new nanoelectronic networks. These new results open up new directions for merging electronics with living tissue, thereby creating promising opportunities in areas ranging from fundamental brain science research to the development of powerful brain-machine interfaces and regenerative medicine.

Magnetic resonance imaging (MRI) contrast agents (CA) are increasingly being used to enhance the contrast between normal and diseased tissues and to help detect various anatomical and functional abnormalities in MRI scans. MRI CAs are pharmaceutical compounds containing paramagnetic or superparamagnetic ions that increases the relaxation rate of nearby water protons and thereby enhancing MRI signal intensity of surrounding tissues. Today, 40-50% of the MRI procedures are performed with contrast agents, majority of them being gadolinium chelate based agents (GBCA). However, for advanced MRI applications such as molecular imaging and blood pool imaging, GBCAs suffer from an inherent high threshold of detectability. Theory suggests that the relaxivity (a quantitative measure of CA efficacy) of GBCA is sub-optimal, and predicts the possibility of developing new contrast agents up to hundred times greater relaxivity that can allow the same clinical MRI performance at substantially lower dosages (micromolar or nanomolar dose) reducing the cost of the procedure. Also, the recent discovery and association with nephrogenic systemic fibrosis in some patients has fostered concern and Food and Drug Administration restrictions on the clinical use of GBCA. Therefore, there is a need for a next-generation T₁ MRI CA that show lower toxicity profile, and possess greater relaxivity than clinical Gd³⁺-based CAs. To address the limitations of gadolinium chelate based contrast agents we have developed a novel high-performance carbon nanostructure-based MRI CA. We have synthesized graphene nanoplatelets and functionalized with dextran (GNP-Dex; small stacks (1-12 layers) of graphene, thickness = 1-5 nanometers, diameters of ~ 100 nm) that are monodispersed, water-soluble and intercalated and co-ordinated with trace amounts of manganese (0.06 weight %). The graphene sheets sequester the manganese to prevent its leakage and amplify its relaxivity. Our previous results show that these nanoparticles exhibit excellent magnetic and relaxometric properties with relaxivity ~ 20X greater than current clinical contrast agents. In this study, we report on safety and efficacy of GNP-Dex as an MRI CA in vivo (Wistar rats). Our results demonstrate that the maximum tolerated dose (MTD) 50 mg/kg le; MTD le; 125mg/kg and eliminate mainly through feces within 24 hours. Histopathological changes were observed at ge;125 mg/kg in the heart, lung, spleen, and kidney. In vivo small animal MRI at 7 Tesla showed significant contrast enhancement at potential diagnostic dosages (5 nmoles/kg) in blood vessels for up to 120 min. Taken together, these results suggest that GNP-Dex formulations are safe, and have excellent potential as a blood pool MRI CA. These results lay the foundation for preclinical safety and efficacy studies of GNP-Dex in large animals, necessary for their eventual clinical translation.

Directed outgrowth is crucial for nerve regeneration. In an effort to facilitate peripheral nerve regeneration by mimicking naturally occurring processes, we have fabricated and chemically modified patterned films and preseeded them with Schwann cells to furnish a combination of physical, chemical and biological cues to dorsal root ganglia. The synergistic effects caused the axons to grow along the direction of the grooves at an accelerated rate. We inserted these patterned substrates into conduits that were implanted at the site of rat sciatic nerve transections and showed significantly enhanced functional recovery compared to conventional entubulization strategies. However, there is no readily available source of Schwann cells for translational use in implants. In order to overcome this drawback and facilitate peripheral nerve regeneration, we have fabricated patterned polymeric conduits and investigated the transdifferentiation of readily accessible bone-marrow derived mesenchymal stem cells to Schwann-cell like phenotypes on these materials. Nanomedicine-based strategies can significantly impact peripheral nerve regeneration as well. Schwann cells, in conjunction with patterned substrates, have been shown to secrete nerve growth factor and facilitate peripheral nerve regeneration. Therefore, we have developed biodegradable polymeric nanoparticles for sustained delivery of nerve growth factor to the regenerating nerves.
We have also extended these approaches to the central nervous system to investigate neural stem cell differentiation on these patterned substrates. Effective control of neural stem cell differentiation on these substrates prior to implantation is crucial for their implementation in a regenerative framework. The synergistic combination of physical, chemical and biological cues was found to promote selective neurogenesis on micropatterned substrates compared to controls. In addition to these cues, we used electrical cues to promote differentiation of neural stem cells preferentially to neuronal fates. This provides a unique method to spatially control the differentiation of neural stem cells on different regions of the same substrate, for subsequent utilization in a regeneration scheme for nerve repair.

The discovery of insulin transformed diabetes mellitus specifically type 1 from a fatal illness into a chronic illness. The development of blood glucose meters and better insulin preparations enabled better diabetes control and demonstrated that better control preserved organ systems affected by the disorder. Subsequently, the development of insulin analogs and continuous subcutaneous insulin delivery resulted in further improvements in glucose control including less hypoglycemia. In the last 16 years, continuous subcutaneous glucose monitoring (CGM) has become available and been developed through several iterations. The glucose: insulin system has been modelled by several engineering groups and has led to the description of various types of control algorithms. During the same period, the FDA has categorized the development of the Artificial Endocrine Pancreas (AEP) as a critical pathway and has approved the use of glucose: insulin simulators as an alternative for preclinical animal studies. Thus, in the last 8 years, more than 30 inpatient clinical trials of AEP control have been conducted and have demonstrated that the AEP is safe and effective and can be tested in the outpatient setting for short periods. A few short duration outpatient studies have confirmed the safety and effectiveness of current AEP control. The stage is set for longer duration studies. However, hypoglycemia and postprandial hyperglycemia continue to occur stressing the need for better CGM, faster acting insulin preparations and better informed control algorithms. CGM currently has several limitations including the inaccuracy during periods of rapid plasma glucose changes, the need for calibration daily, probes that need to be replaced at weekly or sub weekly intervals and periods of missing data. Several groups are attempting to develop better CGM sensors.

Functionalized graphene quantum dots were synthesized by the bottom-up approach through the irradiation of a liquid hydrocarbon precursor using a pulsed laser. The functionalization of these nanoparticles with polyethylene glycol provides higher solubility and enhancement in their fluorescence quantum yield in aqueous solution. Graphene quantum dots were characterized by UV-Vis spectroscopy, transmission electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, PL emission spectroscopy, and Fourier transform IR spectroscopy. We have tested their toxicity in different human cell lines, and they did not affect the cell viability at concentrations up to 500 µg/ml. Moreover, graphene quantum dots were employed as high contrast nanoprobes in confocal and fluorescent microscopy, which make them suitable in imaging. In addition to their use in diagnosis, we tested them in therapeutic strategies such as photodynamic therapy and drug delivery. We found that they generate reactive oxygen species when irradiated at specific excitation wavelength. Taking together their photoactivity and their ability to deliver some insoluble drugs to tumors, make them promising in cancer therapy.

Lyotropic lipid liquid crystalline phases that deviate from the conventional bilayer structure such as cubic, hexagonal, and sponge phases, have been recently recognized as relevant materials for gene and drug delivery. Cubic phases having 3D nanostructured intertwined channels that facilitate co-solubilization of hydrophobic and hydrophilic small molecules are of particular importance, as they comprise negative Gaussian curvature membranes that are known for their ability to efficiently permeate cell membranes.
In this work we will show our recent advances in engineering approaches to stabilize the same topologically rich nanostructures obtained in bulk but now confined onto solid surfaces. We will present a detailed structural characterization by Grazing Incidence Scattering techniques and Atomic Force Microscopy and reveal an incredibly rich phase behavior of thin lipid film/hard surface devices. These new materials have great potential in the field of bioelectronics as well as bioadhesive drug delivery.

Micro/nano-scale technologies can have a significant impact on medicine and biology in the areas of cell manipulation, diagnostics and monitoring. At the convergence of these new technologies and biology, we research for enabling solutions to the real world problems at the clinic. Emerging nano-scale and microfluidic technologies integrated with biology offer innovative possibilities for creating intelligent, mobile medical lab-chip devices that could transform diagnostics and monitoring, tissue engineering and regenerative medicine. In this talk, we will present an overview of our laboratory's work in these areas focused on applications in point-of-care and primary care settings including applications for ovarian cancer detection from urine, rapid CD4 counts for global health, multiple pathogen detection with a focus on viral load from unprocessed whole blood and bedside peritonitis detection for end-stage kidney disease patients going through peritoneal dialysis therapy. These emerging technologies could shape our future creating broadly applicable platforms for scientific discovery, providing clinical solutions for resource-constrained settings in the developing world as well as for primary care settings in the developed world.

In vivo fluorescence imaging in the traditional visible and near-infrared (NIR) windows with wavelengths in the range of 400~900 nm has been limited with sub-optimal imaging resolution at depths of >500 µm, owing to the strong scattering and absorption of fluorescence photons by the turbid biological tissue, as well as the high autofluorescence interference from the tissue in these windows. By exploiting the unique second near-infrared (NIR-II, 1.0~1.7 µm) fluorescence of certain nanomaterials such as single-walled carbon nanotubes (SWNTs) and conjugated copolymer nanoparticles, we have shown that the quality of in vivo fluorescence imaging can be significantly improved with crisper resolution at deeper tissue penetration. In this talk, I will first talk about in vivo mouse hindlimb vascular imaging in an animal model of lower limb ischemia using SWNTs as the NIR-II fluorophore, where we demonstrate microvascular angiography with improved spatial resolution over CT, and hemodynamic measurement with larger dynamic range than ultrasound. Then I will talk about mouse brain vascular imaging with sub-10 µm resolution at millimeter depth inside the mouse brain through intact scalp and skull, using chemically separated SWNTs with fluorescence emission in an optimized NIR-IIa region (1.3~1.4 µm). I will then highlight our recent progress on in vivo fluorescence imaging using large diameter SWNTs in the NIR-IIb window (1.5~1.7 µm), which is the longest-wavelength region ever exploited for fluorescence imaging and thus affords the unprecedented imaging resolution down to ~3 µm at a depth of > 3 mm owing to the minimum amount of scattering among all optical windows. Finally I will discuss the development of new NIR-II fluorescent materials other than SWNTs, in particular conjugated copolymers that allow for ultrafast video-rate imaging and tracking of blood flow in real time, owing to the high brightness of the polymer fluorophore.
References:
1. Guosong Hong, Jerry C. Lee, Joshua T. Robinson, Uwe Raaz, Liming Xie, Ngan F. Huang, John P. Cooke and Hongjie Dai. “Multifunctional in vivo vascular imaging using near-infrared II fluorescence” Nature Medicine2012,18, 1841-1846
2. Guosong Hong, Shuo Diao, Junlei Chang, Alexander L. Antaris, Changxin Chen, Bo Zhang, Su Zhao, Dmitriy N. Atochin, Paul L. Huang, Katrin I. Andreasson, Calvin J. Kuo and Hongjie Dai. “Through Skull Fluorescence Imaging of the Brain in a New Near-Infrared Window” Nature Photonics, accepted
3. Guosong Hong, Yingping Zou, Alexander L. Antaris, Shuo Diao, Di Wu, Kai Cheng, Xiaodong Zhang, Changxin Chen, Bo Liu, Yuehui He, Justin Z. Wu, Jun Yuan, Bo Zhang, Zhimin Tao, Chihiro Fukunaga and Hongjie Dai. “Ultrafast fluorescence imaging in vivo with conjugated polymer fluorophores in the second near-infrared window” Nature Communications2014,5, 4206

Implantable medical devices (e.g., ophthalmological devices, dental implants, prostheses like hips and knees and many others) are making an impact in global health care and quality of life. These medical devices utilize a variety of materials including polymers, metals, ceramics, and some nanocarbons, whose properties are critical in achieving device optimal performance. This performance is based on an adequate host response (“biocompatibility”) against the material-device. Recent literature on the performance of metal and polymer components-based implants in patients has shown failure rates far from acceptable. Therefore the search for novel biocompatible materials for implantable medical devices is urgently needed.
Diamond is being considered as a biomaterial for a new generation of implantable medical devices. It exhibits excellent mechanical and tribological properties, it is extremely resistant to chemical attack by body fluids, and it has been demonstrated to be biocompatible, making it suitable for a variety of implantable medical devices. A novel material named ultrananocrystalline diamond (UNCD), in film form develop an patented by one of the authors (OA) and collaborators, exhibits most features of natural crystalline diamond, including highest hardness (~ 1000 GPa) of any known material, extremely low coefficient of friction (~ 0.02-0.04) and remarkable smoothness (surface roughness = 3-5 nm rms) due to their extremely small grain size (3-5 nm). In addition, UNCD exhibits electrical conductivity when nitrogen is incorporated in the grain boundaries or boron atoms are inserted substitutionally in the diamond lattice. Furthermore, extensive studies involving implantation of UNCD-coated microchips in animal eyes, and UNCD-coated metal simulating dental implants in animal bones, have shown that UNCD exhibits excellent biocompatibility and extreme resistance to chemical attack by body fluids. Due to its multifunctionality, UNCD coatings show a great potential for application to a variety of medical devices, a feature rarely seen in the majority of current biomaterials.
Results will be reported demonstrating that UNCD is a novel bioinert/biocompatible coating for some critical implantable medical devices, namely: 1) UNCD coating for implantable microchip in the eye to restore partial vision to blind people due to genetically induced degeneration of photoreceptors, 2) coating for valves to drain aqueous humor from the eye to treat the condition known as glaucoma, and 3) UNCD coating for a new generation of dental implants.

Graphene has special properties of enhancing Raman signals of molecules. This property, first discovered by Jin Zhang and Xi Ling, will be disused in the context of health-care potential.

Due to their nanoscale, morphology, and chemical composition, the tracking and the quantitative analysis of Carbon NanoTubes (CNTs) in biological samples still represent huge challenges. A new technique for the quantitative and accurate detection of CNTs in various biological samples at different scales (whole organisms to organs) was developed using amphibian larvae exposed to Double-Walled CNTs (DWCNTs) [1]. This technique is based on the dielectric relaxation of ultra-low volume suspensions under a microwave electromagnetic field. CNT concentrations were consequently extracted from complex permittivity measurements at 5 GHz, making possible to quantitatively assess the animal exposure to CNTs. Results show that the DWCNT concentrations in larvae exposed to 10 and 50 mg/L is not significantly different (respectively around 42 and 47 mg/g dry weight), as well as low bioaccumulation factors (respectively 0.005 and 0.001). Furthermore, the majority of DWCNTs are located in the intestines (around 152 mg/g dry weight). These results will be compared to the classical chemical analysis, taking advantage of the presence of residual Co and Mo catalytic by-products in the DWCNT samples.
This work opens new opportunities for the assessment of the environmental impact of CNTs [2-5] but also much more generally in the field of biomedical applications as this method could also be used to assess the amount of CNTs in the body after administration.
References:
[1] E. Flahaut, R. Bacsa, A. Peigney, Ch. Laurent, Chem. Commun., (2003), 1442-1443, "Gram-Scale CCVD Synthesis of Double-Walled Carbon Nanotubes"
[2] F. Bourdiol, F. Mouchet, A. Perrault, I. Fourquaux, L. Datas, C. Gancet, J-C. Boutonnet, E. Pinelli, L. Gauthier, E. Flahaut, Carbon, 54, (2013), 175-191, "Biocompatible polymer-assisted dispersion of multi walled carbon nanotubes in water, application to the investigation of their ecotoxicity using Xenopus laevis amphibian larvae"
[3] L. Verneuil, J. Silvestre, F. Mouchet, E. Flahaut, J-C. Boutonnet, F. Bourdiol, D. Baqué, L. Gauthier, E. Pinelli, Nanotoxicogy, (2014), accepted, "Multi-walled carbon nanotubes, natural organic matter and the benthic diatom Nitzschia palea: “a sticky story"
[4] F. Mouchet, P. Landois, P. Puech, E. Pinelli, E. Flahaut, L. Gauthier, Nanomedicine, 5, (6), (2010), 963-974, "CNT ecotoxicity in amphibians: assessment of multi-walled carbon nanotubes and comparison with double-walled carbon nanotubes"
[5] K. W. Kwok, K. M. Leung, E. Flahaut, J. Cheng, S. H. Cheng, Nanomedicine, 5, (6), (2010), 951-961, "Chronic toxicity of double-walled carbon nanotubes to three marine organisms: Influence of different dispersion methods"

Efforts in biomolecular nanotechnology are increasingly directed towards structural control of supramolecular self-assembly. Developments in this area of research are often inspired by living systems. Peptide-based self-assembly is attracting attention in mimicking intra- and extra-cellular fibrous networks with minimal complexity. It was previously found that using Fmoc-dipeptide gelators creates hydrogel scaffolds that are too hydrophobic for certain types of cells. Co-assembly of Fmoc-dipeptide fibres with a surfactant-like peptide derivative provides a handle for introducing chemical functionality, giving rise to more hydrophilic fibre surfaces that may contain functional peptides into the hydrogels. In this study, we demonstrated a facile supramolecular approach for the formation of functionalized nanofibres by combining the advantages of biocatalytic self-assembly and surfactant/gelator co-assembly. This is achieved by enzymatically triggered reconfiguration of free flowing micellar aggregates of pre-gelator (Fmoc-FYp) and functional surfactants Fmoc-X (X = S, T or RGD, where RGD is a well-known cell adhesion motif) to form nanofibres that become coated with the surfactants. This resulted in the formation of fibres that display the functionality at the surface. Furthermore, by varying enzyme concentration, the gel stiffness and supramolecular organization of building blocks can be varied. Next step would be functionalization of peptide fibres with sugar-amphiphiles (e.g Fmoc-Galactosamine) followed by testing their ability to bind to some carbohydrate-binding proteins (e.g. lectin). These results would give an indication on the ability of these scaffolds to bind to certain types of cells (e.g. hepatocytes).

Although biomedical applications of nanotechnology, which typically involve functionalized nanoparticles, have taken significant strides, biological characterization of unmodified nanoparticles remains underinvestigated. We demonstrated that unmodified gold nanoparticles (AuNPs) inhibited the proliferation of a number of cancer cells in a size- and concentration-dependent manner by abrogating MAPK-signaling. In addition, these AuNPs reversed epithelial-mesenchymal transition (EMT) by reducing secretion of a number of heparin-binding proteins, up-regulating E-Cadherin, and down-regulating Snail, N-Cadherin, and Vimentin. Inhibition of MAPK signaling and reversal of EMT upon AuNP treatment inhibited tumor growth and metastasis in two separate orthotopic models of ovarian cancer. The ability of a single self-therapeutic nanoparticle to abrogate signaling cascades of multiple growth factors is unique and purports possible medical applications as potential antitumor and antimetastatic agent. Potential applications of these unique particles to identify new therapeutic targets will also be discussed.

Understanding cell interaction with one-dimensional (1D) nanomaterials, including nanotubes, nanowires, nanofibers, filamentous bacteria, and certain nanoparticle chains, has fundamental importance to many applications such as biomedical diagnostics, therapeutics, and nanotoxicity. Here we show that there are two fundamental modes of interaction between 1D nanomaterials and lipid membranes: a perpendicular entry mode and a parallel adhering mode. Moreover, cell uptake of 1D nanomaterials via receptor-mediated endocytosis is dominated by a single dimensionless parameter that scales with the membrane tension and radius of the nanomaterial and inversely with the membrane bending stiffness. It is shown that as cell membrane internalizes 1D nanomaterials the uptake follows a near-perpendicular entry mode at small membrane tension but it switches to a near-parallel interaction mode at large membrane tension.

The ability for materials to monitor a wide variety of motion and deformation is attracting increasing attention. Graphene woven fabrics (GWFs) serves as a good candidate for strain sensing due to its special mesh structure composed of woven graphene microribbons (GMRs). They can measure very high strain (up to 10%) and offer high sensitivity with significant resistance changes of 10 times at 2% and 10,000 times at 8%.We assemble three distinct sensors to explore its excellent electromechanical property and promote the practical application of GWFs. Firstly, A flexible and wearable strain sensor is assembled by adhering the GWFs on polymer and medical tape composite film, demonstrating notable resistance change when monitoring weak human motions including hand clenching, phonation, expression change, blink, breath, and pulse. Secondly, a Pre-strained GWFs for highly sensitive torsion sensor with wide dynamic range is illustrated with its tolerance against high torsion up to 800 rad/m and a detection limit as low as 0.3 rad/m. Thirdly, a polymer-coated graphene micro-tube piping (GMP) structure with a cross-linked and interconnected channel network is synthesized for detection and analysis of the pollutants volatile organic compounds (VOCs) in liquid, promoting the instant distinct selectivity within seconds and significantly decrease the response time. Generally, those three sensors based on piezoresistive GWFs exhibit the following common features: ultra-light, relatively good sensitivity, high reversible extensibility, superior physical robustness, easy fabrication, good flexibility and so on, indicating wide potential applications in fields of the displays, robotics, fatigue detection, health care, recreation, industrial production, automotive electronics, virtual reality, hazard warning and so forth.

Reports of asthma, a fatal and chronic inflammatory lung disease, have increased by 21% in the previous decade in the US. Improving asthma patient outcomes requires early diagnosis; yet, children often find it difficult to perform traditional airway function measurements such as sputum induction and bronchoalveolar lavage. The development of an highly sensitive, selective, and portable molecular sensor could provide for more effective, non-invasive diagnosis and promote advanced mediation of asthma via early onset detection. However, conventional methods for detecting these biomarkers, such as enzyme-linked immunosorbent assay (ELISA), are indirect, labor intensive and time consuming. Electronic sensors based on single-atom-thick, sp² graphene has been shown to boast parts-per-billion (ppb) sensitivities, a consequence of the high surface areas of these materials. By modifying graphene surfaces with biorecognition moieties tuned to the target of interest, we have developed arrays of ultrasensitive graphene nanosensors. By functionalizing these sensors with antibody agents for key asthma-related target cytokines, such as TNF-α, IL-4 and IL-5, detection limits as low as 1 pg/mL were achieved. By sensing key cytokines of the disease from exhaled human breath, this graphene nanosensor platform could potentially promote accurate and non-invasive diagnosis of asthma.

As a representative two-dimensional (2D) nanomaterial, graphene has attracted significant attention in the field of ultrasensitive sensors because of its excellent electronic and chemical properties, and large specific surface area. In this talk, three different types of graphene-based materials with unique features, i.e., reduced graphene oxide (rGO), crumpled graphene (CG), and vertically-oriented graphene (VG), will be introduced, together with their applications for versatile biosensors based on two major biosensing mechanisms (electronic sensing and electrochemical sensing). In an electronic biosensor, graphene-based materials are functionalized with gold nanoparticle (NP)-antibody conjugates and used as a conducting channel in a field-effect transistor (FET). Electrical detection of protein binding is accomplished through FET and direct current (dc) measurements. In an electrochemical biosensor, highly electrocatalytic metal oxides are combined with graphene-based materials to offer both a high electrocatalytic activity and a high electrical conductivity for enzymeless biosensing. Biomolecules are then detected through electrochemical measurements such as cyclic voltammetry. The above-mentioned graphene-based platforms will be demonstrated for the detection of Escherichia coli (E. coli) bacteria, glucose, and various proteins.

We developed a novel, electronic biosensor for opioids that consists of an engineered mu receptor protein (MUR) with high binding affinity for opioids, chemically bonded to a graphene field-effect transistor (GFET) to read out ligand binding. The modified MUR that provided chemical recognition was computationally redesigned to enhance its solubility and stability in an aqueous environment. The MUR is a member of the G-protein coupled receptor family, which are the targets of more than 40% of modern pharmaceuticals. A shadow mask process was developed to fabricate arrays of hundreds of GFETs with average mobility of ~1500 cm² V^-1 s^-1 and yield exceeding 98%. Devices were functionalized with engineered MUR using a covalent bonding scheme based on diazonium salts. The biosensor exhibits high sensitivity and selectivity for the target naltrexone, with a detection limit of 10 pg/mL. The combination of these advances in obtaining functional forms of receptor proteins (GPCRs) that can be manipulated outside biomembranes and the new GFET fabrication procedure opens a route to highly sensitive nanosensors, where the recognition element is essentially the biological receptor protein.

We present results on novel graphene-based passive wireless sensor concept that has the potential to be utilized for wireless glucose sensing. The sensors consist of a metal-oxide-graphene variable capacitor (varactor) coupled with an inductor to form a resonant oscillator. Due to the quantum capacitance effect in graphene, the capacitance and thus the resonant frequency can be changed in the presence of an external charge in intimate contact with the graphene [1]. The high carrier mobility and high capacitance per unit area of graphene allow for drastic improvements in the miniaturization of graphene varactor-based passive sensors over microelectromechanical system (MEMS)-based sensors [2].
The design, construction and characterization of multi-finger graphene varactors are described in [3]. Briefly, these devices consist of a metallic gate embedded in SiO₂ and capped with a thin layer (~ 7 nm) of HfO₂ dielectric. Single-layer graphene grown by chemical vapor deposition is then transferred onto the substrate and patterned by photolithography. Finally, metallic contacts to the graphene are patterned and deposited via evaporation. Due to the back-gated construction of these varactors, the graphene is exposed directly to the environment over the gate electrodes thus providing sensitivity of the circuit to the local environment. These devices can further be functionalized with receptors to sense a variety of biomolecules, including glucose.
Essential to understanding the performance of these devices as glucose sensors, their native response to water is critical. Previously, we have demonstrated the operation of non-functionalized graphene varactor sensors as wireless humidity sensors [4]. These sensors showed resonant frequency shifts proportional to relative humidity (RH) ranging from 1% to 97% with a linear frequency shift of 5.7±0.3 kHz / % RH. In this work, we provide additional insight into the effect of humidity on graphene-based capacitive sensors and show that the response involves a complex amalgamation of both oxide and quantum capacitance variation as a function of relative humidity. These results will be critical to understanding the response mechanism of the sensor to glucose and other bio-molecules.
Finally, we will show progress toward functionalizing graphene to sense glucose and will show results of graphene-based glucose sensing using both glucose oxidase [5] and concanavalin A [6] functionalization schemes. This work represents a significant step forward to realizing a new class of compact wireless sensors for in vivo glucose monitoring.
[1] S. J. Koester, Appl. Phys. Lett. 2011; [2] C. Son and B. Ziaie, IEEE Trans. Biomed. Eng., 2008; [3] M. A. Ebrish, et al., Appl. Phys. Lett. 2012; [4] D. A. Deen, et al., IEEE Sens. J. 2014; [5] Y. Huang, et al., Nanoscale 2010.; [6] Y. Chen, et al. ACS Nano 2012.

The need for improved medical sensors based on lab-on-a-chip technologies has increased significantly because of the dramatic growth in the number of people with chronic diseases and the associated costs for their healthcare. Development and initial results of a hybrid plastic microfluidic device with an integrated graphene-protein biosensor chip for use in point-of-care will be presented. The initial prototype is a glucometer that uses optimized glucose oxidase bound to a graphene field effect sensor. Advantages of miniaturization are to save time and reagents, enabling rapid and inexpensive assays and reducing the need for skilled personnel. In addition, home-based sensors must be able to work with a drop of blood or saliva and cannot require large samples of blood plasma if they are going to be distributed. The development of the graphene-based POC devices will be presented using glucose as a target analyte. Technologies required for development of the prototype include modification of the glucose oxidase for improved performance by protein engineering, methods to bind the enzyme to the graphene attached to the silicon oxide surface of sensor chip, and integration into a thermoplastic microfluidic device. Overall performance of the graphene-based glucose sensor as well as the integrated prototype in terms of sensitivity, precision, and accuracy will also be presented.

MicroRNAs (miRNAs) function as regulatory molecules of gene expression in cells. Because of their extensive functional involvement in many cellular processes, under both normal and pathological conditions, they are potentially considered as biomarkers in disease diagnosis and therapeutic targets in drug development. miRNAs are single stranded, evolutionarily conserved non-coding RNAs of 19 to 25 nucleotides in length spliced from hair-pin loop transcripts of pre-miRNAs, which are ~60 to 80 nucleotides in length. miRNAs are also coded by approximately 1% genome in many species, and approximately 3% in human genome. Since miRNAs expression is dysregulated in various pathological conditions including cancer, and the secreted miRNAs are very stable in the blood circulation, precise quantitation of various miRNAs level from the blood at different pathological condition would serve as potential biomarkers to diagnose diseases at the early stage, and also can serve as markers in evaluating response of diseases to various therapy. Hence development of high sensitive, simple, and rapid detection system for multiple miRNAs in a single device would be crucial for molecular biology and clinical diagnostics. To address these challenges, we are developing a multiplex miRNA detection system using graphene biosensors based on electrical impedance spectroscopy.
The sensors composed of graphene, a single monolayer of graphite, in between two gold electrodes. The graphene covers 10 mm (length) × 3 mm (width) area and is functionalized with anti-sense miRNAs using a linker molecule (1-pyrenebutanoic acid succinimidyl ester). The system has been tested using miRNA samples at femtomolar to micromolar concentrations which were be incubated on the graphene surface at 60°C followed by slow cooling to room temperature before washing. Furthermore, analysis using Raman spectroscopy was conducted for comparison with the electrical results.

Pancreas and liver cancer are highly lethal. More than 95% of patients diagnosed with these cancers die from the malignant disease. We have no effective therapies to cure these cancers, except the small number of patients with early-stage localized tumors that can be removed surgically. New therapies that are less toxic than standard chemotherapy or ionizing radiation therapy are urgently needed.
We have developed a novel non-invasive radiofrequency field treatment device. Shortwave radiofrequency energy passes easily through the human body without causing damage or harm. Gold or carbon nanoparticles can be targeted to cancer cells by attaching antibodies or drugs to the surface of the nanoparticles. The targeted nanoparticles attach to cancer-specific proteins on the surface of the cancer cells, are internalized into the cells, and then release heat during non-invasive radiofrequency field treatment. Only targeted cancer cells containing conducting nanoparticles develop thermal cytotoxicity, so there are minimal or no toxic effects to normal cells or organs.
We currently have murine models of pancreas and liver cancer and have identified target ligands on the surface of these cancers not found on normal cells. We deliver the targeted gold or carbon nanoparticles intravenously which allows them to attach and be taken into the cancer cells, and then treat with the radiowave field device to kill the malignant tumors with heat. We have detected no normal tissue or organ toxicities in our preclinical studies. Understanding the barriers to successful delivery of the targeted nanoparticles to optimize the intratumoral distribution of nanoparticles to then permit non-invasive radiofrequency-induced thermal destruction of malignant tumors is now a critical focus.

Over 10,000 new cases of glioblastoma multiforme (GBM) are diagnosed in the United States each year and the expected survival is approximately one year from diagnosis. Challenges to GBM therapy include: (1) tumor inaccessibility and risk of damage to the normal brain; (2) insufficient and heterogeneous blood-brain barrier permeability; (3) resistance to therapy; and (4) aggressive tumor relapse and spread beyond local control. Laser induced interstitial thermotherapy (LITT), which bypasses the blood-brain barrier, has shown some efficacy, but an inability to achieve thermoablative temperatures throughout the target lesion has limited its clinical utility. We are studying methods to increase the efficacy of laser-based treatment using carbon nanotube mediated thermal therapy (CNMTT). CNMTT uses laser energy to excite multiwalled carbon nanotubes (MWCNTs) introduced to the tumor site to increase the delivery of heat to the tumor. However, it is essential to develop of an effective strategy to selectively delivery CNTs to GBM tumors for CNMTT. Convection enhanced delivery (CED) is a local injection technique that bypasses the blood-brain barrier and relies on pressure-driven infusion through an intracranial catheter to distribute macromolecules along a pressure gradient rather than relying only on diffusion. The use of CED for delivery of nanotubes has not previously been tested. Therefore, we investigated the influence of different types of coatings on the transport of CNTs infused by CED into a three dimensional brain mimicking hydrogel. Secondly, delivery and confinement of ablative heat across the entire tumor volume is a current clinical problem and failure to treat at the tumor margins leads to tumor regrowth and patient death. Furthermore, cells exposed to sub-ablative heat stress could develop resistance to subsequent treatment. To determine the effect of sub-ablative heating by CNMTT, we used a forced resistance model where cancer cells were repeatedly exposed to sub-ablative temperatures, either using conventional heating by water-bath or through CNMTT. Our studies indicate that cells previously exposed to conventional heat treatment exhibited increased tolerance compared to control cells when subsequently exposed to sub-ablative temperatures. In contrast, cancer cells do not develop tolerance to heating through CNMTT following prior exposure to sub-ablative temperatures induced by CNMTT. Western blots probing for proteins involved in heat shock response suggest HSP70 is induced in cancer cells through conventional heating but not through CNMTT. These studies may lead to development of a novel thermal ablative treatment for GBM that reduces the drive of refractory GBMs toward therapeutic resistance and aggressive regrowth, resulting in more effective treatment of GBM than current strategies.

Drug resistance in cancer treatment is a challenging problem. The use of nanomaterials in oncology has the potential to offer innovating treatments especially where classical chemical therapy does not succeed. In this work, we are discussing 2 different approaches in terms of mechanism of action of carbon nanotubes, using 2 different kinds of multi-walled carbon nanotubes (MWNTs).
In the first approach, we show how the interaction between MWNTs and protein polymers such as microtubules or actin, both classical anti-cancer targets, can be used for anti-cancer activity. Functionalized MWNTs are actively endocytosed by cancer cells interfering with the dynamics of the microtubule cytoskeleton, behaving as mitotic spindle poisons, leading to proliferative arrest and cell death by apoptosis. We demonstrate how MWNTs are also inhibitors of cancer cell migration in different cell lines as well as in glioblastoma multiforme cells, obtained from surgical specimens. Our data validate MWNTs as future alternatives to the traditional anti-mitotic microtubule-stabilizing anti-cancer drugs [1].
In the second approach, in their raw form, double-walled carbon nanotubes (DWNTs) [2] may still contain small metallic aggregates, left over from the catalytic process, that can give them a redox activity and introduce very low electrochemical potentials able to affect biological systems. Indeed, we found that redox active raw-DWNTs inhibit rat colorectal cancer cell proliferation by blocking cells in the G2 phase through ROS generation by tumor cells. We suggest that raw-DWNTs could also modulate autophagy in tumor cells through induction of intracellular acidification. To the best of our knowledge, this is the first time that DWNTs have been found to inhibit proliferation of cancer cells [3].
References:
[1] L. García- Hevia, J. C. Villegas, L. Álvarez-Montes, J. L. Fernández-Luna, E. Flahaut, L. Rodríguez-
Fernández, J. González, R. Valiente, M. L. Fanarraga, Angewandte Chemie Int. Ed., (2014), accepted, "Inhibition of Cancer Cell Migration by Multiwalled Carbon Nanotubes"
[2] E. Flahaut, R. Bacsa, A. Peigney, Ch. Laurent, Chem. Commun., (2003), 1442-1443, "Gram-Scale CCVD Synthesis of Double-Walled Carbon Nanotubes"
[3] S. Fiorito, E. Flahaut, S. Rapino, F. Paolucci, F. Andreola, N. Moroni, E. Pittaluga, M. Zonfrillo, G. Valenti, A. Mastrofrancesco, F. Groppi, E. Sabbioni, E. Bakalis, F. Zerbetto, A. Serafino, Carbon (2014), accepted, "Redox Active Double Wall Carbon Nanotubes Show Intrinsic Anti-tumoral Effects in vitro"

Covalently functionalized, soluble or well-dispersed SWNT may be used as scaffolds to append multiple ligands for therapeutic applications. Such constructs display unusual pharmacokinetics at the cellular and whole animal scale. SWNT efficiently and rapidly deliver peptides and other appended probes into various cell compartments, depending on the modification and cell type; this mechanism can be used as an approach to induce immune responses to the peptides or cytotoxic activity for other ligands. DNA or analogs can also be covalently appended for delivery. Despite their size, SWNT rapidly clear renally allowing the appending of cytotoxic warheads. A 1,3-cycloaddition was developed using an azomethine ylide generated by reacting paraformaldehyde and a side-chain-BOC-protected, lysine-derived alpha-amino acid, H-Lys(Boc)-OH, with purified SWCNT or C60. This cycloaddition and its lysine adduct provides the benefits of dense, covalent modification, ease of purification, commercial availability of reagents, and pH-dependent solubility of the product. We also describe a strategy to overcome limitations in antibody- and particle-based targeting of tumors that takes advantage of the unique biochemical and pharmacokinetic properties of single-walled carbon nanotubes (SWNT.) The conjugation of SWNT to morpholino oligonucleotide sequences (SWNT-cMORF) confers on them the ability to self-assemble with cancer-selective antibodies appended with complementary oligonucleotides (mAb-MORF) with sub-nanomolar affinity and in physiologic conditions. We synthesized SWNT-cMORF conjugates containing multiple copies of 18-mer oligonucleotides, fluorophores, and radiometal chelates and show that the conjugates can self-assemble onto several cancer cell types previously targeted with mAb-MORF, both in vitro and in mice. Self-assembly promoted target antigen complex internalization, in principle allowing delivery of cytotoxic agents into cells via normally non-internalizing targets. The SWNT-cMORF conjugates were labeled to high specific activities with both alpha- and gamma-emitting isotopes, enabling amplification of therapeutic or diagnostic effectors.

In the hot topic of nanomedicine, semiconductor nanowires, thanks to the possibility of controlling both structure and composition at the atomic scale, allowed to achieve the remarkable goal to merge nanoelectronics and biology (see e.g. Q. Quing et al., Nature Nanotechnol. 9, 142-147 2014 and references therein enclosed). However, despite the outstanding results achieved, some problems concerning the long term biocompatibility of the NWs still remain to be solved, even if very recently silicon/aluminum oxide core shell nanowires presented a long term stability (> 100 days) in physiological solutions (W. Zhou et al., Nano Lett. 14, 1614-1619 2014). In this respect, new cytocompatible semiconductor nanowires can be of interest for the scientific community because they expand the family of nanosystems suitable for nanomedicine applications.
Following that approach, in this work the first evidence of in vitro cytocompatibility of SiC/SiO₂ core shell nanowires is reported (A. Cacchioli et al and G. Salviati., Nano Letters, accepted pending minor revisions). The NWs do not induce in vitro cytotoxic activity and no death or irreversible cell damage are revealed ten days after exposure to NWs from 1 to 100 µg/ml for 72 h. Different internalization mechanisms by adenocarcinomic alveolar basal epithelial cells, monocytic cell line derived from an acute monocytic leukemia, breast cancer cells and normal human-derma fibroblasts are shown. The internalization occurs mainly for macropinocytosis and sporadically by direct penetration in all cell models considered, whereas it occurred for phagocytosis only in monocytic leukemia cells. The cytocompatibility of the nanowires is proved by the analysis of cell proliferation, cell cycle progression and oxidative stress on the cells treated with NWs as compared to controls. Reactive oxygen species generation was detected as an early event that then quickly run out with a rapid decrease only in adenocarcinomic alveolar basal epithelial and human-derm fibroblasts cells. In all the cell lines the intracellular presence of NWs induce the same molecular events but to a different extent: peroxidation of membrane lipids and oxidation of proteins. The NWs do not elicit cytotoxic activity leading to irreversible cellular damages or death
Our findings can be considered relevant for the scientific community in particular because the nanosystem proposed presents three concurrent functionalities: i) enhanced optical activity (F. Fabbri et al and G. Salviati., Nanotechnology 2010 21 345702) that makes the SiC/SiO2 NWs excellent candidates as photosensitizers for Photo Dynamic Therapy after proper functionalization; ii) the well known versatility of the SiO₂ shell to wet chemistry functionalizations makes the NWs suitable for biomolecule-delivery vectors and iii) the cytocompatibility makes them interesting even for future intracellular electrodes for signal transduction

Single-walled carbon nanotubes (SWCNTs) are a family of artificial nanomaterials that display remarkable optical properties. These include sharp and structure-dependent spectral transitions and robust fluorescence at short-wave infrared (SWIR) wavelengths. The physical basis for SWCNT optical properties will be introduced and some related biomedical applications will be described. One is the use of the intrinsic SWIR fluorescence to detect, image, and quantify trace concentrations of SWCNTs in cells, tissues, and organisms. These methods are important for studying nanotube uptake at the cellular level as well as for measuring pharmacokinetics, biodistributions, and clearance pathways in animals. Such information is relevant for investigating possible toxic effects and also for developing nanotube-based diagnostic and therapeutic agents. Another application is aimed at developing novel fluorescent immunoprobes suitable for spectrally multiplexed detection in the SWIR region. Here structure-sorted SWCNTs emitting at specific wavelengths are linked to antibodies and then imaged in SWIR fluorescence microscopy. The long wavelengths of SWCNT emission minimize interference from autofluorescence backgrounds and allow observations in several additional spectral channels for simultaneous imaging of multiple antigen targets in vitro. New methods for purifying and characterizing structurally sorted SWCNT samples will also be discussed.

Multi-walled carbon nanotubes (MWCNTs) are increasingly being developed for a variety of biomedical purposes. Their unique combination of optical, thermal and chemical properties affords the possibility of incorporating multiple diagnostic and therapeutic functions into a single particle. MWCNTs can readily be functionalized to display a high density of diagnostic or therapeutic agents. Active targeting of nanotubes by attachment of ligands with an enhanced binding affinity for cancer specific biological moieties is one strategy which may allow for cancer selective delivery of MWCNTs. Many cancers, including breast cancer, are dependent on aerobic glycolysis for survival (the Warburg effect) and express higher levels of glucose transporters (GLUTs) than normal tissue. To exploit this fact, we developed glycosylated nanotubes to selectively target breast tumors. Two iterations of glycosylated nanotubes were created for this study. In one version, glucosamine was directly conjugated to acid treated MWCNTs using an 1-ethyl-3-(3-dimethylaminopropyl)carboiimide (EDC) based conjugation and termed MWCNT- Gln. To create the second iteration of the nanotubes, glucosamine was conjugated to an N-hydroxysuccinimide ester (NHS) of distearyl phosphotidylcholine-poly(ethylene glycol)(DSPE-PEG) and the DSPE-PEG-glucosamine (DSPE-PEG-Gln) construct was added to a solution of acid oxidized MWCNTs. The DSPE-PEG-Gln self-assembled to coat the MWCNTs through hydrophobic interactions and was termed MWCNT-DSPE-PEG-Gln. The mechanisms by which CNTs are taken up by cells include both energy-dependent internalization (e.g. phagocytosis; clathrin or caveolae mediated endocytosis) and passive diffusion through the plasma membrane (e.g. “nanoneedle” effects). Given the many paths CNTs can use to bind to and enter cells, precisely how targeting increases the binding and/or uptake of actively targeted CNTs remains to be determined. Comparative studies of nanoparticle binding kinetics are complicated because the physical properties that influence cellular interactions of nanoparticles also affect their solution dynamics. To date, direct comparisons of the binding and uptake of PEGylated and uncoated CNTs have not been conducted. Therefore, we compared the binding and uptake of MWCNT-Gln and MWCNT-DSPE-PEG-Gln in breast cancer cells. To delineate the role that glucose transporters (GLUTs) played in the binding and uptake of the targeted nanotubes, the breast cancer cells were treated with inhibitors of sugar transport (excess glucosamine; cytochalasin B) or cytochalasin D, an actin depolymerizing agent that can inhibit macropinocytosis and caveolin-mediated endocytosis with a minimal effect on sugar transport. An in vivo study in mice was conducted to evaluate differences in biodistribution and body clearance between MWCNT-Gln and MWCNT-DSPE-PEG-Gln. These studies lay the foundation for development of a cancer targeting diagnostic agent based upon sugar functionalized MWCNTs.

Our lab at MIT has been interested in how the 1D and 2D electronic structures of carbon nanotubes and graphene respectively can be utilized to advance new concepts in molecular detection. We introduce CoPhMoRe or corona phase molecular recognition¹ as a method of discovering synthetic antibodies, or nanotube-templated recognition sites from a heteropolymer library. We show that certain synthetic heteropolymers, once constrained onto a single-walled carbon nanotube by chemical adsorption, also form a new corona phase that exhibits highly selective recognition for speci#64257;c molecules. To prove the generality of this phenomenon, we report three examples of heteropolymers-nanotube recognition complexes for ribo#64258;avin, L-thyroxine and estradiol. The platform opens new opportunities to create synthetic recognition sites for molecular detection. We have also extended this molecular recognition technique to neurotransmitters, producing the first fluorescent sensor for dopamine. Another area of advancement in biosensor development is the use of near infrared fluorescent carbon nanotube sensors for in-vivo detection². Here, we show that PEG-ligated d(AAAT)₇ DNA wrapped SWNT are selective for nitric oxide, a vasodilator of blood vessels, and can be tail vein injected into mice and localized within the viable mouse liver. We use an SJL mouse model to study liver inflammation in vivo using the spatially and spectrally resolved nIR signature of the localized SWNT sensors. Lastly, we discuss graphene as an interfacial optical biosensor, showing that it possesses two pKa values in alkaline and basic ranges. We use this response to measure dopamine in real time, spatially resolved at the interface with living PC12 cells which efflux dopamine, indicating graphene&’s promise as an interfacial sensor in biology.
Zhang, JQ et. al. Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes. Nature Nanotechnology, 8, 12, 2013, 959-968
Iverson, NM, et. al. In vivo biosensing via tissue-localizable near-infrared-fluorescent single-walled carbon nanotubes. Nature Nanotechnology, 8, 11, 2013, 873-880

We demonstrate a methodology to design ultra-small electrodes using 1D zinc oxide nanostructures. These electrodes offer a number of advantages when employed in electrochemical studies for sensor applications. We have fabricated and characterized the ultra-microelectrode based hybrid devices. ZnO nanorods were prepared on non-silicon electronic circuits using wet chemical processes. The primary reason for use of ultramicroelectrodes and smaller electrodes is the benefit obtained from the enhanced diffusion which takes place. As electrodes decrease in size, radial (3-dimensional) diffusion becomes dominant and results in faster diffusion. This high rate of diffusion at small electrodes enables measurement of kinetics by steady-state methods rather than by transient techniques. By decreasing electrode size from micrometer to nanometer scale, study of faster electrochemical and chemical reactions becomes possible. The electrochemical response of this platform for detection of analytes was recorded using electrochemical impedance spectroscopy (EIS). The influence of aspect ratio of nanorods was quantitatively demonstrated by computing the hysteresis of I-V characteristics. It was identified that 1D nanorods facilitated measurements at steady state conditions mainly due to formation of nanoelectrodes. The nanorods were surface characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), structurally by X-ray diffraction (XRD) methods and electrically using Hall measurements. The nanostructured surface was tested for detection of cortisol, a major indicator of stress level and disease state of patients. Results indicated sensor performance with amplified response and large dynamic range for tested concentrations of cortisol.

Early generation glucose biosensors that utilized glucose oxidase (GOx) demonstrated how the use of carbon-based electrodes can be favorable over platinum electrodes. Initial GOx-based biosensors determined physiological glucose concentrations as a function of the oxidation of H₂O₂ (as produced by GOx), whereby H₂O₂ is oxidised at platinum electrodes. Such devices were proven to be problematic, since other compounds (e.g. acetaminophen and ascorbic acid) are also oxidised at platinum electrodes (at a similar potential to that used for the oxidation of H₂O₂) yielding false-positive results; the use of carbon-based electrodes and electron mediators can efficiently lower interference from unwanted compounds. Furthermore, the use of electron mediators can completely negate the need to quantify glucose concentrations as a function of H₂O₂, since electrons that are gained by GOx (following the enzymatic oxidation of glucose) are shuttled directly to an electrode.
Cholesterol oxidase (ChOx, commonly coupled with cholesterol esterase) is emerging in a similar fashion to the early generation of GOx-based glucose biosensors. Current ChOx-based biosensors mostly quantify cholesterol as a function of the concentration of H₂O₂ that is
produced by ChOx (following the oxidation of cholesterol to cholest-5-en-3-one); research aims towards the development of an electron-mediated ChOx-based bioelectrode.
The development of such carbon-based bioelectrodes has also led towards the creation of enzymatic biological fuel cells (BFCs); the supply of electrons to an electrode (as with mediated GOx-based biosensors) partially fulfils the requirements of an anode within a fuel cell. Enzymatic biocathodes have also been prepared whereby the modification of carbon nanotubes (CNTs) with substrate-like moieties can facilitate the docking/orientation of enzymes to electrodes; electrons can then be directly transferred between an enzyme and an electrode
without the need for an electron mediator.
Resulting enzymatic BFCs can be implanted, and are capable of producing electrical energy from physiological concentrations of glucose (in the presence of dissolved O₂). The development of an electron-mediated ChOx-based bioelectrode may therefore lead towards an implantable enzymatic BFC that can produce electrical energy from the oxidation of cholesterol. This paper will discuss the biosensors and biofuel cell electrodes from the perspective of the carbon nanomaterials used for improving electron transfer.

Nanostructures, such as nanowires, nanobelts, nanosprings and nanotubes, are gaining growing interest in biosensors as they provide high sensitivity, multiplexing, small size and portability. Carbon nanotubes (CNTs) and graphene are one such class of nanostructure material that exhibit superior sensing behavior due to their large surface carbon atoms that are highly responsive to surface adsorption events. Combined with their compatibility with modern microfabrication technologies, facile functionalization with molecular recognition elements make them promising candidates for applications in biosensors for healthcare.
In this presentation I will report the recent results of our group on the development of CNTs, graphene and three-dimensional graphene-CNTs hybrid film based biosensors for healthcare. CNT, graphene and CNT-graphene hybrid were functionalized with biological receptors such as aptamers, antibodies and binding proteins for highly sensitive, selective, rapid, cost-effective and point-of-care monitoring of bacterial and viral pathogens, biomarkers for acute myocardial infarction and psychosocial stress, blood glucose, etc.

Semiconducting single wall carbon nanotube are excellent candidates for detecting various types of chemicals and biological analytes. We have developed a novel, flexible s-SWCNT conductance based sensor platform for instantaneous measurement of pathogens in the environment and for monitoring physiological parameters of human body for applications in the wearable electronic skin and environmental monitoring. These two terminal sensors comprises of highly sensitive and selectively functionalized s-SWCNTs serving as the channel with an active area of <10 mm². s-SWCNTs channels were fabricated using a novel offset printing process while the electrodes were made using conventional lithography process. This multi-scale offset printing process is based on directed assembly and transfer. A reusable damascene template is specifically created for high-yield and high-rate assembly and transfer of nanoelements. Electrophoresis is used to assemble semi-conducting carbon nanotubes with very high degree of control, while transfer printing is used to transfer the assembled nanoelements to a recipient flexible substrate. To ensure electric field uniformity during assembly the reusable templates are designed so that all micro/nano conductive patterns have same electrical potential during the electrophoresis assembly stage. Using this assembly and transfer printing process we demonstrate 100% yield. Selective functionalization is conducted employing an in-house developed enzyme-immobilization technique. The choice of functionalization molecule is determined by the chemo- bio analyte that is to be detected. We show that D-glucose, L-lactate, Urea, Oxytetracycline (OTC), E. coli, and Adenovirus were detected with very high sensitivity, selectivity, stability and repeatability. This developed biosensor platform detects D-glucose, L-lactate, Urea, Oxytetracycline (OTC), E. coli, and Adenovirus over a wide range (0~300 mu;M, 0~100 mM, 0~100 mg/dL, 0-150 mg/L, 0-10⁷ cfu/ml, and 0-10⁷ pfu/ml respectively) in less than 5 secs making them suitable for various applications.

Genomic diagnostic platforms have become extremely important in emerging applications such as infectious disease recognition and environmental monitoring. The state-of the art includes devices which offer either high accuracy (e.g., qPCR) or large multiplexing capabilities (e. g, microarrays) but none, however, exhibits a combination of both. An abundance of clinical conditions are genetically manifested at ultra-low concentrations, especially at early onset, requiring PCR amplification for diagnosis. Continued reliance on PCR amplification represents a significant bottleneck for the application of many of these techniques in point-of-care applications. High purification is often required to remove compounds inhibitory to this amplification. Single-strand bioelectronics direct sequencing approaches which do not require amplification, such as nanopore sequencing, promise to drive down the cost of direct sequencing but will still rely on capture events to determine concentrations. In the case of rare events, because there is no specificity for capture, direct sequencing will have reduced throughput because most of the time the system will be sequencing uninteresting and common genetic materials.
We are developing a label-free assay platform delivering fM sensitivity without amplification and enabling multiplexing comparable to microarray technology. The single-molecule field-effect transistor (smFET) platform is based on an array of individual carbon nanotube devices functionalized with a single capture DNA probe integrated on a silicon complementary metal-oxide-semiconductor (CMOS) amplification platform for parallel and multiplexed measurement. The probe is conjugated through a point defect on the smFET device, which renders the device charge sensitive at the point of attachment and insensitive to nonspecific binding at the rest of nanotube surface. Hybridization of a single DNA molecule modulates the conductance at the 1D channel, allowing the detection of hybridization with a single target molecule at high signal-to-noise ratio. Target concentrations are determined by measuring the time between capture events with the analysis of capture and release kinetics yielding information on binding affinities through temperature-controlled studies. The kinetic studies allow the identification of single-nucleotide polymorphisms and other mismatches by measuring the bound dwell time at the different temperatures.
The CMOS array supports the measurement of 6000 smFET devices and bandwidths for measurement of more than 10 MHz. Integration with CMOS provides for both reduced parasitic capacitances in interfacing to the smFET devices and scalable integration of many parallel devices. Signal levels for the smFET devices exceed 10 nA, more than seven orders of magnitude higher than the signals produced by single-molecule fluorescence, demonstrating the significant advantages of bioelectronic approaches for single-molecule diagnostics.

Cryopreservation of living cells has the potential to create a great impact on modern regenerative, transfusion and reproductive medicine. Current cryopreservation methods utilize bulk volumes, causing cryo-injury to the cells, subsequently resulting in irreversible disruption of cell morphology, mechanics, and function. Here, we report the preservation of human red blood cell (RBC) morphology, mechanics, and function following vitrification in nanoliter volumes using an innovative cryo-printing approach. This approach integrates naturally-occuring cryo-inks, where ectoine acts as a key component. Results indicate preservation of RBC morphology and submembranous spectrin-actin network, as well as the preservation of membrane stiffness, cell deformability, and membrane flickering as crucial mechanical properties of RBC. Finally, we verify maintenance of essential RBC function by quantifying: phosphorylation of band-3 protein, which associates with the organization and function of RBC membrane; expression of complement receptor 1, which mediates adhesion of immune complexes to RBCs; and intracellular levels of nitric oxide and reactive oxygen species, which serve as typical measures of blood flow regulation and cell homeostasis.

Introduction: Inorganic nanomaterials have been used to reinforce biodegradable polymeric scaffolds for bone tissue engineering applications [1,2]. These scaffolds should be porous, mechanically strong and biocompatible. In this study, we report the efficacy and in vitro cytocompatibility of several inorganic one-dimensional (boron nitride nanotubes (BNNTs), tungsten disulfide nanotubes (WSNTs)) and two-dimensional (boron nitride nanoplatelets (BNNPs), molybdenum sulfide nanoplatelets (MSNPs)) nanomaterials as reinforcing agents to enhance mechanical properties of biodegradable and biocompatible polymer polypropylene(fumarate) (PPF) for bone tissue engineering. We also investigate cell spreading and protein adsorptive properties of these nanoparticle reinforced scaffolds.
Materials and Methods: Scaffolds (porosity > 80 vol. %) were prepared by dispersing 0.2 wt. % BNNTs, BNNPs, MSNPs and WSNTs in PPF, crosslinking at 60°C and salt leaching (to induce porosity). Structure and compressive mechanical properties of scaffolds were studied. Then, MC3T3 pre-osteoblasts were cultured on nanocomposite scaffolds and in vitro cellularity and cell spreading were studied. Finally, fetal bovine serum (FBS) adsorption was characterized.
Results and discussion: Although as a result of introducing 80% porosity to the structure no significant increase in mechanical properties was observed for BNNP, MSNP and WSNT nanocomposites, BNNTs caused a significant increase in mechanical strength compared to the PPF control (~18.5% and ~124.7% increase in compressive modulus and offset yield strength, respectively). This increase can be correlated to presence of BNNTs with high mechanical strength (compressive modulus of BNNT is ~ 600 GPa). Structural characterization showed a scaffold with interconnected > 80 volume % porosity. Confocal images showed infiltration of MC3T3 cells inside the scaffolds after 5 days of incubation at 37°C whereas SEM showed cell spreading and cytoplasmic extensions for cellular attachment. An excellent cell viability was observed in all scaffolds where greater than 50% of initially seeded cells were viable after one day of incubation. Up to 25% increase in FBS adsorption (compared to PPF) was observed for nanocomposite scaffolds after 9 days incubation, and can be attributed to the increase in surface energy of the scaffolds due to the presence of nanomaterials.
Conclusions: Using high strength inorganic nanomaterials like BNNTs we were able to significantly reinforce PPF polymer scaffolds. The scaffolds show excellent cellularity, cell attachment and spreading, and improved protein adsorption. The results taken together suggest that one- and two- dimensional inorganic nanomaterials are promising materials to reinforce biodegradable polymeric scaffolds for tissue engineering applications.
References
[1]. Lalwani, G., et al., Biomacrolecules, 2013. 14(3): p. 900-90.
[2]. Lalwani, G., et al., Acta Biomaterialia, 2013 .9(9): p.8365-73.

Vertically-oriented graphene (VG), also referred to as "carbon nanowall" (CNW), is a two-dimensional “graphitic” platelet that is typically oriented vertically on a substrate. The unique vertical orientation and open structure make VG an ideal sensing material as its entire surface is accessible by analyte molecules. A sensitive and selective field-effect transistor (FET) biosensor with VG sheets labeled with gold nanoparticle (NP)-antibody conjugates is reported. VG sheets are directly grown on the sensor electrode using a plasma-enhanced chemical vapor deposition (PECVD) method and work as the sensing channel. This approach can serve as a simple and one-step method to prepare graphene-based sensor devices and can be a better alternative route to drop-casting methods that are commonly used to fabricate graphene and carbon nanotube devices. Because the VG is directly grown on the sensor electrode, it is believed that this platform could achieve higher stability and repeatability than the drop-casting method, which in general has a high noise level and poor stability due to the weak binding between the graphene sheet and the sensor electrode. In this study, the binding of probe and target proteins (Immunoglobulin G) will induce a significant change in the electrical conductivity of the sensor. The new protein sensors show high sensitivity (down to ~2 ng/ml or 13 pM) and selectivity to specific proteins and are especially attractive for large-scale fabrication of sensor devices since the graphene growth and the assembly are accomplished in one step. The sensitivity of the VG sensor is also among the best of carbon nanomaterial (e.g., carbon nanotube and graphene)-based protein sensors, which have a detection limit typically from fM to nM. Previous studies have shown that the FET sensor signal is largely dependent on the initial sensor resistance and varies from sensor to sensor. With the PECVD method, multiple sensors can be fabricated in one batch under the same growth conditions; therefore, it is possible to control the initial resistance level of the sensor device and minimize variations in the sensor signal. Moreover, by designing the substrate (sensor electrode) patterns, VG could grow along the patterns and create various sensor structures for different applications. The new VG-based sensing platform could serve as a general platform for biomolecular detection because of its easy fabrication and controllable sensor structure.

The emergence of 2-dimensional (2D) materials could herald numerous advanced scientific methodologies for both fundamental and applied research. These ultrathin materials can be functionalized and, thus, have the potential to make new devices and sensors that are both highly efficient and sensitive. In addition to being mechanically robust, the 2D materials can be engineered to provide sensor architectures that further increase their inherent high surface area by creating 3D geometries using layer by layer assembly to make stacked devices that could potentially be transparent. The increased sensor surface area would deliver increased signal-to-noise and sensitivity. In this talk, I will discuss highly sensitive and selective electrochemical detection of bio-analytes using some of these engineered 2D materials such as graphene nano-ribbons, fluorinated graphene, and molybdenum disulfide. It is found that surface moieties, defects and surface charges in these ultra-thin layers result in enhanced electron transfer kinetics between the electrodes and biomolecules. This in turn results in an oxidation or reduction of biomolecules with a high peak current, indicating the possible uses of 2D materials for various point-of-care devices. A novel stable 3D electrode geometry has been found to have enhanced heterogeneous electron transfer properties compared to our 2D electrodes and provides evidence that electrode geometry and surface area could significantly impact the performance of biosensors. These results will also be discussed during the talk.

Serum glucose, cholesterol, triglyceride and HbA1C monitoring are all valuable tools in the health management of the aging population especially given the increase in diabetes and cardiovascular diseases. Even for glucose monitoring, the challenges in obtaining sufficiently accurate and reliable measurements are so significant (1), that the FDA is contemplating more stringent standards. Freckmann et al (2) have compared 43 blood glucose self-monitoring systems. Out of this 34 systems were completely assessed, and 27 (79.4%) of these 34 systems fulfill the minimal accuracy requirements of the standard DIN EN ISO 15197:2003. Only 18 (52.9%) of 34 systems fulfilled the minimal accuracy requirements if tighter criteria of the current draft revision of ISO 15197 are considered. None of them meet the even more stringent requirement of ISO 2012 and FDA. Because inaccurate systems bear the risk of false therapeutic decisions, rising health care costs, there is an urgent compelling need for significantly enhanced BG monitoring systems for PC applications. POC tests for other biomedically important analytes are generally even less accurate. The proposed system uses advanced graphene protein microfluidic chip to transduce enzymatic binding into electrical signals that can be read and processed by a stand-alone system or even a cell-phone. These new biosensor chips will be housed in a plastic microfluidic system for sample acquisition, preparation, and distribution to four separate biosensing chips. This approach will improve accuracy because it reduces operator errors, calibration problems, and strip-to-strip variability, while increasing sensor sensitivity/specificity with the option to use redundant sensors for improved statistical confidence. Typically CNT and graphene-based sensors are several orders of magnitude greater in their sensitivity compared to standard approaches such as those used in glucose sensors. The prototype device will be used in clinical testing for glycohemoglobin (HgbA1c) and lipid profile will be performed on groups of patients who will be divided based on age, gender, types of treatments they are on and co-morbidities criteria. Simultaneous venipuncture blood draw will be done for comparable tracking of accuracy of the results. The data will be analyzed to assess both the specificity and sensitivity of the tests as well as to determine the accuracy and speed of obtaining results . Subjects will be divided into 2 arms - one the “Diabetes group” and one the “ Lipid group”. Each of these groups will then be subdivided based on the above-mentioned criteria to perform preliminary clinical trials of the first prototype.
David C. Klonoff,J. Clinical Endoc. Metabolism 96, 2968, 2011
G. Freckmann et al., J. Diabetes Sci. Tech. 6, 1060, 2012

Fibrils formed by proteins are vital components for cells. However, selective formation of xenogenous nanofibrils of small molecules on mammalian cells has yet to be observed. Here we report an unexpected observation of hydrogel/nanonets of a small d-peptide derivative in pericellular space. Surface and secretory phosphatases dephosphorylate a precursor of a hydrogelator to trigger the self-assembly of the hydrogelator and to result in pericellular hydrogel/nanonets selectively around the cancer cells that overexpress phosphatases. Cell-based assays confirm that the pericellular hydrogel/nanonets block cellular mass exchange to induce apoptosis of cancer cells, including multidrug-resistance (MDR) cancer cells, MES-SA/Dx5. Pericellular hydrogel/nanonets of small molecules to exhibit distinct functions illustrates a fundamentally new way to engineer molecular assemblies spatiotemporally in cellular microenvironment for inhibiting cancer cell growth and even metastasis.

Through evolutionary pressure, bacteria have developed resistance to many biocidal compounds, including commercial antibiotics and silver nanoparticles. Thus, biofilms that develop on implanted medical devices, like intravenous catheters, are challenging to fight and cause grave health concerns. New approaches to delay the onset of biofilm formation with less evolutionary pressure on pathogens are a priority. The theory of mechanobiology is that fundamental cellular processes are modulated by substrate stiffness. In this study, we investigate if this theory applies to biofilm development by exploring how bacteria interact on compliant surfaces. Poly(ethylene glycol) (PEG) hydrogels were synthesized as a function of polymer concentration, resulting in six hydrogels having Young&’s moduli ranging from 44.00 - 6,500 kPa. In our first study, Escherichia coli K12 (E. coli) attachment was found to inversely correlate with hydrogel stiffness. Using confocal microscopy, colony development and early biofilm growth were quantified on the PEG hydrogels, as well as on agar hydrogel controls. It was determined that the total number of adhered cells on the soft hydrogel was 15 fold lower than the stiff after 2 hours and 5 fold lower after 24 hours. A second approach, which featured a flow-cell, assessed the weak rolling adhesion of E. coli and Staphyloccus aureus on the soft and stiff PEG hydrogels. Bacterial cell rolling is produced by shear forces overcoming reversible physiochemical binding forces of a bacterium to a surface. Stick-and-roll behavior has been indicated as a physiological advantage of bacteria leading to enhanced surface colonization and biofilm formation, a mode of transport inadvertently enhanced by non-adhesive antifouling surfaces. Understanding how bacteria overcome antifouling and biocidal materials will significantly aid in future design of functional material surfaces.

Nanomaterials such as multiwalled carbon nanotubes (MWCNTs) have the potential to play a significant role in transforming how cancer is treated. MWCNTs generate substantial heat when exposed to a near infrared laser which allows far greater heat generation and localization within a tumor target than laser irradiation alone. Carbon nanotube mediated thermal therapy (CNMTT) is effective for treating bulk cancer and stem cell-like cancer populations which may enhance treatment efficacy, reduce disease recurrence and prevent the development of therapeutic resistance. Laser energy can be delivered by inserting a fiber optic laser directly into the tumor via a catheter and heat delivered by interstitial lasers is now being used as a focal cytoreductive treatment for brain tumors. We are studying methods to increase the efficacy of laser-based treatment using CNMTT. Our current research aims to address several issues needed for successful clinical application of CNMTT for treatment of brain tumors including:
(i) development of an effective strategy to selectively delivery CNTs to tumors and distribute them throughout the tumor volume;
(ii) optimizing parameters for generating a uniform and predictable temperature distribution in the targeted area following NIR exposure with minimal heat spread to neighboring healthy tissue;
(iii) elimination of bulk cancer and stem-cell like cancer cells at the margin of the targeted area;
(iv) acute and long-term toxicological investigation of CNTs;
(v) understanding the roles of treatment induced stress in the development of tolerance to CNMTT and disease recurrence.
We assess the role of nanotube coating materials in both the blood compatibility and tumor interstitial transport of carbon nanotubes. We use magnetic resonance thermometry to experimentally validate modeling methodology that will assist in optimizing the application of carbon nanotubes in thermal tumor ablation by allowing precise application of ablative heat temperatures with minimal spillover to surrounding structures. Cancer cells previously exposed to conventional heat treatment exhibit increased tolerance compared to control cells when subsequently exposed to sub-ablative temperatures. However, repeated exposure to CNMTT does not induce similar tolerance. Our data suggest that unlike conventional heat treatment, CNMTT can simultaneously eliminate both the differentiated cells that constitute the bulk of a tumor along with the cancer stem cells that drive tumor growth and recurrence. Furthermore, we demonstrate specificity of binding/uptake by mammalian cells of large nanoparticles like MWCNTs can be mediated by interaction with glucose transporters that are overexpressed on many cancer cells.

Early detection and effective treatment of cancer are vitally important for millions of people around the world. Advanced diagnostic techniques such as magnetic resonance imaging have been very successful and current cancer treatments such as surgery and chemotherapy have saved many lives. However, these diagnostic and therapeutic methods have respective disadvantages. Another major issue is cancer is often diagnosed and treated too late: for many people with the symptoms, cancer has already invaded other parts of the body and drugs can only achieve limited therapeutic effects. In recent years, nanotechnology has advanced rapidly and has made a profound impact in the medical field. Nanomedicine emerged a decade ago and involves the use of nanotechnologies to detect and treat diseases or even to identify and stop potential sources of diseases before they get started. Nanoparticles have attracted great attention for their potential as diagnostic and/or therapeutic tools in oncology due to their unique properties. Combining diagnosis with therapy in multifunctional nanodevices, which are termed “theranostics”, can provide great benefits to both patients and nations&’ healthcare systems. Design and development of novel theranostics is a major direction in cancer nanotechnology. The new generation of theranostics should have greatly improved capability for early cancer detection and provide multiple functions for cancer cell targeting, sensing/imaging, anti-cancer drug storage and release, and possibly other therapies. Different inorganic nanoparticles (NPs) can provide the basic unit for theranostics, and gold nanoparticles (AuNPs) and gold-silver nanoparticles (Au-Ag NPs) are particularly appealing due to their good biocompatibility and unique optical properties. Highly branched AuNPs or Au-Ag NPs can provide very high enhancement factors for surface enhanced Raman scattering (SERS) signals, enabling early detection of cancer cells because of the high sensitivity of SERS for detections down to single molecules. For AuNPs and Au-Ag NPs, the ability to control their size and shape during synthesis for achieving the desired plasmonic property and their surface conjugation with macromolecules (e.g., antibody) allow for selective, early detection of cancer cells as well as killing of cancer cells through photothermal effect. With either a polymer shell or core, metal-polymer nanohybrids can be formed as novel theranostics, with anti-cancer drug being stored in the polymeric component. This paper presents our investigations into novel theranostics based on AuNPs or Au-Ag NPs, which provide functions of cancer cell targeting, imaging, anticancer drug delivery, photothermal therapy, etc. The synthesis of theranostics, their structural features and designed functions are discussed. The biological assessment of some of these nanodevices is also presented.

Cisplatin is a first-line chemotherapy agent clinically used for treating a variety of cancers, but suffers from dose-limiting side effects, particularly nephrotoxicity. A variety of nanotechnology drug delivery materials have been developed for administering cisplatin in a targeted or localized delivery fashion to mitigate these side effects, such as using nano- and microparticles, liposomes, films, and meshes. Due to its hydrophilicity, however, cisplatin often exhibits burst-release kinetics from many of these materials. Here we extend the use of three-dimensional, superhydrophobic electrospun meshes to fabricate a cisplatin-nanofiber delivery depot that exhibits linear, sustained release of drug under the rather aggressive release conditions of 10% serum and shaking at 37 °C. Cisplatin concentration as a function of incubation duration is measured by flameless atomic absorption spectroscopy and shows a linear release profile over >70 days. The slow, sustained release of cisplatin from these meshes is due to a marked reduction in the wettability of the mesh compared to non-superhydrophobic meshes (water contact angle of ~151° versus ~135°, respectively). Sustained release of cisplatin from the nanofiber network is further evaluated for in vitro efficacy against the Lewis Lung Carcinoma (LLC) cell line and achieves prolonged efficacy against these cancer cells compared to non-superhydrophobic cisplatin-loaded meshes. Finally, superhydrophobic cisplatin-eluting meshes are investigated for their ability to prevent tumor recurrence after surgical resection of LLC tumor allografts, which delays tumor recurrence to a greater extent than that achieved with systemic treatment.

Significant improvement in chemotherapy for cancer treatment has prompted the development of anticancer drugs. Camptothecin (CPT) has appealed a great deal of interest as a promising anticancer drug. Previous researches reported about the antitumor activity of CPT for treating certain types of cancer cells. However, its poor water solubility and lactone ring hydrolysis of CPT has been hampered the clinical application of CPT. The major problem of conventional drug delivery is that CPT is easily carboxylated under physiological conditions and binds with human serum albumin and loss of antitumor activity. Therefore, the encapsulation of CPT is needed to overcome its intrinsic characteristics and achieve application as an antitumor treatment. In order to overcome these drawbacks, various types of drug carrier systems have been widely studied. Among various nanomaterials, silica based nanomaterials garnered considerable interest as drug delivery agent because of low toxicity, large surface area, and easy synthetic process. The typical example is the mesoporous silica based drug delivery system. However, mesoporous silica nanomaterials have relatively low drug loading efficiency than that of hollow structure. Therefore, there is a requirement for silica-based nanomaterials with an extensive hollow cavity to deliver a large amount of drug to cancer cells. To improve drug loading capacity, hollow nanoparticle (HNP) with a size of 50 nm is used as CPT delivery agent. It is known that HNP with 50 nm diameter is favored in cellular uptake.
In this study, silica-titania hollow nanoparticles (HNPs) with 50 nm were fabricated by using the dissolution and redeposition method, and encapsulated CPT for anticancer treatment. The HNP is further modified with anti-[human epidermal growth factor receptor 2] monoclonal antibody (herceptin) to target human breast cancer SK-BR-3 cells. The herceptin modified HNP was called as HER-HNP. The extensive hollow cavity of HNPs (ca. 30 nm) allowed the loading of a large amount of CPT. The solvent dependent release profile of CPT from CPT-loaded HER-HNP (HER-HNP-CPT) behavior was differently shown in hydrophobic and hydrophilic environment. The apoptosis/necrosis and viability of HER-HNP-CPT-treated cells were investigated to evaluate in vitro antitumor efficacy. The viability of HER-HNP-CPT treated SK-BR-3 decreased to 60%, which demonstrated highly efficient chemotherapy. However, CPT loaded into HNP or HER-HNP had no significant effects on the viability of macrophages. Collectively, HER-HNPs are very suitable for application in anticancer therapy. A HER-HNP-CPT drug delivery system offers a new opportunity for effective antitumor treatment of HER positive tumor cells. Furthermore, our HER-HNP-CTP system proposes new positive tumor cells and can be expanded to practical applications with further studies.