B I O Microchip devices for the Study of Single vesicle Fusion events

Neurotransmitters and hormones are stored within secretory vesicles inside the cell and release their contents in a quantal event by fusion with the plasma membrane. We develop and fabricate microchip devices that we apply in cell experiments for electrochemical measurements of quantal release events from adrenal chromaffin cells. Surface patterned electrodes on transparent surfaces are applied to relate the fusion events to fluorescence changes observed under a microscope with simultaneous electrochemical detection. CMOS microchip devices are used for highly parallel detection of quantal release events from a large number of cells. Summary of Research: Adrenal chromaffin cells release adrenaline or noradrenaline in response to stimulation. These molecules are stored in secretory vesicles inside the cell and are released by fusion of the secretory vesicles with the plasma membrane. Single vesicle release events, also termed quantal release events, can be detected as an amperometric current using a carbon fiber or microfabricated electrode [1]. We use microfabricated electrode arrays to characterize the spatiotemporal relation between conformational changes of the fusion complex protein SNAP-25, located on the plasma membrane, and the opening of a fusion pore. In these experiments a SNAP-25 construct named SCORE (SNARE COmplex REporter) [2] is used, which incorporates the fluorescent protein groups CFP and Venus. In cells, this construct responds to stimulation with a small change in fluorescence resonance energy transfer (FRET). The construct was expressed in adrenal chromaffin cells and individual fusion events were spatiotemporally correlated with the FRET changes. The results indicate that the conformational change indicated by the SCORE constructs precedes fusion by ~ 90 ms (Figure 1). We developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes [3]. Surface patterned microelectrodes were fabricated from indium-tin-oxide (ITO), nitrogen-doped Figure 1: Representative amperometric spikes (top) measured with three different transparent electrode materials(bottom). diamond-like carbon (DLC) deposited on top of ITO, or very thin (12-17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not BIOLOGICAL APPLICATIONS 11 2011-2012 CNF RESEARCH ACCOMPLISHMENTS B I O as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release [3] (Figure 2). To test pharmacological and molecular manipulations of the fusion complex and other regulators of transmitter release in a high throughput approach, we develop an electrochemical CMOS sensor array. The CMOS IC incorporating a 10 × 10 array of potentiostats as well as timing and readout circuitry (Figure 3a) was fabricated at MOSIS, and 15 μm × 15 μm AlCu contacts were included in the design to serve as interconnection between electrode material and underlying integrated amplifier (Figure 3b). Post-fabrication of the Pt electrode array was performed photolithographically. A schematic of the geometry with an aligned image of a single electrode of the array (Figure 3c) is shown on expanded scale in Figure 3d. Figure 4: Live chromaffin cell recording of neurotransmitter release. Cells were cultured on-chip 24 hrs prior to the recording. Twelve of 100 electrodes are shown. The amperometric traces from each electrode measured after KCl stimulation are superimposed. Figure 2: TIR-FRET imaging of SCORE is correlated with fusion events. Average FRET change from 903 events is centered at release site (A) and occurs at time of fusion (B). Temporal event correlation reveals a 90 ms delay between FRET change and fusion (C). Figure 3: Amperometric CMOS amplifier array. Array surface with working electrodes and underlying circuitry after post-fabrication (a). Individual electrode/ potentiostat array element focused at the underlying in-pixel potentiostat, consisting of nine transistors (b), and on the surface working electrode (c) aligned with schematic of electrode topology (d). For live-cell measurement of single vesicle release events, chromaffin cells were plated on the biosensor (Figure 4). Out of the twelve electrodes shown in Figure 4, eight electrodes were fully or partially covered by chromaffin cells. After a high-k+ stimulation, many amperometric events were recorded by most of cell-covered electrodes as well as adjacent electrodes (Figure 4). Each amperometric spike corresponds to quantal release of the contents from a single vesicle. This parallel recording of amperometric spikes confirmed the biosensor’s capability to record quantal release events simultaneously from live-cells over the typical recording time (~ 150 s) with sub-millisecond resolution and pico-ampere current resolution that is comparable to conventional lownoise amplifiers.