Bratten C D, Cobbold P H, Cooper J M
Department of Electronics and Electrical Engineering, University of Glasgow, U.K.
Anal Chem. 1998 Mar 15;70(6):1164-70. doi: 10.1021/ac970982z.
To study the cellular events surrounding the formation of purines in cardiac ischemia, we have micromachined a micrometer-scale titer chamber containing an integrated electrochemical sensor, capable of measuring analytes produced by a single heart cell. The analytical procedure involves the determination of metabolites via the amperometric detection of enzymically generated hydrogen peroxide, measured at a platinized microelectrode, poised at a suitably oxidizing potential, equivalent to +420 mV vs Ag/AgCl. Signals were recorded as current-time responses and were integrated to give a total charge (Q) attributable to the reaction under investigation. The amount of analyte produced by the cell was subsequently quantified by the addition of a known amount of calibrant. As a consequence, by using a cascade of three enzymes (adenosine deaminase, nucleotide phosphorylase, and xanthine oxidase), we were able to show that, after rigor contracture had been induced in a single myocyte, adenosine (but not inosine) only reached the extracellular space after the cell membrane had been permeabilized by detergent. These data, which could only be obtained unambiguously by using this single-cell methodology, have provided us with information on the origin of ischemic adenosine which challenges the established assumption that purine release is an early retaliatory response from intact anoxic myocytes.
为了研究心脏缺血过程中嘌呤形成周围的细胞事件，我们用微机械加工制造了一个包含集成电化学传感器的微米级滴定室，该传感器能够测量单个心脏细胞产生的分析物。分析过程包括通过在镀铂微电极上对酶促产生的过氧化氢进行安培检测来测定代谢物，该微电极保持在适当的氧化电位，相对于Ag/AgCl为 +420 mV。信号记录为电流 - 时间响应，并进行积分以得到归因于所研究反应的总电荷（Q）。随后通过添加已知量的校准物来定量细胞产生的分析物的量。因此，通过使用三种酶（腺苷脱氨酶、核苷酸磷酸化酶和黄嘌呤氧化酶）的级联反应，我们能够表明，在单个心肌细胞诱导出现强直收缩后，只有在细胞膜被去污剂通透后，腺苷（而非肌苷）才会到达细胞外空间。这些数据只能通过这种单细胞方法明确获得，它们为我们提供了关于缺血腺苷来源的信息，这对嘌呤释放是完整缺氧心肌细胞早期报复性反应这一既定假设提出了挑战。
