Amperometry is a powerful method to record quantal release events from chromaffin cells and is widely used to assess how specific drugs modify quantal size, kinetics of release, and early fusion pore properties. Surface-modified CMOS-based electrochemical sensor arrays allow simultaneous recordings from multiple cells. A reliable, low-cost technique is presented here for efficient targeting of single cells specifically to the electrode sites. An SU-8 microwell structure is patterned on the chip surface to provide insulation for the circuitry as well as cell trapping at the electrode sites. A shifted electrode design is also incorporated to increase the flexibility of the dimension and shape of the microwells. The sensitivity of the electrodes is validated by a dopamine injection experiment. Microwells with dimensions slightly larger than the cells to be trapped ensure excellent single-cell targeting efficiency, increasing the reliability and efficiency for on-chip single-cell amperometry measurements. The surface-modified device was validated with parallel recordings of live chromaffin cells trapped in the microwells. Rapid amperometric spikes with no diffusional broadening were observed, indicating that the trapped and recorded cells were in very close contact with the electrodes. The live cell recording confirms in a single experiment that spike parameters vary significantly from cell to cell but the large number of cells recorded simultaneously provides the statistical significance.

Current research into the function of carbonic anhydrases in cell physiology emphasizes the role of membrane-bound carbonic anhydrases, such as carbonic anhydrase IX that has been identified in malignant tumors and is associated with extracellular acidification as a response to hypoxia. We present here a mass spectrometric method to determine the extent to which total carbonic anhydrase activity is due to extracellular carbonic anhydrase in whole cell preparations. The method is based on the biphasic rate of depletion of 18 O from CO 2 measured by membrane inlet mass spectrometry. The slopes of the biphasic depletion are a sensitive measure of the presence of carbonic anhydrase outside and inside of the cells. This property is demonstrated here using suspensions of human red cells in which external carbonic anhydrase was added to the suspending solution. It is also applied to breast and prostate cancer cells which both express exofacial carbonic anhydrase IX. Inhibition of external carbonic anhydrase is achieved by use of a membrane impermeant inhibitor that was synthesized for this purpose, p-aminomethylbenzenesulfonamide attached to a polyethyleneglycol polymer.Keywords carbonic anhydrase; mass spectrometry; carbon dioxide; stable isotope; enzyme activityThe zinc metalloenzyme carbonic anhydrase (CA) catalyzes the hydration of carbon dioxide to form bicarbonate and a proton [1,2]. There are 16 isozymes of CA in the α-class of mammalian carbonic anhydrases that play a role in respiration, formation of secretory fluids, acid-base balance, and other physiological functions [3]. The isozyme CA IX is of particular interest since it is found in few normal tissues but its expression is induced in certain tumor cells by hypoxia [4,5]. CA IX is a transmembrane protein the catalytic site of which is external to the cell and is shown to play a role in extracellular acidification associated with cell Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. proliferation and metastasis [6][7][8][9]. Its inhibition may be of benefit in anticancer therapies for several types of cancer, including breast and prostate cancer. NIH Public AccessThe inhibition of carbonic anhydrases by sulfonamides is well studied in terms of its biochemical, physiological, and medical aspects [3,10,11]. Oxygen-18 and carbon-13 labeled bicarbonate was prepared by dissolving KH 13 CO 3 (99% 13 C) in enriched water (up to 90% 18 O enrichment). This solution was allowed to come to isotopic equilibrium overnight, after which water was removed by vacuum distillation. The resulting product was stored at room temp...

A potentiostat circuit for the application of bipolar electrode voltages and detection of bidirectional currents using a microelectrode array is presented. The potentiostat operates as a regulated-cascode amplifier for positive input currents, and as an active-input regulated-cascode mirror for negative input currents. This topology enables constant-potential amperometry and fast-scan cyclic voltammetry (FSCV) at microelectrode arrays for parallel recording of quantal release events, electrode impedance characterization, and high-throughput drug screening. A 64-channel FSCV detector array, fabricated in a 0.5-μm, 5-V CMOS process, is also demonstrated. Each detector occupies an area of 45 μm × 30 μm and consists of only 14 transistors and a 50-fF integrating capacitor. The system was validated using prerecorded input stimuli from actual FSCV measurements at a carbon-fiber microelectrode.

Highlights d Exocytotic transmitter release is an electrodiffusion process d Narrow fusion pores are cation selective d Fusion pore ion selectivity decreases as the fusion pore expands

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