UCLA

The ability to monitor simultaneously the electrical activity of neurons and inter-neuronal chemical signaling is expected to lead to an effective means to investigate the complex circuitry underlying brain function. Great advancements have been made in recording electrical activity from large numbers of interconnected neurons concurrently through few-millisecond-timescale measurements. However, corresponding tools to monitor chemical neurotransmission with such high spatiotemporal resolution have yet to emerge. The development of such tools has been particularly challenging because neurotransmitter concentrations are typically low, in the nanomolar to few micromolar ranges. In addition, the neurotransmitter of interest must be detected selectively against the complex background of brain extracellular fluid. The spatiotemporal shortcomings of neurochemical sensors has made problematic the faithful recording of neurotransmitter dynamics, and certainly has made it challenging, if not impossible, to correlate chemical signaling with neuronal activity. In this dissertation, work is presented on the fabrication and characterization of higher performance microsensors for neurotransmitter detection that better address this need.

A key breakthrough described in this thesis was the several-fold improvement in response time and sensitivity of selective electroenzymatic sensors for glutamate and choline (as surrogate for acetylcholine). Previous devices based on microelectrode arrays (MEAs) of electroenzymatic sensing sites on silicon or ceramic microprobes demonstrated the potential utility of such tools for the monitoring of glutamate and other neurotransmitters in vivo during behavioral studies. However, less than optimal sensitivity and response time limited the neuroscience applications of these promising research tools. In this work, the optimization of sensor construction was guided by a detailed mathematical model that prescribed optimal coatings of both permselective films and immobilized enzyme layers so as to maximize performance while maintaining selectivity against interfering species. These design modifications led to glutamate sensors with a ~6-fold sensitivity enhancement, ~10-fold reduction in response time, as well as high-performance choline sensors with remarkable sensitivity and response times in the millisecond range that are near the theoretical performance limits predicted by the model. Importantly, these results were attained without compromising detection limit or selectivity. The much faster response times are expected to allow more faithful recording of neurotransmitter signaling dynamics in vivo. Further, great improvements in sensitivity will enable neurochemical recording electrodes to be reduced to cellular dimension and will permit the application of higher density, multiplexed MEAs for simultaneous monitoring of multiple analytes and for facile integration of electrical recording sites. The improved capabilities of these optimized glutamate and choline sensors has been validated by neuroscience collaborators studying the reward-seeking behavior of laboratory rats. By using our microsensors, they were able to rapidly, clearly and continuously monitor cholinergic and glutamatergic transmissions in the area of the brain associated with cognition and uncover the roles that the neurotransmitters acetylcholine and glutamate play.

Multianalyte sensing microprobes also were created to provide neuroscientists with a new tool to unravel the interplay of multiple neurochemicals in vivo. Redox enzymes used in electroenzymatic sensors are most commonly immobilized on microelectrodes by manually spreading a mixture of enzyme and bovine serum albumin (BSA) on an electrode surface coated with permselective films followed by crosslinking with glutaraldehyde. This manual approach clearly becomes problematic when the MEA feature size is less than or equal to ~100 μm. A polydimethylsiloxane (PDMS) microstamping method was developed to enable precise transfer of enzyme onto targeted microelectrodes. Two model enzymes, glucose oxidase and choline oxidase, were successfully stamped onto different sites on the same microprobe to create a dual sensor. The resulting dual sensor showed the expected response to glucose and choline on the appropriate microsensor sites without cross-talk. The performance of the stamped sensor was further improved through the use of a polycation-functionalized zwitterionic polymer, poly(2-methacryloyloxyethyl phosphorylcholine)-g-poly(allylamine hydrochloride), as an alternative enzyme immobilization matrix to BSA. This specially designed polymer addressed the problem of pattern spreading found when using BSA by contributing a stronger intermolecular force, thereby allowing deposition of a much thicker yet more finely defined enzyme pattern. The increase in enzyme loading led to a choline sensor with two-fold improvement in both sensitivity and detection limit. This successful enzyme stamping approach is expected to contribute to neuroscience by enabling the simultaneous recording of multiple neurochemicals in close proximity in vivo.

A highly flexible PDMS-based microprobe also was developed to address the long-standing challenge of relative shear motion at the probe-tissue interface that is attributed to the mechanical mismatch between the typically very stiff probe and the surrounding soft neural tissue. The PDMS microprobe was designed with internal channels filled with gallium (Ga) metal. Ga is solid at room temperature but melts at body temperature. Thus, using Ga in probe construction made possible the deep insertion of probes into rat brains under cooled conditions but these implanted devices later become highly flexible upon Ga melting. This sensor design serves as an attractive platform for chronic in vivo studies with freely moving animals where probe micromotion must be minimized to avoid gliosis and permit long term neurochemical recordings.

Finally, the dependency on the existence of appropriate redox enzymes has always been the major limitation on applicability of electroenzymatic sensor technology. A second class of sensing devices were developed using aptamers as the biological recognition elements. Si microprobes with an array of aptamer-based field-effect transistors (FETs) were created. Since aptamers carry a great deal of charge, a change in conformation of its negatively charged phosphodiester backbone upon binding with target in close proximity to semiconductor channel enables signal transduction and amplification by the underlying FET. These aptamer-based FET microprobes exhibit an unprecedented detection limit of ~10 fM (10-14 M) to serotonin that is substantially lower than all other analytical methods (typically in the range of 10-9 – 10-6 M), thereby providing a potential alternative route to detection of neurotransmitters, especially those for which no redox enzymes exist and that are present in the brain at very low concentration. Our main contribution to this project was to help Dr. Anne Andrews group miniaturizing their well-developed aptamer-based FETs in micro-range for in vivo application.