Electrochemical Redox Cycling Behavior of Gold Nanoring Electrodes Microfabricated on a Silicon Micropillar.

We report the microfabrication and characterization of concentric gold nanoring electrodes (Au NREs), which were fabricated by patterning two gold nanoelectrodes on the same silicon (Si) micropillar tip. Au NREs of 165 ± 10 nm in width were micropatterned on a 6.5 ± 0.2 µm diameter 80 ± 0.5 µm height Si micropillar with an intervening ~ 100 nm thick hafnium oxide insulating layer between the two nanoelectrodes. Excellent cylindricality of the micropillar with vertical sidewalls as well as a completely intact layer of a concentric Au NRE including the entire micropillar perimeter has been achieved as observed via scanning electron microscopy and energy dispersive spectroscopy data. The electrochemical behavior of the Au NREs was characterized by steady-state cyclic voltammetry and electrochemical impedance spectroscopy. The applicability of Au NREs to electrochemical sensing was demonstrated by redox cycling with the ferro/ferricyanide redox couple. The redox cycling amplified the currents by 1.63-fold with a collection efficiency of > 90% on a single collection cycle. The proposed micro-nanofabrication approach with further optimization studies shows great promise for the creation and expansion of concentric 3D NRE arrays with controllable width and nanometer spacing for electroanalytical research and applications such as single-cell analysis and advanced biological and neurochemical sensing.

Brain-Implantable Multifunctional Probe for Simultaneous Detection of Glutamate and GABA Neurotransmitters: Optimization and In Vivo Studies.

Imbalances in levels of glutamate (GLU) and gamma-aminobutyric acid (GABA) and their sub-second signaling dynamics occur in several brain disorders including traumatic brain injury, epilepsy, and Alzheimer's disease. The present work reports on the optimization and in vivo testing of a silicon (Si) multifunctional biosensor probe for sub-second simultaneous real-time detection of GLU and GABA. The Si probe features four surface-functionalized platinum ultramicroelectrodes (UMEs) for detection of GLU and GABA, a sentinel site, and integrated microfluidics for in-situ calibration. Optimal enzyme concentrations, size-exclusion phenylenediamine layer and micro spotting conditions were systematically investigated. The measured GLU sensitivity for the GLU and GABA sites were as high as 219 ± 8 nA µM-1 cm-2 (n = 3). The measured GABA sensitivity was as high as 10 ± 1 nA µM-1 cm-2 (n = 3). Baseline recordings (n = 18) in live rats demonstrated a useful probe life of at least 11 days with GLU and GABA concentrations changing at the levels of 100's and 1000's of µM and with expected periodic bursts or fluctuations during walking, teeth grinding and other activities and with a clear difference in the peak amplitude of the sensor fluctuations between rest (low) and activity (higher), or when the rat was surprised (a reaction with no movement). Importantly, the probe could improve methods for large-scale monitoring of neurochemical activity and network function in disease and injury, in live rodent brain.

Nafion and Multiwall Carbon Nanotube Modified Ultrananocrystalline Diamond Microelectrodes for Detection of Dopamine and Serotonin.

Neurochemicals play a critical role in the function of the human brain in healthy and diseased states. Here, we have investigated three types of microelectrodes, namely boron-doped ultrananocrystalline diamond (BDUNCD), nafion-modified BDUNCD, and nafion-multi-walled carbon nanotube (MWCNT)-modified BDUNCD microelectrodes for long-term neurochemical detection. A ~50 nm-thick nafion-200-nm-thick MWCNT-modified BDUNCD microelectrode provided an excellent combination of sensitivity and selectivity for the detection of dopamine (DA; 6.75 µA µM-1 cm-2) and serotonin (5-HT; 4.55 µA µM-1 cm-2) in the presence of excess amounts of ascorbic acid (AA), the most common interferent. Surface stability studies employing droplet-based microfluidics demonstrate rapid response time (<2 s) and low limits of detection (5.4 ± 0.40 nM). Furthermore, we observed distinguishable DA and 5-HT current peaks in a ternary mixture during long-term stability studies (up to 9 h) with nafion-MWCNT-modified BDUNCD microelectrodes. Reduced fouling on the modified BDUNCD microelectrode surface offers significant advantages for their use in long-term neurochemical detection as compared to those of prior-art microelectrodes.

Brain-Implantable Multifunctional Probe for Simultaneous Detection of Glutamate and GABA Neurotransmitters.

Glutamate (GLU) and gamma-aminobutyric acid (GABA) are neurotransmitters (NTs) with an essential role in signal transmission in the brain. Brain disorders, such as epilepsy, Alzheimer's and Parkinson's diseases, and traumatic brain injury can be linked to imbalances in the GLU-GABA homeostasis that occurs in sub-second to seconds time frames. Current measurement techniques can detect these two NT concentrations simultaneously only in vitro. The present work reports on the fabrication of a silicon multifunctional biosensor microarray probe for sub-second simultaneous GLU-GABA detection in real-time, with excellent analyte sensitivity and selectivity and in vivo capabilities. The novel Si probes feature four surface-functionalized platinum ultramicroelectrodes (UMEs) for simultaneous amperometric detection of GLU and GABA with a sentinel, and a built-in microfluidic channel for the introduction of neurochemicals in the proximity of the UMEs. The microchannel also allows functioning of an On-Demand In-situ Calibrator that runs in-situ biosensor calibration. The probe exhibited excellent robustness at insertion in agarose-gel brain-tissue-mimicking test, and remarkably high hydrogen peroxide sensitivity (a by-product of GLU-GABA enzyme biosensor) with values on the order of 5000 nA µM -1 cm -2 and maximum sensitivities of 204±15 nA µM -1 cm -2 and 37±7 nA µM -1 cm -2 for GLU and GABA, respectively. Furthermore, the limit of detection of the biosensors reached as low as 7 nM, 165 nM and 750 nM for H 2 O 2, GLU and GABA, respectively and a temporal resolution of hundreds of milliseconds during in vivo studies using freely moving rats.

Surface Fouling of Ultrananocrystalline Diamond Microelectrodes during Dopamine Detection: Improving Lifetime via Electrochemical Cycling.

In this work, we report the electrochemical response of a boron-doped ultrananocrystalline diamond (BDUNCD) microelectrode during long-term dopamine (DA) detection. Specifically, changes to its electrochemical activity and electroactive area due to DA byproducts and surface oxidation are studied via scanning electron microscopy, energy dispersive spectroscopy, electrochemical impedance spectroscopy, and silver deposition imaging (SDI). The fouling studies with amperometry (AM) and fast scan cyclic voltammetry (FSCV) methods suggest that the microelectrodes are heavily fouled due to poor DA-dopamine- o-quinone cyclization rates followed by a combination of polymer formation and major changes in their surface chemistry. SDI data confirms the presence of the insulating polymer with sparsely distributed tiny electroactive regions. This resulted in severely distorted DA signals and a 90% loss in signal starting as early as 3 h for AM and a 56% loss at 6.5 h for FSCV. This underscores the need for cleaning of the fouled microelectrodes if they have to be used long-term. Out of the three in vivo suitable electrochemical cycling cleaning waveforms investigated, the standard waveform (-0.4 V to +1.0 V) provides the best cleaned surface with a fully retained voltammogram shape, no hysteresis, no DA signal loss (a 90 ± 0.72 nA increase), and the smallest charge transfer resistance value of 0.4 ± 0.02 MΩ even after 6.5 h of monitoring. Most importantly, this is the same waveform that is widely used for in vivo detection with carbon fiber microelectrodes. Future work to test these microelectrodes for more than 24 h of DA detection is anticipated.

An enzyme-based electrochemical biosensor probe with sensitivity to detect astrocytic versus glioma uptake of glutamate in real time in vitro.

Glutamate, a major excitatory neurotransmitter in the central nervous system, is essential for regulation of thought, movement, memory, and other higher functions controlled by the brain. Dysregulation of glutamate signaling is associated with severe neuropathological conditions, such as epilepsy, and glioma, a form of brain cancer. Glutamate signals are currently detected by several types of neurochemical probes ranging from microdialysis-based to enzyme-based carbon fiber microsensors. However, an important technology gap exists in the ability to measure glutamate dynamics continuously, and in real time, and from multiple locations in the brain, which limits our ability to further understand the involved spatiotemporal mechanisms of underlying neuropathologies. To overcome this limitation, we developed an enzymatic glutamate microbiosensor, in the form of a ceramic-substrate enabled platinum microelectrode array, that continuously, in real time, measures changes in glutamate concentration from multiple recording sites. In addition, the developed microbiosensor is almost four-fold more sensitive to glutamate than enzymatic sensors previously reported in the literature. Further analysis of glutamate dynamics recorded by our microbiosensor in cultured astrocytes (control condition) and glioma cells (pathological condition) clearly distinguished normal versus impaired glutamate uptake, respectively. These results confirm that the developed glutamate microbiosensor array can become a useful tool in monitoring and understanding glutamate signaling and its regulation in normal and pathological conditions. Furthermore, the developed microbiosensor can be used to measure the effects of potential therapeutic drugs to treat a range of neurological diseases.

A Novel Microbiosensor Microarray for Continuous ex Vivo Monitoring of Gamma-Aminobutyric Acid in Real-Time.

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter that is essential for normal brain function. It is involved in multiple neuronal activities, including plasticity, information processing, and network synchronization. Abnormal GABA levels result in severe brain disorders and therefore GABA has been the target of a wide range of drug therapeutics. GABA being non-electroactive is challenging to detect in real-time. To date, GABA is detected mainly via microdialysis with a high-performance liquid chromatography (HPLC) system that employs electrochemical (EC) and spectroscopic methodology. However, these systems are bulky and unsuitable for real-time continuous monitoring. As opposed to microdialysis, biosensors are easy to miniaturize and are highly suitable for in vivo studies; they selectively oxidize GABA into a secondary electroactive product (usually hydrogen peroxide, H2O2) in the presence of enzymes, which is then detected by amperometry. Unfortunately, this method requires a rather cumbersome process with prereactors and relies on externally applied reagents. Here, we report the design and implementation of a GABA microarray probe that operates on a newly conceived principle. It consists of two microbiosensors, one for glutamate (Glu) and one for GABA detection, modified with glutamate oxidase and GABASE enzymes, respectively. By simultaneously measuring and subtracting the H2O2 oxidation currents generated from these microbiosensors, GABA and Glu can be detected continuously in real-time in vitro and ex vivo and without the addition of any externally applied reagents. The detection of GABA by this probe is based upon the in-situ generation of α-ketoglutarate from the Glu oxidation that takes place at the Glu microbiosensor. A GABA sensitivity of 36 ± 2.5 pA µM-1cm-2, which is 26-fold higher than reported in the literature, and a limit of detection of 2 ± 0.12 µM were achieved in an in vitro setting. The GABA probe was successfully tested in an adult rat brain slice preparation. These results demonstrate that the developed GABA probe constitutes a novel and powerful neuroscientific tool that could be employed in the future for in vivo longitudinal studies of the combined role of GABA and Glu (a major excitatory neurotransmitter) signaling in brain disorders, such as epilepsy and traumatic brain injury, as well as in preclinical trials of potential therapeutic agents for the treatment of these disorders.

Detection of neurochemicals with enhanced sensitivity and selectivity via hybrid multiwall carbon nanotube-ultrananocrystalline diamond microelectrodes.

Abnormal neurochemical signaling is often the underlying cause of brain disorders. Electrochemical microsensors are widely used to monitor neurochemicals with high spatial-temporal resolution. However, they rely on carbon fiber microelectrodes that often limit their sensing performance. In this study, we demonstrate the potential of a hybrid multiwall carbon nanotube (MWCNT) film modified boron-doped ultrananocrystalline diamond (UNCD) microelectrode (250 µm diameter) microsensor for improved detection of dopamine (DA) in the presence of common interferents. A series of modified microelectrodes with varying film thicknesses were microfabricated by electrophoretic deposition (EPD) and characterized by scanning electron microscopy, x-ray photoelectron spectroscopy, electrochemical impedance spectroscopy (EIS) and silver deposition imaging. Using cyclic voltammetry, the 100-nm "thin" film microelectrode produced the most favorable combination of DA sensitivity value of 36 ±2% µA/µM/cm2 with a linear range of 33 nM to 1 µM and a limit of detection (LOD) of 9.5 ± 1.2% nM. The EIS spectra of these microelectrodes revealed three regions with inhomogeneous pore geometry and differing impedance values and electrochemical activity, which was found to be film thickness dependent. Using differential pulse voltammetry, the modified microelectrode showed excellent selectivity by exhibiting three distinct peaks for the DA, serotonin and excess ascorbic acid in a ternary mixture. These results provide two key benefits: first, remarkable improvements in DA sensitivity (>125-fold), selectivity (>2000-fold) and LOD (>180-fold), second, these MWCNTs can be selectively coated with a simple, scalable and low cost EPD process for highly multiplexed microsensor technologies. These advances offer considerable promise for further progress in chemical neurosciences.

Enabling long term monitoring of dopamine using dimensionally stable ultrananocrystalline diamond microelectrodes.

Chronic dopamine (DA) monitoring is a critical enabling technology to identify the neural basis of human behavior. Carbon fiber microelectrodes (CFM), the current gold standard electrode for in vivo fast scan cyclic voltammetry (FSCV), rapidly loses sensitivity due to surface fouling during chronic neural testing. Periodic voltage excursions at elevated anodic potentials regenerate fouled CFM surfaces but they also chemically degrade the CFM surfaces. Here, we compare the dimensional stability of 150 µm boron-doped ultrananocrystalline diamond (BDUNCD) microelectrodes in 1X PBS during 'electrochemical cleaning' with a similar-sized CFM. Scanning electron microscopy and Raman spectroscopy confirm the exceptional dimensional stability of BDUNCD after 40 h of FSCV cycling (~8 million cycles). The fitting of electrochemical impedance spectroscopy data to an appropriate circuit model shows a 2x increase in charge transfer resistance and an additional RC element, which suggests oxidation of BDUNCD electrode surface. This could have likely increased the DA oxidation potential by ~34% to +308 mV. A 2x increase in BDUNCD grain capacitance and a negligible change in grain boundary impedance suggests regeneration of grains and the exposure of new grain boundaries, respectively. Overall, DA voltammogram signals were reduced by only ~20%. In contrast, the CFM is completely etched with a ~90% reduction in the DA signal using the same cleaning conditions. Thus, BDUNCD provides a robust electrode surface that is amenable to repeated and aggressive cleaning which could be used for chronic DA sensing.

Nanocrystalline Diamond Electrodes: Enabling electrochemical microsensing applications with high reliability and stability.

THE DIAMOND (D) IS ONE OF the most precious materials in the world with unmatched physical and chemical properties, such as hardness, extreme chemical stability, high thermal conductivity, the highest acoustic velocity of any material, an extremely low friction coefficient when smooth, and nearly unmatched biocompatibility [1]. The carbon (C) atoms in Ds are tetrahedrally coordinated, i.e., each C atom is bonded to four others in the D lattice. This bonding is referred to as sp 3 bonding, and the strength and configuration of these bonds provide Ds with these unmatched fundamental properties and characteristics. Realizing these properties of the D in a C-based film that can easily be integrated into functional engineering systems and deployed in many applications has been a challenge for several decades. This is of primary concern in microelectronics, sensing, and hard-coating applications.

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.

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.

Characterization of carbon nanofiber electrode arrays using electrochemical impedance spectroscopy: effect of scaling down electrode size.

We report here how the electrochemical impedance spectra change as (i) electrode size is reduced to nanometer scale and (ii) spacing between vertically aligned carbon nanofiber (VACNF) electrodes is varied. To study this, we used three types of electrodes: standard microdisks (100 microm Pt, 10 microm Au, and 7 microm glassy carbon), randomly grown (RG) VACNFs where spacing between electrodes is not fixed, and electron beam patterned VACNF nanoelectrode arrays (pNEAs) where electrode spacing is fixed at 1 microm. As the size of the microdisk electrode is reduced, the spectrum changed from a straight line to a semicircle accompanied by huge noise. Although a semicircle spectrum can directly indicate the electron transfer resistance (R(ct)) and thus is useful for biosensing applications, the noise from electrodes, particularly from those with diameters < or =10 microm, limits sensitivity. In the case of VACNFs, the electrode spacing controls the type of spectrum, that is, a straight line for RG VACNFs and a semicircle for pNEAs. In contrast to microdisks, pNEAs showed almost insignificant noise even at small perturbations (10 mV). Second, only pNEAs showed linearity as the amplitude of the sinusoidal signal was increased from 10 to 100 mV. The ability to apply large amplitudes reduces the stochastic errors, provides high stability, and improves signal-to-noise (S/N) ratio. This new class of nanoelectrochemical system using carbon pNEAs offers unique properties such as semicircle spectra that fit into simple circuits, high S/N ratio, linearity, and tailor-made spectra for specific applications by controlling electrode size, spacing, and array size.

Wafer-scale fabrication of patterned carbon nanofiber nanoelectrode arrays: a route for development of multiplexed, ultrasensitive disposable biosensors.

One of the major limitations in the development of ultrasensitive electrochemical biosensors based on one-dimensional nanostructures is the difficulty involved with reliably fabricating nanoelectrode arrays (NEAs). In this work, we describe a simple, robust and scalable wafer-scale fabrication method to produce multiplexed biosensors. Each sensor chip consists of nine individually addressable arrays that uses electron beam patterned vertically aligned carbon nanofibers (VACNFs) as the sensing element. To ensure nanoelectrode behavior with higher sensitivity, VACNFs were precisely grown on 100 nm Ni dots with 1 microm spacing on each micro pad. Pretreatments by the combination of soaking in 1.0 M HNO(3) and electrochemical etching in 1.0M NaOH dramatically improved the electrode performance, indicated by the decrease of redox peak separation in cyclic voltammogram (DeltaE(p)) to approximately 100 mV and an approximately 200% increase in steady-state currents. The electrochemical detection of the hybridization of DNA targets from E. coli O157:H7 onto oligonucleotide probes were successfully demonstrated. The 9 arrays within the chip were divided into three groups with triplicate sensors for positive control, negative control and specific hybridization. The proposed method has the potential to be scaled up to NxN arrays with N up to 10, which is ideal for detecting a myriad of organisms. In addition, such sensors can be used as a generic platform for many electroanalysis applications.