Harvey A. Fishman

Ion Channels and Lipid Bilayer Membranes Under High Potentials Using Microfabricated Apertures

Membrane-bound proteins are important from both a scientific and a technological point of view. However, their study and application require a stable lipid bilayer to maintain protein function. Here we provide a method for producing lipid bilayers across apertures on a silicon substrate using a Langmuir–Blodgett technique commonly used with Teflon films. These bilayers display the same properties as those across apertures in Teflon in terms of capacitance, conductance, noise, and protein insertion and function. In addition, the bilayers remain stable at higher transmembrane potentials than those in Teflon, typically remaining unbroken over ±400 mV. These properties are demonstrated by the insertion of the staphylococcal protein pore α-hemolysin into pre-formed bilayers, and subsequent current recording at high potentials (±400 mV).

Building thick photoresist structures from the bottom up

Micromachining has pushed the limits of photolithography in the vertical direction. We demonstrate a new technique for producing structures in thick photoresists, with excellent contact alignment and high aspect ratios using a standard UV source. In this technique, we exposed the resist from the bottom by defining the mask as part of the substrate. By doing so, we successfully created high aspect ratio and multiple-layer structures. Additionally, the structures have a slight inward taper, a very useful feature for molding. This technique is a new way of thinking about photolithography, allowing novel structures and simplified processing.

Patch-Clamp Detection of Neurotransmitters in Capillary Electrophoresis

Gamma-aminobutyrate acid, L-glutamate, and N-methyl-D-aspartate were separated by capillary electrophoresis and detected by the use of whole-cell and outside-out patch-clamp techniques on freshly dissociated rat olfactory interneurons. These neuroactive compounds could be identified from their electrophoretic migration times, unitary channel conductances, and power spectra that yielded corner frequencies and mean single-channel conductances characteristic for each of the different agonist-receptor interactions. This technique has the sensitivity to observe the opening of a single ion channel for agonists separated by capillary electrophoresis.

Towards a Neurotransmitter-Based Retinal Prosthesis Using an Inkjet Print-head

Electronic chips that provide a patterned stimulus to cells in the retina may provide a viable treatment for age-related macular degeneration. A surrogate MEMS device, in the form of a print-head from a desktop printer, has been used to eject a pattern of neurotransmitters (bradykinin) onto living rat pheochromocytoma (PC12) cells. Fluorescent calcium imaging was used to measure the patterned stimulation of individual cells. The chemical stimulation of cells by directed microfluidic delivery may have applications in retinal prosthetic devices, and in other prosthetic implants in the nervous system.

Microcolumn sample injection by spontaneous fluid displacement

The withdrawal of a capillary structure from a sample solution causes a droplet to be formed at the end of the capillary. Because of the interfacial pressure difference across the curved surface of the droplet, the droplet is driven into the entrance of the capillary, thereby causing injection of the sample. Assuming negligible sample penetration by diffusive or convective mixing, this injection is intrinsically the smallest possible for a capillary. Moreover, the injection volume can be varied by changing the shape of the capillary structure, specifically the outer diameter of the capillary. This injection method eliminates the need for external pressure differences, applied fields across the capillary, or precise timing, thus offering several advantages over conventional procedures. Studies using capillary electrophoresis as the separation procedure show that approximately 3.5 nl (66 microns I.D. capillary) sample volumes can be injected by hand with a reproducibility of 5.8 +/- 0.7% R.S.D. Parameters that affect the variability of the injection are discussed.

Carbon Nanotube Bucky Paper as an Artificial Support Membrane for Retinal Cell Transplantation

BACKGROUND AND OBJECTIVE Transplantation of epithelial cells on a substrate to rescue diseased retinal cells is an experimental therapy for age-related macular degeneration. Carbon nanotube bucky paper was tested for cell transplantation into the retina. MATERIALS AND METHODS Bucky paper was prepared and human RPE cells cultured on its surface demonstrating its utility as a cell transplantation substrate. Bucky paper was implanted underneath 9 rabbit retinas using a standard 3-port pars plana vitrectomy and subretinal bleb. A 1 mm retinotomy was created through which Bucky paper precut to fit was inserted with the subretinal forceps, into the subretinal bleb. The retina was reattached by airfluid exchange. RESULTS By light microscopy, RPE cells demonstrated normal morphology and growth patterns on the bucky paper surface. Scanning electron microscopy confirmed a confluent monolayer of cells, and indicated the formation of microvilli on the apical surface. Bucky paper remained flat in the subretinal space after 2 weeks, the retina fully attached without edema or inflammation. CONCLUSION Bucky paper possesses the necessary attributes for therapeutic cell transplantation in the eye.

Fabrication of a carbon nanotube protruding electrode array for a retinal prosthesis

Implantable retinal prosthetic devices consisting of microelectrode arrays are being built in attempts to restore vision. Current retinal prostheses use metal planar electrodes. We are developing a novel electro-neural interface using carbon nanotube (CNT) bundles as flexible, protruding microelectrodes. We have synthesized vertically self-assembled, multi-walled CNT bundles by thermal chemical vapor deposition. Using conventional silicon-based micro-fabrication processes, these CNT bundles were integrated onto pre-patterned circuits. CNT protruding electrodes have significant potentials in providing safer stimulation for retinal prostheses. They could also act as recording units to sense electrical and chemical activities in neural systems for fundamental neuroscience research.

Attracting retinal cells to electrodes for high-resolution stimulation

Development of the electronic retinal prosthesis for restoration of sight in patients suffering from the degenerative retinal diseases faces many technological challenges. To achieve significant improvement in the low vision patients the visual acuity of 20/80 would be desirable, which corresponds to the pixel size of 20μm in the retinal implant. Stimulating current strongly (quadratically) depends on distance between electrode and cell. To achieve uniformity in stimulation thresholds, to avoid erosion of the electrodes and overheating of tissue, and to reduce the cross-talk between the neighboring pixels the neural cells should not be separated from electrodes by more than a few micrometers. Achieving such a close proximity along the whole surface of an implant is one of the major obstacles for the high resolution retinal implant. To ensure proximity of cells and electrodes we have developed a technique that prompts migration of retinal cells towards stimulating sites. The device consists of a multilayered membrane with an array of perforations of several (5-15) micrometers in diameter in which addressable electrodes can be embedded. In experiments in-vitro using explants of the whole retina of P7 rats, and in-vivo using adult rabbits and RCS rats the retinal tissue grew into the pores when membranes were positioned on the sub-retinal side. Histology has demonstrated that migrating cells preserve synaptic connections with cells outside the pores, thus allowing for signal transduction into the retina above the implant. Intimate proximity of cells to electrodes achieved with this technique allows for reduction of the stimulation current to 2μA at the 10μm electrode. A 3mm disk array with 18,000 pixels can stimulate cells with 0.5 ms pulses at 50Hz while maintaining temperature rise at the implant surface below 0.3°C. Such an implant can, in principle, provide spatial resolution geometrically corresponding to the visual acuity of 20/80 in a visual field of 10°.

Pushing the limits of artificial vision

The field of neural prostheses has made significant strides toward restoring damaged or lost nervous system function for millions of people. These advances have led to the development of selective electrical interfaces that communicate directly with nerve cells to modulate neural processes rationally. The first neural prosthesis developed was the cardiac pacemaker, which has become pervasive in society. The pacemaker also provides the promise that such an approach can address higher order neural dysfunctions. Technologies borrowed from the semiconductor industry have allowed researchers to micro fabricate devices with active elements on the length scale of nerve cells. This size capability has led to the development of the cochlear implant, which has restored hearing in thousands of deaf patients. Restoring more complex sensory functions, such is vision, provides greater engineering challenges, but achieving them is within our grasp. Current approaches to achieving artificial vision are discussed, along with strategies being used to push the capability limits of these prostheses even further.

Novel Interface to Biological Systems for Retinal Prosthetics

The development of retinal prostheses requires a method for interconnecting an imaging system to the retina. Such a system must be able to individually address and stimulate retinal neurons, a significant advance from current technology. As a step toward this goal, we present a novel electronic-to-biologic interface using microfabricated apertures in a silicon substrate. Apertures are created in a thin silicon nitride membrane, after which the surface is appropriately modified to support cell growth. Excitable cells are seeded on the device and imaged using Ca 2+ -sensitive fluorescent dyes in either an inverted or confocal microscope. Using rat pheochromocytoma (PC12) cells, we show the ability to stimulate locally through the apertures. The device allows for the stimulation of cells at precise locations, a necessary requirement for future high-resolution retinal prostheses.