Microfabricated Devices to Study Single Vesicle Release

Many cell types, including neurons, chromaffin cells of the adrenal adrenal gland and mast cells, release transmitter molecules stored in ‘vesicles’ by a process called exocytosis. In this process, an intracellular vesicle loaded with molecules to be secreted, fuses with the cell membrane, forming an initial opening called a fusion pore, and emits its contents into the extracellular space. The process of fusion pore formation and expansion is not well understood. We are developing microchip devices that detect exocytosis of oxidizable molecules from single vesicles electrochemically, to better understand the process of exocytosis and the role of drugs in the modulation of loading and release of single vesicles. Summary: Release of the catecholamines dopamine, adrenaline, and noradrenaline from single vesicles can be detected amperometrically as a brief spike of oxidation current using carbon fiber electrodes [1] as well as microfabricated platinum electrochemical detector (ECD) arrays on microscope cover-glasses to obtain also spatial information about the release sites [2]. The ECD arrays detect quantal noradrenaline release events of single vesicles from chromaffin cells. Release positions were determined based on the relative fraction of molecules that is detected by each of the four electrodes of the array and verified by simultaneous fluorescence microscopy [3]. The time course of the signal was analyzed to determine the release kinetics from single vesicles and apparent diffusion constants of the molecules. We found that adrenaline molecules leave their vesicles more quickly then previously thought, but diffuse much slower near the cell surface (D = 0.8-1.3x10-6cm2/s) then they do in bulk media (D = 6x10-6cm2/s) [3]. This method opens the door to experiments where we can observe the actions of fluorescently labeled molecules involved in exocytosis, while concurrently recording and then co-localizing electrochemical information from the same events. The new generation of detectors uses SiO2 as the insulating layer instead of photoresist, which makes the detectors much more robust. They can be cleaned multiple times using solvents or HCl and reused. It also eliminates fluorescence background of the photoresist. The new ECD arrays measure release using three instead of four electrodes, using the fourth electrode at a retracted position to measures noise for later subtraction. The features of this new design are finer than in the old ECD arrays (minimum feature ~0.9 μm), which makes the use of projection lithography necessary (5x-stepper). Various drugs affect release from single vesicles (also called quantal release). As an example, the drug L-Dopa, used to treat Parkinson’s disease, increases quantal size, while drugs such as reserpine and amphetamines decrease quantal size. Using patch amperometry, a method developed in our laboratory [4], we found that changes in quantal size are associated with changes in vesicle size [5]. Since many drugs affect quantal size, we are developing amperometric electrode active pixel arrays with integrated electronics that can perform high throughput measurements of quantal size and the effect of drugs on the spatio-temporal properties of single vesicle exocytosis. The design involves a regulated cascode amplifier (RCA) pixel circuit which allows the determination of electrical charge released due to the oxidation with two electrons transferred per oxidised molecule. In our circuit, since the electrode is held at a reference potential, common to all pixels, we use a scheme called share-buffered direct injection that has been employed in infrared imaging systems [6, 7]. Our circuit provides pico-amp resolution and can detect currents up to 1nA-600pA. Pt electrodes were deposited on the CMOS die using FEI 611 FIB system at the CNF. References: [1] Wightman, R.M., et al., Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc. Natl. Acad. Sci. USA, 1991. 88: p. 10754-10758. [2] Dias, A.F., et al., An electrochemical detector array to study cell biology on the nanoscale. Nanotechnology, 2002. 13: p. 285-289. [3] Hafez, I., et al., Electrochemical imaging of fusion pore openings using electrochemical detector (ECD) arrays. Proceedings of the National Academy of Sciences, USA, 2005: p. in revision. [4] Dernick, G., et al., Patch Amperometry high resolution measurements of single vesicle fusion and release. Nature Methods, 2005: p. in revision. [5] Gong, L.W., G. Alvarez De Toledo, and M. Lindau, Secretory vesicles membrane area is regulated in tandem with quantal size in chromaffin cells. Journal of Neuroscience, 2003. 23(21): p. 7917-7921. [6] Hsieh, C.-C., et al., Focal-plane-arrays and CMOS readout techniques of infrared imaging systems. IEEE Transactions on Circuits and Systems for Video Technology, 1997. 7(4): p. 594-605. [7] Wu, C.-Y., et al., A new share-buffered direct-injection readout structure for infrared detector. SPIE, 1993. 2020(Infrared Technology XIX): p. 57-64.