David A. Borkholder

Plasma-etched neural probes

Abstract A new method is presented for microfabricating silicon-based neural probes that are designed for neurobiology research. Such probes provide unique capabilities to record high-resolution signals simultaneously from multiple, precisely defined locations within neural tissue. The fabrication process utilizes a plasma etch to define the probe outline, resulting in sharp tips and compatibility with standard CMOS processes. A low-noise amplifier array has been fabricated through the MOSIS service to complete a system that has been used in multiple successful physiological experiments.

Portable cell-based biosensor system for toxin detection

Abstract A portable cell-based biosensor has been developed and characterized. The prototype system relies on extracellular recording from excitable cells cultured over an array of platinized gold microelectrodes. Extracellular potentials were bandpass filtered between 80 Hz to 2.8 kHz and amplified with a selectable gain of either 1000 or 5000. The input-referred noise level of the system was only 8.7 μV RMS in the laboratory setting, reaching only 10.6 μV RMS in an outdoor environment, more than sufficient for measurement of extracellular potentials from excitable cells. The system also incorporates a feedback control system for temperature regulation and a 36-channel multiplexer for selection of up to four output channels for simultaneous display. Wherever possible, low-cost ‘off-the-shelf’ components were utilized in this prototype biosensor design. Using this system, extracellular recordings from chick myocardiocytes were performed under both laboratory and outdoor conditions.

Microelectrode arrays for stimulation of neural slice preparations

A planar 6 x 6 array of iridium electrodes with four reference electrodes has been developed for use with neural tissue preparations. Precise knowledge of the relative locations of the array elements allows for spatial neurophysiological analyses. The 10 microns diameter platinized iridium electrodes on a 100 microns pitch have been used to stimulate acutely prepared slices of spinal cord from free-ranging rodents. An intracellular recording from a single neuron in the substantia gelatinosa (SG) using the whole-cell, tight-seal technique allowed low noise, high resolution studies of excitatory or inhibitory electrical responses of a given neuron to inputs from the primary afferent fibers or from stimulation by individual electrodes of the array. The resulting maps of responses provide an indication of the interconnectivity of neural processes. The pattern emerging is that of limited interconnectivity in the SG from areas surrounding a recorded neuron but with strong excitatory or inhibitory effects from those oriented in a longitudinal (rostral-caudal) direction relative to the neuron. The observations to date suggest the neurons of the SG are arranged in sets of independent networks, possibly related to sensory modality and input from particular body regions.

Description and demonstration of a CMOS amplifier-based-system with measurement and stimulation capability for bioelectrical signal transduction

An extracellular recording system incorporating an electrode array and an amplifier/stimulator CMOS chip is described and characterized. Important features of this custom VLSI chip include 16 instrumentation amplifiers with a gain of 50 and the incorporation of a cross-point array allowing designation of an extracellular microelectrode as either a stimulator or sensor. The planar array consisted of 32 microelectrodes, 14 microns in diameter, and four larger reference electrodes. Microelectrodes, interconnecting traces, and bond pads were patterned with a 500-nm layer of gold. The interconnecting traces were passivated with a 1-micron thick layer of silicon nitride to provide chemical and electrical insulation and microelectrode impedance was lowered utilizing electrode position of platinum black. The amplifier exhibited a nearly flat frequency response with high pass and low pass corner frequencies of 0.7 Hz and 50 kHz, respectively. The input referred noise over the 50 kHz bandwidth was 12-16 microVRMS, well below the magnitude of previously reported extracellular potentials. Crosstalk between neighboring channels resulted in an output signal below the amplifier noise level, even for relatively large extracellular potentials. Using this system, extracellular recording were demonstrated yielding typical peak-to-peak biopotentials of magnitude 0.9-2.1 mV and 100-400 microV for chick cardiac myocytes and rat spinal cord neurons, respectively. The key components of this extracellular recording system can be manufactured using industry standard thin film photolithographic techniques.

Cell-based sensor microelectrode array characterized by imaging x-ray photoelectron spectroscopy, scanning electron microscopy, impedance measurements, and extracellular recordings

We are developing a cell-based biosensor consisting of a planar microelectrode array that allows detection of extracellular potentials and their modulation in the presence of toxins or other active agents. To improve cell–electrode coupling, the microelectrodes were electroplated with platinum black. We report on the use of imaging x-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), impedance measurements, and extracellular recordings to assess the effectiveness of this procedure. SEM provided highly detailed images of the shape and structure of well-formed deposits of thickness on the order of 1 μm or more. Because of its inherent high surface sensitivity, imaging XPS could reveal the presence of platinum deposits that were too thin to be detected by SEM. For typical, well-plated microelectrodes, impedance measurements showed reductions in the electrical resistance at 100 Hz from roughly 60 MΩ or more 1 MΩ. The overall electronic coupling of biopotentials to the microelectrodes wa...

Microsystems Technologies for Drug Delivery to the Inner Ear

The inner ear represents one of the most technologically challenging targets for local drug delivery, but its clinical significance is rapidly increasing. The prevalence of sensorineural hearing loss and other auditory diseases, along with balance disorders and tinnitus, has spurred broad efforts to develop therapeutic compounds and regenerative approaches to treat these conditions, necessitating advances in systems capable of targeted and sustained drug delivery. The delicate nature of hearing structures combined with the relative inaccessibility of the cochlea by means of conventional delivery routes together necessitate significant advancements in both the precision and miniaturization of delivery systems, and the nature of the molecular and cellular targets for these therapies suggests that multiple compounds may need to be delivered in a time-sequenced fashion over an extended duration. Here we address the various approaches being developed for inner ear drug delivery, including micropump-based devices, reciprocating systems, and cochlear prosthesis-mediated delivery, concluding with an analysis of emerging challenges and opportunities for the first generation of technologies suitable for human clinical use. These developments represent exciting advances that have the potential to repair and regenerate hearing structures in millions of patients for whom no currently available medical treatments exist, a situation that requires them to function with electronic hearing augmentation devices or to live with severely impaired auditory function. These advances also have the potential for broader clinical applications that share similar requirements and challenges with the inner ear, such as drug delivery to the central nervous system.

High-performance, low-voltage electroosmotic pumps with molecularly thin silicon nanomembranes

Significance Electroosmotic pumps (EOPs) are a class of pumps in which fluid is driven through a capillary or porous media within an electric field. Current research on EOPs concerns the development of new materials in which high electroosmotic flow rates can be achieved for low voltages. Such pumps could be used for portable microfluidic devices. Porous nanocrystalline silicon (pnc-Si) is a material that is formed into a 15-nm-thick nanomembrane. pnc-Si membranes are shown here to have high electroosmotic flow rates at low applied voltages due to the high electric fields achieved over the ultrathin membrane. A prototype EOP was designed using pnc-Si membranes and shown to pressurize fluid through capillary tubing at voltages as low as 250 mV. We have developed electroosmotic pumps (EOPs) fabricated from 15-nm-thick porous nanocrystalline silicon (pnc-Si) membranes. Ultrathin pnc-Si membranes enable high electroosmotic flow per unit voltage. We demonstrate that electroosmosis theory compares well with the observed pnc-Si flow rates. We attribute the high flow rates to high electrical fields present across the 15-nm span of the membrane. Surface modifications, such as plasma oxidation or silanization, can influence the electroosmotic flow rates through pnc-Si membranes by alteration of the zeta potential of the material. A prototype EOP that uses pnc-Si membranes and Ag/AgCl electrodes was shown to pump microliter per minute-range flow through a 0.5-mm-diameter capillary tubing with as low as 250 mV of applied voltage. This silicon-based platform enables straightforward integration of low-voltage, on-chip EOPs into portable microfluidic devices with low back pressures.

Murine Intracochlear Drug Delivery: Reducing Concentration Gradients within the Cochlea

Direct delivery of compounds to the mammalian inner ear is most commonly achieved by absorption or direct injection through the round window membrane (RWM), or infusion through a basal turn cochleostomy. These methods provide direct access to cochlear structures, but with a strong basal-to-apical concentration gradient consistent with a diffusion-driven distribution. This gradient limits the efficacy of therapeutic approaches for apical structures, and puts constraints on practical therapeutic dose ranges. A surgical approach involving both a basal turn cochleostomy and a posterior semicircular canal canalostomy provides opportunities for facilitated perfusion of cochlear structures to reduce concentration gradients. Infusion of fixed volumes of artificial perilymph (AP) and sodium salicylate were used to evaluate two surgical approaches in the mouse: cochleostomy-only (CO), or cochleostomy-plus-canalostomy (C+C). Cochlear function was evaluated via closed-system distortion product otoacoustic emissions (DPOAE) threshold level measurements from 8 to 49 kHz. AP infusion confirmed no surgical impact to auditory function, while shifts in DPOAE thresholds were measured during infusion of salicylate and AP (washout). Frequency dependent shifts were compared for the CO and C+C approaches. Computer simulations modeling diffusion, volume flow, interscala transport, and clearance mechanisms provided estimates of drug concentration as a function of cochlear position. Simulated concentration profiles were compared to frequency-dependent shifts in measured auditory responses using a cochlear tonotopic map. The impact of flow rate on frequency dependent DPOAE threshold shifts was also evaluated for both surgical approaches. Both the C+C approach and a flow rate increase were found to provide enhanced response for lower frequencies, with evidence suggesting the C+C approach reduces concentration gradients within the cochlea.

Round window membrane intracochlear drug delivery enhanced by induced advection.

Delivery of therapeutic compounds to the inner ear via absorption through the round window membrane (RWM) has advantages over direct intracochlear infusions; specifically, minimizing impact upon functional hearing measures. However, previous reports show that significant basal-to-apical concentration gradients occur, with the potential to impact treatment efficacy. Here we present a new approach to inner ear drug delivery with induced advection aiding distribution of compounds throughout the inner ear in the murine cochlea. Polyimide microtubing was placed near the RWM niche through a bullaostomy into the middle ear cavity allowing directed delivery of compounds to the RWM. We hypothesized that a posterior semicircular canalostomy would induce apical flow from the patent cochlear aqueduct to the canalostomy due to influx of cerebral spinal fluid. To test this hypothesis, young adult CBA/CaJ mice were divided into two groups: bullaostomy approach only (BA) and bullaostomy+canalostomy (B+C). Cochlear function was evaluated by distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) thresholds during and after middle ear infusion of salicylate in artificial perilymph (AP), applied near the RWM. The mice recovered for 1week, and were re-tested. The results demonstrate there was no significant impact on auditory function utilizing the RWM surgical procedure with or without the canalostomy, and DPOAE thresholds were elevated reversibly during the salicylate infusion. Comparing the threshold shifts for both methods, the B+C approach had more of a physiological effect than the BA approach, including at lower frequencies representing more apical cochlear locations. Unlike mouse cochleostomies, there was no deleterious auditory functional impact after 1week recovery from surgery. The B+C approach had more drug efficacy at lower frequencies, underscoring potential benefits for more precise control of delivery of inner ear therapeutic compounds.

Implantable micropump technologies for murine intracochlear infusions

Due to the very small size of the mouse inner ear, 600 nL volume, developing effective, controlled infusion systems is quite challenging. Key technologies have been created to minimize both size and power for an implantable pump for murine intracochlear infusions. A method for coupling fine capillary tubing to microfluidic channels is presented which provides low volume, biocompatible interconnects withstanding pressures as high as 827 kPa (120 psi) and consuming less than 20 nL of volume exiting in-plane with the pump. Surface micromachined resistive bridges integrated into the flow channel for anemometry based flow rate measurement have been optimized for low power operation in the ultra-low flow rate regime. A process for creation of deformable diaphragms over pump chambers with simultaneous coating of the microfluidic channels has been developed allowing integration of a biocompatible fluid flow path. These advances represent enabling capabilities for a drug delivery system suitable for space constrained applications such as subcutaneous implantation in mice.