A double potential pulse scheme is reported for observation of cholesterol efflux from the plasma membrane of a single neuron cell. Capillary Pt disk microelectrodes having a thin glass insulator allow the 10 μm diameter electrode and cell to be viewed under optical magnification. The electrode, covalently functionalized with cholesterol oxidase, is positioned in contact with the cell surface resulting in enzyme catalyzed cholesterol oxidation and efflux of cholesterol from the plasma membrane at the electrode contact site. Enzymatically generated hydrogen peroxide accumulates at the electrode/cell interface during a 5 s hold-time and is oxidized during application of a potential pulse. A second, replicate potential pulse is applied 0.5 s after the first potential pulse to gauge background charge prior to significant accumulation of hydrogen peroxide. The difference in charge passed between the first and second potential pulse provides a measure of hydrogen peroxide generated by the enzyme and is an indication of the cholesterol efflux. Control experiments for bare Pt microelectrodes in contact with the cell plasma membrane show difference charge signals in the range of about 7-10 pC. Enzyme-modified electrodes in contact with the plasma membrane show signals in the range of 16-26 pC.

中文翻译：

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双电位脉冲计时库仑法用于检测盘铂微电极处的等离子膜胆固醇流出

报告了双电位脉冲方案用于观察胆固醇从单个神经元细胞的质膜流出。具有薄玻璃绝缘体的毛细管 Pt 圆盘微电极允许在光学放大倍率下观察 10 μm 直径的电极和细胞。用胆固醇氧化酶共价功能化的电极与细胞表面接触，导致酶催化的胆固醇氧化和胆固醇从电极接触部位的质膜流出。酶促产生的过氧化氢在 5 秒的保持时间内积聚在电极/细胞界面，并在施加电位脉冲期间被氧化。应用第二个复制电位脉冲 0。在第一个电位脉冲后 5 秒，在过氧化氢大量积聚之前测量背景电荷。第一次和第二次电势脉冲之间传递的电荷差异提供了酶产生的过氧化氢的量度，并且是胆固醇流出的指示。与细胞质膜接触的裸铂微电极的对照实验显示约 7-10 pC 范围内的不同电荷信号。与质膜接触的酶修饰电极显示 16-26 pC 范围内的信号。与细胞质膜接触的裸铂微电极的对照实验显示约 7-10 pC 范围内的不同电荷信号。与质膜接触的酶修饰电极显示 16-26 pC 范围内的信号。与细胞质膜接触的裸铂微电极的对照实验显示约 7-10 pC 范围内的不同电荷信号。与质膜接触的酶修饰电极显示 16-26 pC 范围内的信号。