Nanoporous Gold Biointerfaces: Modifying Nanostructure to Control Neural Cell Coverage and Enhance Electrophysiological Recording Performance.

Nanostructured neural interface coatings have significantly enhanced recording fidelity in both implantable and in vitro devices. As such, nano-porous gold (np-Au) has shown promise as a multifunctional neural interface coating due, in part, to its ability to promote nanostructure-mediated reduction in astrocytic surface coverage while not affecting neuronal coverage. The goal of this study is to provide insight into the mechanisms by which the np-Au nanostructure drives the differential response of neurons versus astrocytes in an in vitro model. Utilizing microfabricated libraries that display varying feature sizes of np-Au, it is demonstrated that np-Au influ-ences neural cell coverage through modulating focal adhesion formation in a feature size-dependent manner. The results here show that surfaces with small (≈30 nm) features control astrocyte spreading through inhibition of focal adhesion formation, while surfaces with large (≈170 nm and greater) features control astrocyte spreading through other mechanotransduction mechanisms. This cellular response combined with lower electrical impedance of np-Au electrodes significantly enhances the fidelity and stability of electrophysiological recordings from cortical neuronglia co-cultures relative to smooth gold electrodes. Finally, by leveraging the effect of nanostructure on neuronal versus glial cell attachment, the use of laser-based nanostructure modulation is demonstrated for selectively patterning neurons with micrometer spatial resolution.

Utilizing dynamic laser speckle to probe nanoscale morphology evolution in nanoporous gold thin films.

This paper demonstrates the use of dynamic laser speckle autocorrelation spectroscopy in conjunction with the photothermal treatment of nanoporous gold (np-Au) thin films to probe nanoscale morphology changes during the photothermal treatment. Utilizing this spectroscopy method, backscattered speckle from the incident laser is tracked during photothermal treatment and both the characteristic feature size and annealing time of the film are determined. These results demonstrate that this method can successfully be used to monitor laser-based surface modification processes without the use of ex-situ characterization.

Substrate Topography Guides Pore Morphology Evolution in Nanoporous Gold Thin Films.

This paper illustrates the effect of substrate topography on morphology evolution in nanoporous gold (np-Au) thin films. One micron-high silicon ridges with widths varying between 150 nm to 50 µm were fabricated and coated with 500 nm-thick np-Au films obtained by dealloying sputtered gold-silver alloy films. Analysis of scanning electron micrographs of the np-Au films following dealloying and thermal annealing revealed two distinct regimes where the ratio of film thickness to ridge width determines the morphological evolution of np-Au films.

Polymer Bioelectronics: A Solution for Both Stimulating and Recording Electrodes.

The advent of closed-loop bionics has created a demand for electrode materials that are ideal for both stimulating and recording applications. The growing complexity and diminishing size of implantable devices for neural interfaces have moved beyond what can be achieved with conventional metallic electrode materials. Polymeric electrode materials are a recent development based on polymer composites of organic conductors such as conductive polymers. These materials present exciting new opportunities in the design and fabrication of next-generation electrode arrays which can overcome the electrochemical and mechanical limitations of conventional electrode materials. This review will examine the recent developments in polymeric electrode materials, their application as stimulating and recording electrodes in bionic devices, and their impact on the development of soft, conformal, and high-density neural interfaces.

Electroactive Polymers for On-Demand Drug Release.

Conductive materials have played a significant role in advancing society into the digital era. Such materials are able to harness the power of electricity and are used to control many aspects of daily life. Conductive polymers (CPs) are an emerging group of polymers that possess metal-like conductivity yet retain desirable polymeric features, such as processability, mechanical properties, and biodegradability. Upon receiving an electrical stimulus, CPs can be tailored to achieve a number of responses, such as harvesting energy and stimulating tissue growth. The recent FDA approval of a CP-based material for a medical device has invigorated their research in healthcare. In drug delivery, CPs can act as electrical switches, drug release is achieved at a flick of a switch, thereby providing unprecedented control over drug release. In this review, recent developments in CP as electroactive polymers for voltage-stimuli responsive drug delivery systems are evaluated. The review demonstrates the distinct drug release profiles achieved by electroactive formulations, and both the precision and ease of stimuli response. This level of dynamism promises to yield "smart medicines" and warrants further research. The review concludes by providing an outlook on electroactive formulations in drug delivery and highlighting their integral roles in healthcare IoT.

A Pilot In Vivo Study of Flexible Fully Polymeric Nerve Cuff Electrodes.

Recent trends in the field of bioelectronics have been focused on the development of electrodes that facilitate safe and efficient stimulation of nervous tissues. Novel conducting polymer (CP) based materials, such as flexible and fully polymeric conductive elastomers (CEs), constitute a promising alternative to improve on the limitations of current metallic devices. This pilot study demonstrates the performance of tripolar CE-based peripheral nerve cuffs compared to current commercial tripolar platinum-iridium (PtIr) nerve cuffs in vivo. CE and metallic cuff devices were implanted onto rodent sciatic nerves for a period of 8 weeks. Throughout the entire study, the CE device demonstrated improved charge transfer and electrochemical safety compared to the PtIr cuff, able to safely inject 2 to 3 times more charge. In comparison to the commercial control, the CE cuff was able to record in the in vivo setting with reduced noise and produced smaller voltages at all simulation levels. CE technologies provide a promising alternative to metallic devices for the development of bioelectronics with enhanced chronic device functionality.

Controlled electroactive release from solid-state conductive elastomer electrodes.

This work highlights the development of a conductive elastomer (CE) based electrophoretic platform that enables the transfer of charged molecules from a solid-state CE electrode directly to targeted tissues. Using an elastomer-based electrode containing poly (3,4-ethylenedioxythiophene) nanowires, controlled electrophoretic delivery of methylene blue (MB) and fluorescein (FLSC) was achieved with applied voltage. Electroactive release of positively charged MB and negatively charged FLSC achieved 33.19 ± 6.47 µg release of MB and 22.36 ± 3.05 µg release of FLSC, a 24 and 20-fold increase in comparison to inhibitory voltages over 1 h. Additionally, selective, and sequential release of the two oppositely charged molecules from a single CE device was demonstrated, showing the potential of this device to be used in multi-drug treatments.

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology.

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. This work describes the preparation of rat sciatic nerves for ex vivo neurophysiology, including buffer preparation, animal procedures, equipment setup and neurophysiological recording. This work provides an overview of the different types of experiments possible with this method. The outlined method aims to provide 6 h of stimulation and recording on extracted peripheral nerve tissue in tightly controlled conditions for optimal consistency in results. Results obtained using this method are A-fibre compound action potentials (CAP) with peak-to-peak amplitudes in the millivolt range over the entire duration of the experiment. CAP amplitudes and shapes are consistent and reliable, making them useful to test and compare new electrodes to existing models, or the effects of interventions on the tissue, such as the use of chemicals, surgical alterations, or neuromodulatory stimulation techniques. Both conventional commercially available cuff electrodes with platinum-iridium contacts and custom-made conductive elastomer electrodes were tested and gave similar results in terms of nerve stimulus strength-duration response.

Mind the gap: State-of-the-art technologies and applications for EEG-based brain-computer interfaces.

Brain-computer interfaces (BCIs) provide bidirectional communication between the brain and output devices that translate user intent into function. Among the different brain imaging techniques used to operate BCIs, electroencephalography (EEG) constitutes the preferred method of choice, owing to its relative low cost, ease of use, high temporal resolution, and noninvasiveness. In recent years, significant progress in wearable technologies and computational intelligence has greatly enhanced the performance and capabilities of EEG-based BCIs (eBCIs) and propelled their migration out of the laboratory and into real-world environments. This rapid translation constitutes a paradigm shift in human-machine interaction that will deeply transform different industries in the near future, including healthcare and wellbeing, entertainment, security, education, and marketing. In this contribution, the state-of-the-art in wearable biosensing is reviewed, focusing on the development of novel electrode interfaces for long term and noninvasive EEG monitoring. Commercially available EEG platforms are surveyed, and a comparative analysis is presented based on the benefits and limitations they provide for eBCI development. Emerging applications in neuroscientific research and future trends related to the widespread implementation of eBCIs for medical and nonmedical uses are discussed. Finally, a commentary on the ethical, social, and legal concerns associated with this increasingly ubiquitous technology is provided, as well as general recommendations to address key issues related to mainstream consumer adoption.

Stretchable, Fully Polymeric Electrode Arrays for Peripheral Nerve Stimulation.

There is a critical need to transition research level flexible polymer bioelectronics toward the clinic by demonstrating both reliability in fabrication and stable device performance. Conductive elastomers (CEs) are composites of conductive polymers in elastomeric matrices that provide both flexibility and enhanced electrochemical properties compared to conventional metallic electrodes. This work focuses on the development of nerve cuff devices and the assessment of the device functionality at each development stage, from CE material to fully polymeric electrode arrays. Two device types are fabricated by laser machining of a thick and thin CE sheet variant on an insulative polydimethylsiloxane substrate and lamination into tubing to produce pre-curled cuffs. Device performance and stability following sterilization and mechanical loading are compared to a state-of-the-art stretchable metallic nerve cuff. The CE cuffs are found to be electrically and mechanically stable with improved charge transfer properties compared to the commercial cuff. All devices are applied to an ex vivo whole sciatic nerve and shown to be functional, with the CE cuffs demonstrating superior charge transfer and electrochemical safety in the biological environment.

Flexible Nanowire Conductive Elastomers for Applications in Fully Polymeric Bioelectronic Devices.

Soft, flexible polymer-based bioelectronics are a promising approach to minimize the chronic inflammatory reactions associated with metallic devices, impairing long-term device reliability and functionality. This work demonstrates the fabrication of conductive elastomers (CEs) consisting of chemically synthesized poly(3,4-ethylenedioxythiophene) (PEDOT) nanowires embedded within a polyurethane (PU) elastomeric matrix, resulting in soft and flexible, fully polymeric electrode materials. Increasing PEDOT nanowire loadings resulted in an improvement in electrochemical properties and conductivity, an increased Young's modulus and reduced strain at failure. Nanowire CEs were also found to have significantly improved electrochemical performance compared to one of the standard electrode materials, platinum (Pt). Indirect in vitro cytocompatibility test was carried out to investigate the effect of leachable substances from the CE on primary rodent cells. Nanowire CEs provide a promising alternative to metals for the fabrication of soft bioelectronics.

Model-based geometrical optimisation and in vivo validation of a spatially selective multielectrode cuff array for vagus nerve neuromodulation.

BACKGROUND: Neuromodulation by electrical stimulation of the human cervical vagus nerve may be limited by adverse side effects due to stimulation of off-target organs. It may be possible to overcome this by spatially selective stimulation of peripheral nerves. Preliminary studies have shown this is possible using a cylindrical multielectrode human-sized nerve cuff in vagus nerve selective neuromodulation. NEW METHOD: The model-based optimisation method for multi-electrode geometric design is presented. The method was applied for vagus nerve cuff array and suggested two rings of 14 electrodes, 3 mm apart, with 0.4 mm electrode width and separation and length 0.5-3 mm, with stimulation through a pair in the same radial position on the two rings. The electrodes were fabricated using PDMS-embedded stainless steel foil and PEDOT: pTS coating. RESULTS: In the cervical vagus nerve in anaesthetised sheep, it was possible to selectively reduce the respiratory breath rate (RBR) by 85 ± 5% without affecting heart rate, or selectively reduce heart rate (HR) by 20 ± 7% without affecting respiratory rate. The cardiac- and pulmonary-specific sites on the nerve cross-sectional perimeter were localised with a radial separation of 105 ± 5 degrees (P < 0.01, N = 24 in 12 sheep). CONCLUSIONS: Results suggest organotopic or function-specific organisation of neural fibres in the cervical vagus nerve. The optimised electrode array demonstrated selective electrical neuromodulation without adverse side effects. It may be possible to translate this to improved treatment by electrical autonomic neuromodulation for currently intractable conditions.

Optimisation of bioimpedance measurements of neuronal activity with an ex vivo preparation of Cancer pagurus peripheral nerves.

BACKGROUND: In mammals, fast neural Electrical Impedance Tomography (EIT) can image the myelinated component of the compound action potentials (CAP) using a nerve cuff. If applied to unmyelinated fibres this has great potential to improve selective neuromodulation ("electroceuticals") to avoid off-target effects. Previously, bioimpedance recordings were averaged from unmyelinated crab leg nerve fibres, but the signal to noise ratio (SNR) needs improving. NEW METHOD: Currently, functional non-invasive neuronal imaging is accomplished through surface electrodes or genetically expressed indicators that provide good spatial, but poor temporal, resolution. Here is an improved method for bioimpedance measurements from a model of unmyelinated fibres to enable optimisation through improvement of the 1) signal processing measurement paradigm, 2) neurophysiology, and 3) electrode-nerve interface. RESULTS: For bioimpedance recordings, the recruitment and necessity of the CAP was quantified and saline significantly improved the SNR. An improved protocol resulted in averaging not being required, as sequentially recorded traces produced bioimpedance changes of -0.232 ± 0.064% that did not show phase or timing related artefacts. COMPARISON WITH EXISTING METHOD: Here, two bioimpedance traces displayed an SNR of ≥3:1, while previously over >100 averages were required with greater inter-experimental variability. 10 paired traces were averaged for an SNR of ≥9:1, or near real-time measurement. CONCLUSIONS: This method facilitates further studies aiming to enable non-invasive localization of fascicular activity in unmyelinated fibres within peripheral nerves. This technique could ultimately produce the first 3-D tomographic images to help guide selective neuromodulation using bioelectric devices.

Electrode fabrication and interface optimization for imaging of evoked peripheral nervous system activity with electrical impedance tomography (EIT).

OBJECTIVE: Non-invasive imaging techniques are undoubtedly the ideal methods for continuous monitoring of neural activity. One such method, fast neural electrical impedance tomography (EIT) has been developed over the past decade in order to image neural action potentials with non-penetrating electrode arrays. APPROACH: The goal of this study is two-fold. First, we present a detailed fabrication method for silicone-based multiple electrode arrays which can be used for epicortical or neural cuff applications. Secondly, we optimize electrode material coatings in order to achieve the best accuracy in EIT reconstructions. MAIN RESULTS: The testing of nanostructured electrode interface materials consisting of platinum, iridium oxide, and PEDOT:pTS in saline tank experiments demonstrated that the PEDOT:pTS coating used in this study leads to more accurate reconstruction dimensions along with reduced phase separation between recording channels. The PEDOT:pTS electrodes were then used in vivo to successfully image and localize the evoked activity of the recurrent laryngeal fascicle from within the cervical vagus nerve. SIGNIFICANCE: These results alongside the simple fabrication method presented here position EIT as an effective method to image neural activity.

Nanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity.

Quantitative analysis of fluorescence signals from cells reacted with fluorescently labeled probes is a widely-used method for assessing cell biology. This method has become especially powerful for screening novel nanostructured materials for their influence on cell behavior. However, the effect of nanostructured surface on fluorescence intensity has largely been ignored, which likely leads to erroneous conclusions about cell behavior. This paper investigates this possibility by using fibroblasts cultured on nanoporous gold (np-Au) as a model nanostructured material system. We found that fibroblasts stained for f-actin using phalloidin conjugated with common fluorophores display different levels of fluorescence on np-Au, planar gold, and glass, suggesting different levels of f-actin composition. However, direct quantification via western blots indicates that the actin expression is the same across all conditions. We further investigated whether the fluorescence intensity depended on np-Au feature size, complementing the findings with reflection dark field measurements from different np-Au surfaces. Overall, our experimental measurements in agreement with our electrodynamic simulations suggest that nanostructured surfaces alter the fluorescence intensity of fluorophores by modulating both the excitation and light emission processes. We conclude that comparison of fluorescence on materials with different nanostructures should be done with a quantification method decoupled from the nanostructure's influence.

Engineering on-chip nanoporous gold material libraries via precision photothermal treatment.

Libraries of nanostructured materials on a single chip are a promising platform for high throughput and combinatorial studies of structure-property relationships in the fields of physics and biology. Nanoporous gold (np-Au), produced by an alloy corrosion process, is a nanostructured material specifically suited for such studies because of its self-similar thermally induced coarsening behavior. However, traditional heat application techniques for the modification of np-Au are bulk processes that cannot be used to generate a library of different pore sizes on a single chip. Here, laser micro-processing offers an attractive solution to this problem by providing a means to apply energy with high spatial and temporal resolution. In the present study we use finite element multiphysics simulations to predict the effects of laser mode (continuous-wave vs. pulsed) and thermal conductivity of the supporting substrate on the local np-Au film temperatures during photothermal annealing. Based on these results we discuss the mechanisms by which the np-Au network is coarsened. Thermal transport simulations predict that continuous-wave mode laser irradiation of np-Au thin films on a silicon substrate supports the widest range of morphologies that can be created through photothermal annealing of np-Au. Using the guidance provided by simulations, we successfully fabricate an on-chip material library consisting of 81 np-Au samples of 9 different morphologies for use in the parallel study of structure-property relationships.

Mechanisms of Reduced Astrocyte Surface Coverage in Cortical Neuron-Glia Co-cultures on Nanoporous Gold Surfaces.

Nanoporous gold (np-Au) is a promising multifunctional material for neural electrodes. We have previously shown that np-Au nanotopography reduces astrocyte surface coverage (linked to undesirable gliosis) while maintaining high neuronal coverage in a cortical primary neuron-glia co-culture model as long as two weeks in vitro. Here, we investigate the potential influence of secreted soluble factors from cells grown on np-Au on the cell type-specific surface coverage of cells grown on conventional tissue culture plastic and test the hypothesis that secretion of factors is responsible for inhibiting astrocyte coverage on np-Au. In order to assess whether factors secreted from cells grown on np-Au surfaces reduced surface coverage by astrocytes, we seeded fresh primary rat neuron-glia co-cultures on conventional polystyrene culture dishes, but maintained the cells in conditioned media from co-cultures grown on np-Au surfaces. After one week in vitro, a preferential reduction in astrocyte surface coverage was not observed, suggesting that soluble factors are not playing a role. In contrast, four hours after cell seeding there were a significant number of non-adhered, yet still viable, cells for the cultures on np-Au surfaces. We hypothesize that the non-adherent cells are mainly astrocytes, because: (i) there was no difference in neuronal cell coverage between np-Au and pl-Au for long culture durations and (ii) neurons are post-mitotic and not expected to increase in number upon attaching to the surface. Overall, the results suggest that the np-Au topography leads to preferential neuronal attachment shortly after cell seeding and limits astrocyte-specific np-Au surface coverage at longer culture durations.

Nanoporous gold as a neural interface coating: effects of topography, surface chemistry, and feature size.

Designing neural interfaces that maintain close physical coupling of neurons to an electrode surface remains a major challenge for both implantable and in vitro neural recording electrode arrays. Typically, low-impedance nanostructured electrode coatings rely on chemical cues from pharmaceuticals or surface-immobilized peptides to suppress glial scar tissue formation over the electrode surface (astrogliosis), which is an obstacle to reliable neuron-electrode coupling. Nanoporous gold (np-Au), produced by an alloy corrosion process, is a promising candidate to reduce astrogliosis solely through topography by taking advantage of its tunable length scale. In the present in vitro study on np-Au's interaction with cortical neuron-glia co-cultures, we demonstrate that the nanostructure of np-Au achieves close physical coupling of neurons by maintaining a high neuron-to-astrocyte surface coverage ratio. Atomic layer deposition-based surface modification was employed to decouple the effect of morphology from surface chemistry. Additionally, length scale effects were systematically studied by controlling the characteristic feature size of np-Au through variations in the dealloying conditions. Our results show that np-Au nanotopography, not surface chemistry, reduces astrocyte surface coverage while maintaining high neuronal coverage and may enhance neuron-electrode coupling through nanostructure-mediated suppression of scar tissue formation.