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Nanofluidic technology for chemical neurostimulation

Jones, P. D. (2017).

Dissertation, Mathematisch-Naturwissenschaftliche Fakultät, Eberhard Karls Universität Tübingen. 129 Seiten.

The goal of this work was to develop technology for local chemical stimulation of neurons. Recent years have seen impressive treatment of neurodegenerative diseases by electrical neuroprostheses, including restoration of vision by retinal implants. Despite their success, these prostheses interact by unnatural mechanisms of electrical neurostimulation, which cannot fully mimic biological specificity or resolution. Stimulation by artificial chemical signals could provide a more natural means of restoring neurological functions, but technology for precise, high-resolution chemical release remains primitive in comparison with microelectronics. Current micro- or nanofluidic technology cannot control chemical release with sufficient precision to imitate synaptic release.

This work has focused on two main topics. First, the investigation of hydrophobically gated nanopores has been pursued towards developing precise nanovalves for absolute, diffusion-free control of chemical release. Second, a platform for in vitro chemical stimulation of cells or tissues by chemical release from nanopores integrated with microfluidic control and microelectrodes has been developed. In support of these topics, issues of nanofluidic chemical control and future chemical neuroprostheses have been investigated.

Hydrophobically gated nanopores may provide ideal control for high-resolution chemical release, but previous reports in artificial nanopores suffered from limited reversibility and reproducibility. These challenges were compounded by limited understanding of the physical mechanisms governing the observed behaviour. Extensive literature review was carried out to understand the liquid–vapour behaviour in artificial hydrophobic nanopores. The contributing mechanisms are distinct from hydrophobic gating in biological nanopores with dimensions an order of magnitude smaller. Electrowetting – more specifically, electromechanical force on the liquid–vapour surface – was identified as the principal mechanism. Models of electrowetting in nanopores were developed, including for nanopores with integrated gate electrodes for individual control of nanopores in contact with a shared reservoir. Because reversibility of hydrophobic gating in large hydrophobic nanopores requires trapped bubbles, a novel mechanism was proposed to trap bubbles in circumferential cavities within nanopores. Hydrophobic gating behaviour was investigated by current–voltage recordings of silicon nitride (SiNx) nanopores modified with monofunctional hydrophobic silanes and gold nanopores modified with hydrophobic thiols. Differences observed in SiNx–silane and gold–thiol nanopores demonstrated the importance of material selection for precise, stable nanopore fabrication, and suggested that molecular effects contribute to electrowetting behaviour. Limited stability of thiol-coated gold prevented investigation of electrowetting by integrated electrodes. Through analysis of theoretical and experimental results, next experiments towards reversible hydrophobic gating were proposed.

A nanopore-based in vitro chemical stimulation platform was developed for biological experiments with localized chemical release. A nanopore/microelectrode array (NPMEA) was produced based on established microelectrode arrays (MEAs). MEAs are widely used for in vitro neuroscience research, and established systems for cell culture and tissue preparations can be transferred to the NPMEA. Although many microfluidic platforms for chemical stimulation of cells have been reported, none have reached submicrometre dimensions. Integrating nanofluidic structures introduced unique challenges, but also opened new possibilities for nanofluidic control of chemical release. The NPMEA integrated focusedion-beam-milled nanopores by dry bonding with microfluidic channels produced by photolithography. The process was designed to ensure future compatibility with alternative nanopore designs, such as hydrophobically gated nanopores. The resulting NPMEA has 29 microelectrodes for electrophysiological recording and stimulation, and 30 nanopores individually addressed by microfluidic channels and electrodes. A robust microfluidic connector was produced to connect the 30 microfluidic channels, which surpassed the complexity of many microfluidic applications. The NPMEA was applied in first biological experiments towards proof of local nanopore-based chemical stimulation, but a positive demonstration has not yet been obtained.

This work presents concrete steps towards precise, high-resolution chemical stimulation of neurons. Challenges remain before hydrophobically gated nanopores may be achieved, and nanopore-based chemical release from the developed in vitro system must be verified with biological experiments. The path towards chemical neuroprostheses extends far into the future, but the continuation of this work will be moving in the right direction.

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