Microfabricated Devices For Direct Measurements Of Quantal Transmitter Release From Living Cells
Neurotransmitters are released in packets or quanta from vesicles that fuse with the cell membrane. The machinery proteins called Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptor (SNARE) proteins are known to mediate the vesicle fusion. The quantal release of neurotransmitters from fusion of single vesicles can be monitored using an electrochemical electrode by the oxidation of neurotransmitters which cause a transfer of electrons into the electrode. The resulting oxidation current reveals amperometric spikes that provide information about the frequency of release events, the number of released transmitter molecules from a single vesicle (quantal size) and the kinetics of quantal release events. While carbon fiber electrode (CFE) is most widely used as an electrochemical electrode, microfabricated planar electrodes made of platinum or gold are being developed as a more convenient way to interface with cells. Also, large arrays of planar electrodes can be easily fabricated, which enable high-throughput recording from living cells. In this dissertation, microfabricated devices for direct measurements of transmitter secretion from living cells are introduced. Sensing electrodes can be fabricated on the surface of complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC), which incorporate all the essential signal processing circuits in the chip. Such post-processing of CMOS chips is called post-fabrication. Using IC technology and post-fabrication, an electrochemical sensor array was developed that is capable of 100 parallel recordings. The IC sensor array was able to directly interface with living chromaffin cells to measure the quantal release of neurotransmitter. The biosensor was capable of resolving small (0.7 pA) and fast amperometric spikes reporting release from individual vesicles. The effect of a Parkinson's disease drug called L-dopa was tested using the IC biosensor. The amperometric recordings of L-dopa treated cells revealed an increase in the amount of neurotransmitters release per vesicle by 73% which agrees with the known effect of L-dopa. An alternative and improved post-fabrication technique called tape-off is demonstrated in the following chapter. Tape-off is a dry and self-aligning method, which does not rely on photolithography for microfabrication and exhibits an excellent production yield and time reduction. Post-fabricated biosensor arrays with up to 320 electrodes exhibited stable performance with an electrolyte in contact and successfully measured oxidation of dopamine molecules. Measurement of quantal neurotransmitter release from living cells using the biosensor array fabricated using the tape-off method was demonstrated. Other potential applications of this new technique are discussed. In chapter 4, a portable and cost effective data acquisition system for the IC biosensor is presented that incorporates analog-to-digital converters (ADCs) and universal serial bus (USB)-communication. This data acquisition system outperforms other commercially available ADCs and allows the portable operation of the IC biosensor as it is fully powered through the USB connection. The functionality of the complete system was validated by measuring and storing dopamine oxidation data from the IC biosensor array. Finally, the use of a conducting polymer, poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS), is used as detecting electrode material is demonstrated. Various architectures were evaluated to optimize the performance of PEDOT:PSS microelectrodes in direct contact with electrolyte and cells and the successful recording of the neurotransmitter oxidation released from chromaffin cells is shown. This result represents a new capability for organic electronics that could lead to devices that interface nervous system in novel ways with unique properties of polymer such as softness.
Biosensor; Exocytosis; CMOS Integrated Circuit
Brown, William J; Baird, Barbara Ann
Ph.D. of Biophysics
Doctor of Philosophy
dissertation or thesis