Glassy carbon microelectrode arrays enable voltage-peak separated simultaneous detection of dopamine and serotonin using fast scan cyclic voltammetry.

Progress in real-time, simultaneous in vivo detection of multiple neurotransmitters will help accelerate advances in neuroscience research. The need for development of probes capable of stable electrochemical detection of rapid neurotransmitter fluctuations with high sensitivity and selectivity and sub-second temporal resolution has, therefore, become compelling. Additionally, a higher spatial resolution multi-channel capability is required to capture the complex neurotransmission dynamics across different brain regions. These research needs have inspired the introduction of glassy carbon (GC) microelectrode arrays on flexible polymer substrates through carbon MEMS (C-MEMS) microfabrication process followed by a novel pattern transfer technique. These implantable GC microelectrodes provide unique advantages in electrochemical detection of electroactive neurotransmitters through the presence of active carboxyl, carbonyl, and hydroxyl functional groups. In addition, they offer fast electron transfer kinetics, capacitive electrochemical behavior, and wide electrochemical window. Here, we combine the use of these GC microelectrodes with the fast scan cyclic voltammetry (FSCV) technique to optimize the co-detection of dopamine (DA) and serotonin (5-HT) in vitro and in vivo. We demonstrate that using optimized FSCV triangular waveform at scan rates ≤700 V s-1 and holding and switching at potentials of 0.4 and 1 V respectively, it is possible to discriminate voltage reduction and oxidation peaks of DA and 5-HT, with 5-HT contributing distinct multiple oxidation peaks. Taken together, our results present a compelling case for a carbon-based MEA platform rich with active functional groups that allows for repeatable and stable detection of electroactive multiple neurotransmitters at concentrations as low as 1.1 nM.

Subdiffusive motion of bacteriophage in mucosal surfaces increases the frequency of bacterial encounters.

Bacteriophages (phages) defend mucosal surfaces against bacterial infections. However, their complex interactions with their bacterial hosts and with the mucus-covered epithelium remain mostly unexplored. Our previous work demonstrated that T4 phage with Hoc proteins exposed on their capsid adhered to mucin glycoproteins and protected mucus-producing tissue culture cells in vitro. On this basis, we proposed our bacteriophage adherence to mucus (BAM) model of immunity. Here, to test this model, we developed a microfluidic device (chip) that emulates a mucosal surface experiencing constant fluid flow and mucin secretion dynamics. Using mucus-producing human cells and Escherichia coli in the chip, we observed similar accumulation and persistence of mucus-adherent T4 phage and nonadherent T4∆hoc phage in the mucus. Nevertheless, T4 phage reduced bacterial colonization of the epithelium >4,000-fold compared with T4∆hoc phage. This suggests that phage adherence to mucus increases encounters with bacterial hosts by some other mechanism. Phages are traditionally thought to be completely dependent on normal diffusion, driven by random Brownian motion, for host contact. We demonstrated that T4 phage particles displayed subdiffusive motion in mucus, whereas T4∆hoc particles displayed normal diffusion. Experiments and modeling indicate that subdiffusive motion increases phage-host encounters when bacterial concentration is low. By concentrating phages in an optimal mucus zone, subdiffusion increases their host encounters and antimicrobial action. Our revised BAM model proposes that the fundamental mechanism of mucosal immunity is subdiffusion resulting from adherence to mucus. These findings suggest intriguing possibilities for engineering phages to manipulate and personalize the mucosal microbiome.

Glassy Carbon Neural Interface for Chronic Epidural Stimulation in Rats with Cervical Spinal Cord Injury.

There is growing evidence on the efficacy of electrical stimulation delivered via spinal neural interfaces to improve functional recovery following spinal cord injury. For such interfaces, carbon-based neural arrays are fast becoming recognized as a compelling material and platform due to biocompatibility and long-term electrochemical stability. Here, we introduce the design, fabrication, and in vivo characterization of a novel cervical epidural implant with carbon-based surface electrodes. Through finite element analysis and mechanical load tests, we demonstrated that the array could safely withstand loads applied to it during implantation and natural movement of the subject with minimal stress levels. Furthermore, the long-term in vivo performance of this neural array consisting of glassy carbon surface electrodes was investigated through acute and chronic spinal motor evoked potential recordings and electrode impedance tests in rats. We demonstrated stable stimulation performance for at least four weeks in a rat model of spinal cord injury. Lastly, we found that impedance measurements on all carbon-based spinal arrays were generally stable over time up to four weeks after implantation, with a slight increase in impedance in subsequent weeks when tested in spinally injured rats. Taken together, this study demonstrated the potential for carbon-based electrodes as a spinal neural interface to accelerate both mechanistic research and functional restoration in animal models of spinal cord injury.

Graphene on glassy carbon microelectrodes demonstrate long-term structural and functional stability in neurophysiological recording and stimulation.

Objective.There is a growing interest in the use of carbon and its allotropes for microelectrodes in neural probes because of their inertness, long-term electrical and electrochemical stability, and versatility. Building on this interest, we introduce a new electrode material system consisting of an ultra-thin monoatomic layer of graphene (Gr) mechanically supported by a relatively thicker layer of glassy carbon (GC).Approach.Due to its high electrical conductivity and high double-layer capacitance, Gr has impressive electrical and electrochemical properties, two key properties that are useful for neural recording and stimulation applications. However, because of its two-dimensional nature, Gr exhibits a lack of stiffness in the transverse direction and hence almost non-existent flexural and out-of-plane rigidity that will severely limit its wider use. On the other hand, GC is one of carbon's important allotropes and consists of three-dimensional microstructures of Gr fragments with a natural molecular similarity to Gr. Further, GC has exceptional chemical inertness, good electrical properties, high electrochemical stability, purely capacitive charge injection, and fast surface electrokinetics coupled with lithography patternability. This makes GC an ideal candidate for addressing Gr's lack of out-of-plane rigidity through providing a matching sturdier and robust mechanical backing. Combining the strengths of these two allotropes of carbon, we introduce a new neural probe that consists of ∼1 nm thick layer of patterned Gr microelectrodes supported by another layer of 3-5µm thick patterned GC.Main results. We present the fabrication technology for the newGr on GC(graphene on glassy carbon) microelectrodes and the accompanying pattern transfer technology on flexible substrate and report on the bond between these two allotropes of carbon through FTIR, surface morphology through SEM, topography through atomic force microscopy, and microstructure imaging through scanning transmission electron microscopy. A long-term (18 weeks)in vivostudy of the use of theseGr on GCmicroelectrodes assessed the quality of the electrocorticography-based neural signal recording and stimulation through electrophysiological measurements. The probes were demonstrated to be functionally and structurally stable over the 18 week period with minimal glial response-the longest reported so far for Gr-based microelectrodes.Significance.TheGr on GCmicroelectrodes presented here offers a compelling case for expanding the potentials of Gr-based technology in the broad areas of neural probes.

Carbon-based neural electrodes: promises and challenges.

Neural electrodes are primary functional elements of neuroelectronic devices designed to record neural activity based on electrochemical signals. These electrodes may also be utilized for electrically stimulating the neural cells, such that their response can be simultaneously recorded. In addition to being medically safe, the electrode material should be electrically conductive and electrochemically stable under harsh biological environments. Mechanical flexibility and conformability, resistance to crack formation and compatibility with common microfabrication techniques are equally desirable properties. Traditionally, (noble) metals have been the preferred for neural electrode applications due to their proven biosafety and a relatively high electrical conductivity. Carbon is a recent addition to this list, which is far superior in terms of its electrochemical stability and corrosion resistance. Carbon has also enabled 3D electrode fabrication as opposed to the thin-film based 2D structures. One of carbon's peculiar aspects is its availability in a wide range of allotropes with specialized properties that render it highly versatile. These variations, however, also make it difficult to understand carbon itself as a unique material, and thus, each allotrope is often regarded independently. Some carbon types have already shown promising results in bioelectronic medicine, while many others remain potential candidates. In this topical review, we first provide a broad overview of the neuroelectronic devices and the basic requirements of an electrode material. We subsequently discuss the carbon family of materials and their properties that are useful in neural applications. Examples of devices fabricated using bulk and nano carbon materials are reviewed and critically compared. We then summarize the challenges, future prospects and next-generation carbon technology that can be helpful in the field of neural sciences. The article aims at providing a common platform to neuroscientists, electrochemists, biologists, microsystems engineers and carbon scientists to enable active and comprehensive efforts directed towards carbon-based neuroelectronic device fabrication.

Epi-Intra neural probes with glassy carbon microelectrodes help elucidate neural coding and stimulus encoding in 3D volume of tissue.

OBJECTIVE: In this study, we demonstrate practical applications of a novel 3-dimensional neural probe for simultaneous electrophysiological recordings from the surface of the brain as well as deep intra-cortical tissue. We used this 3D probe to investigate signal propagation mechanisms between neuronal cells and their responses to stimuli in a 3D fashion. APPROACH: This novel probe leverage 2D thin-film microfabrication technique to combine an epi-cortical (surface) and an intra-cortical (depth) microelectrode arrays (Epi-Intra), that unfold into an origami 3D-like probe during brain implantation. The flexible epi-cortical component conforms to the brain surface while the intra-cortical array is reinforced with stiffer durimide polymer layer for ease of tissue penetration. The microelectrodes are made of glassy carbon material that is biocompatible and has low electrochemical impedance that is important for high fidelity neuronal recordings. These recordings were performed on the auditory region of anesthetized European starling songbirds during playback of conspecific songs as auditory stimuli. MAIN RESULTS: The Epi-Intra probe recorded broadband activity including local field potentials (LFPs) signals as well as single-unit activity and multi-unit activity from both surface and deep brain. The majority of recorded cellular activities were stimulus-locked and exhibited low noise. Notably, while LFPs recorded on surface and depth electrodes did not exhibit strong correlation, composite receptive fields (CRFs)-extracted from individual neuron cells through a non-linear model and that are cell-dependent-were correlated. SIGNIFICANCE: These findings demonstrate that CRFs extracted from Epi-Intra recordings are excellent candidates for neural coding and for understanding the relationship between sensory neuronal responses and their stimuli (stimulus encoding). Beyond CRFs, this novel neural probe may enable new spatiotemporal 3D volumetric mapping to address, with cellular resolution, how the brain coordinates function.

Glassy carbon microelectrodes minimize induced voltages, mechanical vibrations, and artifacts in magnetic resonance imaging.

The recent introduction of glassy carbon (GC) microstructures supported on flexible polymeric substrates has motivated the adoption of GC in a variety of implantable and wearable devices. Neural probes such as electrocorticography and penetrating shanks with GC microelectrode arrays used for neural signal recording and electrical stimulation are among the first beneficiaries of this technology. With the expected proliferation of these neural probes and potential clinical adoption, the magnetic resonance imaging (MRI) compatibility of GC microstructures needs to be established to help validate this potential in clinical settings. Here, we present GC microelectrodes and microstructures-fabricated through the carbon micro-electro-mechanical systems process and supported on flexible polymeric substrates-and carry out experimental measurements of induced vibrations, eddy currents, and artifacts. Through induced vibration, induced voltage, and MRI experiments and finite element modeling, we compared the performances of these GC microelectrodes against those of conventional thin-film platinum (Pt) microelectrodes and established that GC microelectrodes demonstrate superior magnetic resonance compatibility over standard metal thin-film microelectrodes. Specifically, we demonstrated that GC microelectrodes experienced no considerable vibration deflection amplitudes and minimal induced currents, while Pt microelectrodes had significantly larger currents. We also showed that because of their low magnetic susceptibility and lower conductivity, the GC microelectrodes caused almost no susceptibility shift artifacts and no eddy-current-induced artifacts compared to Pt microelectrodes. Taken together, the experimental, theoretical, and finite element modeling establish that GC microelectrodes exhibit significant MRI compatibility, hence demonstrating clear clinical advantages over current conventional thin-film materials, further opening avenues for wider adoption of GC microelectrodes in chronic clinical applications.

Ultra-Capacitive Carbon Neural Probe Allows Simultaneous Long-Term Electrical Stimulations and High-Resolution Neurotransmitter Detection.

We present a new class of carbon-based neural probes that consist of homogeneous glassy carbon (GC) microelectrodes, interconnects and bump pads. These electrodes have purely capacitive behavior with exceptionally high charge storage capacity (CSC) and are capable of sustaining more than 3.5 billion cycles of bi-phasic pulses at charge density of 0.25 mC/cm2. These probes enable both high SNR (>16) electrical signal recording and remarkably high-resolution real-time neurotransmitter detection, on the same platform. Leveraging a new 2-step, double-sided pattern transfer method for GC structures, these probes allow extended long-term electrical stimulation with no electrode material corrosion. Cross-section characterization through FIB and SEM imaging demonstrate strong attachment enabled by hydroxyl and carbonyl covalent bonds between GC microstructures and top insulating and bottom substrate layers. Extensive in-vivo and in-vitro tests confirmed: (i) high SNR (>16) recordings, (ii) highest reported CSC for non-coated neural probe (61.4 ± 6.9 mC/cm2), (iii) high-resolution dopamine detection (10 nM level - one of the lowest reported so far), (iv) recording of both electrical and electrochemical signals, and (v) no failure after 3.5 billion cycles of pulses. Therefore, these probes offer a compelling multi-modal platform for long-term applications of neural probe technology in both experimental and clinical neuroscience.

In Vivo Dopamine Detection and Single Unit Recordings Using Intracortical Glassy Carbon Microelectrode Arrays.

In this study, we present a 4-channel intracortical glassy carbon (GC) microelectrode array on a flexible substrate for the simultaneous in vivo neural activity recording and dopamine (DA) concentration measurement at four different brain locations (220µm vertical spacing). The ability of GC microelectrodes to detect DA was firstly assessed in vitro in phosphate-buffered saline solution and then validated in vivo measuring spontaneous DA concentration in the Striatum of European Starling songbird through fast scan cyclic voltammetry (FSCV). The capability of GC microelectrode arrays and commercial penetrating metal microelectrode arrays to record neural activity from the Caudomedial Neostriatum of European starling songbird was compared. Preliminary results demonstrated the ability of GC microelectrodes in detecting neurotransmitters release and recording neural activity in vivo. GC microelectrodes array may, therefore, offer a new opportunity to understand the intimate relations linking electrophysiological parameters with neurotransmitters release.

Multilayer poly(3,4-ethylenedioxythiophene)-dexamethasone and poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate-carbon nanotubes coatings on glassy carbon microelectrode arrays for controlled drug release.

The authors present an electrochemically controlled, drug releasing neural interface composed of a glassy carbon (GC) microelectrode array combined with a multilayer poly(3,4-ethylenedioxythiophene) (PEDOT) coating. The system integrates the high stability of the GC electrode substrate, ideal for electrical stimulation and electrochemical detection of neurotransmitters, with the on-demand drug-releasing capabilities of PEDOT-dexamethasone compound, through a mechanically stable interlayer of PEDOT-polystyrene sulfonate (PSS)-carbon nanotubes (CNT). The authors demonstrate that such interlayer improves both the mechanical and electrochemical properties of the neural interface, when compared with a single PEDOT-dexamethasone coating. Moreover, the multilayer coating is able to withstand 10 × 106 biphasic pulses and delamination test with negligible change to the impedance spectra. Cross-section scanning electron microscopy images support that the PEDOT-PSS-CNT interlayer significantly improves the adhesion between the GC substrate and PEDOT-dexamethasone coating, showing no discontinuities between the three well-interconnected layers. Furthermore, the multilayer coating has superior electrochemical properties, in terms of impedance and charge transfer capabilities as compared to a single layer of either PEDOT coating or the GC substrate alone. The authors verified the drug releasing capabilities of the PEDOT-dexamethasone layer when integrated into the multilayer interface through repeated stimulation protocols in vitro, and found a pharmacologically relevant release of dexamethasone.

Highly Stable Glassy Carbon Interfaces for Long-Term Neural Stimulation and Low-Noise Recording of Brain Activity.

We report on the superior electrochemical properties, in-vivo performance and long term stability under electrical stimulation of a new electrode material fabricated from lithographically patterned glassy carbon. For a direct comparison with conventional metal electrodes, similar ultra-flexible, micro-electrocorticography (µ-ECoG) arrays with platinum (Pt) or glassy carbon (GC) electrodes were manufactured. The GC microelectrodes have more than 70% wider electrochemical window and 70% higher CTC (charge transfer capacity) than Pt microelectrodes of similar geometry. Moreover, we demonstrate that the GC microelectrodes can withstand at least 5 million pulses at 0.45 mC/cm2 charge density with less than 7.5% impedance change, while the Pt microelectrodes delaminated after 1 million pulses. Additionally, poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT-PSS) was selectively electrodeposited on both sets of devices to specifically reduce their impedances for smaller diameters (<60 µm). We observed that PEDOT-PSS adhered significantly better to GC than Pt, and allowed drastic reduction of electrode size while maintaining same amount of delivered current. The electrode arrays biocompatibility was demonstrated through in-vitro cell viability experiments, while acute in vivo characterization was performed in rats and showed that GC microelectrode arrays recorded somatosensory evoked potentials (SEP) with an almost twice SNR (signal-to-noise ratio) when compared to the Pt ones.

Investigation Into the Effects of Nucleotide Content on the Electrical Characteristics of DNA Plasmid Molecular Wires.

In this study, we investigate the effect of nucleotide content on the conductivity of plasmid length DNA molecular wires covalently bound to high aspect-ratio gold electrodes. The DNA wires were all between [Formula: see text] in length (>6000bp), and contained either 39%, 53%, or 64% GC base-pairs. We compared the current-voltage (I-V) and frequency-impedance characteristics of the DNA wires with varying GC content, and observed statistically significantly higher conductivity in DNA wires containing higher GC content in both AC and DC measurement methods. Additionally, we noted that the conductivity decreased as a function of time for all DNA wires, with the impedance at 100 Hz nearly doubling over a period of seven days. All readings were taken in humidity and temperature controlled environments on DNA wires suspended above an insulative substrate, thus minimizing the effect of experimental and environmental factors as well as potential for nonlinear alternate DNA confirmations. While other groups have studied the effect of GC content on the conductivity of nanoscale DNA molecules (<50bp), we were able to demonstrate that nucleotide content can affect the conductivity of micrometer length DNA wires at scales that may be required during the fabrication of DNA-based electronics. Furthermore, our results provide further evidence that many of the charge transfer theories developed from experiments using nanoscale DNA molecules may still be applicable for DNA wires at the micro scale.

AC electrical characterisation and insight to charge transfer mechanisms in DNA molecular wires through temperature and UV effects.

In this study, AC characterisation of DNA molecular wires, effects of frequency, temperature and UV irradiation on their conductivity is presented. λ-DNA molecular wires suspended between high aspect-ratio electrodes exhibit highly frequency-dependent conductivity that approaches metal-like behaviour at high frequencies (∼MHz). Detailed temperature dependence experiments were performed that traced the impedance response of λ-DNA until its denaturation. UV irradiation experiments where conductivity was lost at higher and longer UV exposures helped to establish that it is indeed λ-DNA molecular wires that generate conductivity. The subsequent renaturation of λ-DNA resulted in the recovery of current conduction, providing yet another proof of the conducting DNA molecular wire bridge. The temperature results also revealed hysteretic and bi-modal impedance responses that could make DNA a candidate for nanoelectronics components like thermal transistors and switches. Further, these experiments shed light on the charge transfer mechanism in DNA. At higher temperatures, the expected increase in thermal-induced charge hopping may account for the decrease in impedance supporting the 'charge hopping mechanism' theory. UV light, on the other hand, causes damage to GC base-pairs and phosphate groups reducing the path available both for hopping and short-range tunneling mechanisms, and hence increasing impedance--this again supporting both the 'charge hopping' and 'tunneling' mechanism theories.