The effect of electrode size and surface heterogeneity on electrochemical properties of ultrananocrystalline diamond microelectrode.

We report here the effect of electrode size on electrochemical properties of boron-doped ultrananocrystalline diamond (UNCD) microelectrodes using electrochemical impedance spectroscopy (EIS). By reducing microelectrode size from 250-µm to 10-µm diameter (D), the shape of impedance spectra changes from linear line to two-arcs. The fitting of experimental data to electrochemical circuit model suggests that each arc likely corresponds to UNCD grains and grain boundary phases. The two phases become separable as a result of microelectrode size reduction. In addition, for D ≤ 100-µm, microstructural and morphological defects/heterogeneities of grain boundaries and the presence of surface oxygen are also revealed in the spectra. The microelectrode size reduction specifically affect the impedance of the grain boundaries, e.g. for ultramicroelectrodes, UMEs (D ≤ 25-µm), as the grain boundary impedance increases by ~30-fold. Thus, at UMEs, the grain-grain boundary properties are revealed more sensitively in the spectra. Atomic force microscopy, scanning electron microscopy, Raman spectroscopy and surface profilometry measurements were performed to study the influence of microfabrication on surface properties. A significant increase in surface roughness after microfabrication shows that heterogeneities as observed in the spectra are not only due to intrinsic UNCD properties but also arises from microfabrication.

Characterization of ultrananocrystalline diamond microsensors for in vivo dopamine detection.

We show the technical feasibility of coating and micro patterning boron-doped ultrananocrystalline diamond (UNCD®) on metal microwires and of applying them as microsensors for the detection of dopamine in vivo using fast-scan cyclic voltammetry. UNCD electrode surface consistently generated electrochemical signals with high signal-to-noise ratio of >800 using potassium ferrocyanide-ferricyanide redox couple. Parylene patterned UNCD microelectrodes were effectively applied to detect dopamine reliably in vitro using flow injection analysis with a detection limit of 27 nM and in the striatum of the anesthetized rat during electrical stimulation of dopamine neurons.

Ultrananocrystalline diamond tip integrated onto a heated atomic force microscope cantilever.

We report a wear-resistant ultrananocrystalline (UNCD) diamond tip integrated onto a heated atomic force microscope (AFM) cantilever and UNCD tips integrated into arrays of heated AFM cantilevers. The UNCD tips are batch-fabricated and have apex radii of approximately 10 nm and heights up to 7 µm. The solid-state heater can reach temperatures above 600 °C and is also a resistive temperature sensor. The tips were shown to be wear resistant throughout 1.2 m of scanning on a single-crystal silicon grating at a force of 200 nN and a speed of 10 µm s(-1). Under the same conditions, a silicon tip was completely blunted. We demonstrate the use of these heated cantilevers for thermal imaging in both contact mode and intermittent contact mode, with a vertical imaging resolution of 1.9 nm. The potential application to nanolithography was also demonstrated, as the tip wrote hundreds of polyethylene nanostructures.

A quantitative study of detection mechanism of a label-free impedance biosensor using ultrananocrystalline diamond microelectrode array.

It is well recognized that label-free biosensors are the only class of sensors that can rapidly detect antigens in real-time and provide remote environmental monitoring and point-of-care diagnosis that is low-cost, specific, and sensitive. Electrical impedance spectroscopy (EIS) based label-free biosensors have been used to detect a wide variety of antigens including bacteria, viruses, DNA, and proteins due to the simplicity of their detection technique. However, their commercial development has been hindered due to difficulty in interpreting the change in impedance upon antigen binding and poor signal reproducibility as a result of surface fouling and non-specific binding. In this study, we develop a circuit model to adequately describe the physical changes at bio functionalized surface and provide an understanding of the detection mechanism based on electron exchange between electrolyte and surface through pores surrounding antibody-antigen. The model was successfully applied to extract quantitative information about the bio surface at different stages of surface functionalization. Further, we demonstrate boron-doped ultrananocrystalline diamond (UNCD) microelectrode array (3 × 3 format, 200 µm diameter) improves signal reproducibility significantly and increases sensitivity by four orders of magnitude. This study marks the first demonstration of UNCD array based biosensor that can reliably detect a model Escherichia coli K12 bacterium using EIS, positioning this technology for rapid adoption in point-of-use applications.

Control of Nanoscale Environment to Improve Stability of Immobilized Proteins on Diamond Surfaces.

Immunoassays for detection of bacterial pathogens rely on the selectivity and stability of bio-recognition elements such as antibodies tethered to sensor surfaces. The search for novel surfaces that improve the stability of biomolecules and assay performance has been pursued for a long time. However, the anticipated improvements in stability have not been realized in practice under physiological conditions because the surface functionalization layers on commonly used substrates, silica and gold, are themselves unstable on time scales of days. In this paper, we show that covalent linking of antibodies to diamond surfaces leads to substantial improvements in biological activity of proteins as measured by the ability to selectively capture cells of the pathogenic bacterium Escherichia coli O157:H7 even after exposure to buffer solutions at 37 °C for extended periods of time, approaching 2 weeks. Our results from ELISA, XPS, fluorescence microscopy, and MD simulations suggest that by using highly stable surface chemistry and controlling the nanoscale organization of the antibodies on the surface, it is possible to achieve significant improvements in biological activity and stability. Our findings can be easily extended to functionalization of micro and nanodimensional sensors and structures of biomedical diagnostic and therapeutic interest.

Surface functionalization of thin-film diamond for highly stable and selective biological interfaces.

Carbon is an extremely versatile family of materials with a wide range of mechanical, optical, and mechanical properties, but many similarities in surface chemistry. As one of the most chemically stable materials known, carbon provides an outstanding platform for the development of highly tunable molecular and biomolecular interfaces. Photochemical grafting of alkenes has emerged as an attractive method for functionalizing surfaces of diamond, but many aspects of the surface chemistry and impact on biological recognition processes remain unexplored. Here we report investigations of the interaction of functionalized diamond surfaces with proteins and biological cells using X-ray photoelectron spectroscopy (XPS), atomic force microscopy, and fluorescence methods. XPS data show that functionalization of diamond with short ethylene glycol oligomers reduces the nonspecific binding of fibrinogen below the detection limit of XPS, estimated as > 97% reduction over H-terminated diamond. Measurements of different forms of diamond with different roughness are used to explore the influence of roughness on nonspecific binding onto H-terminated and ethylene glycol (EG)-terminated surfaces. Finally, we use XPS to characterize the chemical stability of Escherichia coli K12 antibodies on the surfaces of diamond and amine-functionalized glass. Our results show that antibody-modified diamond surfaces exhibit increased stability in XPS and that this is accompanied by retention of biological activity in cell-capture measurements. Our results demonstrate that surface chemistry on diamond and other carbon-based materials provides an excellent platform for biomolecular interfaces with high stability and high selectivity.

Wear-resistant diamond nanoprobe tips with integrated silicon heater for tip-based nanomanufacturing.

We report exceptional nanoscale wear and fouling resistance of ultrananocrystalline diamond (UNCD) tips integrated with doped silicon atomic force microscope (AFM) cantilevers. The resistively heated probe can reach temperatures above 600 degrees C. The batch fabrication process produces UNCD tips with radii as small as 15 nm, with average radius 50 nm across the entire wafer. Wear tests were performed on substrates of quartz, silicon carbide, silicon, or UNCD. Tips were scanned for more than 1 m at a scan speed of 25 mum s(-1) at temperatures ranging from 25 to 400 degrees C under loads up to 200 nN. Under these conditions, silicon tips are partially or completely destroyed, while the UNCD tips exhibit little or no wear, no signs of delamination, and exceptional fouling resistance. We demonstrate nanomanufacturing of more than 5000 polymer nanostructures with no deterioration in the tip.

Rapid thermal lysis of cells using silicon-diamond microcantilever heaters.

This paper presents the design and application of microcantilever heaters for biochemical applications. Thermal lysis of biological cells was demonstrated as a specific example. The microcantilever heaters, fabricated from selectively doped single crystal silicon, provide local resistive heating with highly uniform temperature distribution across the cantilevers. Very importantly, the microcantilever heaters were coated with a layer of 100 nm thick electrically insulating ultrananocrystalline diamond (UNCD) layer used for cell immobilization on the cantilever surface. Fibroblast cells or bacterial cells were immobilized on the UNCD/cantilever surfaces and thermal lysis was demonstrated via optical fluorescence microscopy. Upon electrical heating of the cantilever structures to 93 degrees C for 30 seconds, fibroblast cell and nuclear membrane were compromised and the cells were lysed. Over 90% of viable bacteria were also lysed after 15 seconds of heating at 93 degrees C. This work demonstrates the utility of silicon-UNCD heated microcantilevers for rapid cell lysis and forms the basis for other rapid and localized temperature-regulated microbiological experiments in cantilever-based lab on chip applications.

In vitro and in vivo evaluation of ultrananocrystalline diamond for coating of implantable retinal microchips.

In this work, ultrananocrystalline diamond (UNCD) thin films were evaluated for use as hermetic and bioinert coatings for a retinal microchip. These films were deposited on highly conductive Si substrates at different temperatures (from 400 to 800 degrees C), using microwave plasma enhanced chemical vapor deposition with argon-rich Ar/CH4 gas mixtures and different relative amounts of hydrogen (0-20%). Scanning electron microscopy studies showed that all the films are dense and continuous. Results of cyclic voltammetry test revealed that when there was <2% of hydrogen in the plasma, the film obtained renders the surface electrochemically inactive, with very low leakage currents ( approximately 4 x 10(-7) A/cm2 at +/-5 V). In addition, in vivo tests of the UNCD-coated Si samples were performed by implanting them in the eyes of rabbits for 4-6 months within the eye physiological environment. According to all these results, it was concluded that UNCD is a promising candidate for use as the encapsulating coatings for implantable retinal microelectronic devices.

Novel ultrananocrystalline diamond probes for high-resolution low-wear nanolithographic techniques.

A hard, low-wear probe for contact-mode writing techniques, such as dip-pen nanolithography (DPN), was fabricated using ultrananocrystalline diamond (UNCD). Molding within anisotropically etched and oxidized pyramidal pits in silicon was used to obtain diamond tips with radii down to 30 nm through growth of UNCD films followed by selective etching of the silicon template substrate. The probes were monolithically integrated with diamond cantilevers and subsequently integrated into a chip body obtained by metal electroforming. The probes were characterized in terms of their mechanical properties, wear, and atomic force microscopy imaging capabilities. The developed probes performed exceptionally well in DPN molecular writing/imaging mode. Furthermore, the integration of UNCD films with appropriate substrates and the use of directed microfabrication techniques are particularly suitable for fabrication of one- and two-dimensional arrays of probes that can be used for massive parallel fabrication of nanostructures by the DPN method.

Surface functionalization of ultrananocrystalline diamond films by electrochemical reduction of aryldiazonium salts.

The surface functionalization of ultrananocrystalline diamond (UNCD) thin films via the electrochemical reduction of aryl diazonium cations is described. The one-electron-transfer reaction leads to the formation of solution-based aryl radicals, which in turn react with the UNCD surface forming stable covalent C-C bonds. Cyclic voltammetry (CV), X-ray photoelectron spectroscopy (XPS), ac impedance spectroscopy, and contact angle measurements have been employed to characterize the organic overlayer and estimate the surface coverage. The grafting of 3,5-dichlorophenyl groups renders the UNCD surface hydrophobic, whereas the attachment of 4-aminophenyl groups makes the surface relatively hydrophilic. The surface coverage, estimated from the electrochemical and XPS measurements, is as high as 70% of a compact monolayer. The aminophenyl terminated surface was obtained by electrochemical reduction of the tethered nitrophenyl groups. This two-step approach yields a UNCD surface with functional moieties available for the potential covalent coupling of a wide variety of biomolecules (e.g., DNA and proteins).

DNA-modified nanocrystalline diamond thin-films as stable, biologically active substrates.

Diamond, because of its electrical and chemical properties, may be a suitable material for integrated sensing and signal processing. But methods to control chemical or biological modifications on diamond surfaces have not been established. Here, we show that nanocrystalline diamond thin-films covalently modified with DNA oligonucleotides provide an extremely stable, highly selective platform in subsequent surface hybridization processes. We used a photochemical modification scheme to chemically modify clean, H-terminated nanocrystalline diamond surfaces grown on silicon substrates, producing a homogeneous layer of amine groups that serve as sites for DNA attachment. After linking DNA to the amine groups, hybridization reactions with fluorescently tagged complementary and non-complementary oligonucleotides showed no detectable non-specific adsorption, with extremely good selectivity between matched and mismatched sequences. Comparison of DNA-modified ultra-nanocrystalline diamond films with other commonly used surfaces for biological modification, such as gold, silicon, glass and glassy carbon, showed that diamond is unique in its ability to achieve very high stability and sensitivity while also being compatible with microelectronics processing technologies. These results suggest that diamond thin-films may be a nearly ideal substrate for integration of microelectronics with biological modification and sensing.