Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany.
Analyst. 2013 Aug 7;138(15):4260-5. doi: 10.1039/c3an00809f. Epub 2013 Jun 11.
Large arrays of femtoliter-sized chambers were etched into the surface of fused silica slides to enclose and observe hundreds of single horseradish peroxidase (HRP) molecules in parallel. Individual molecules of HRP oxidize the fluorogenic substrate Amplex Red to fluorescent resorufin in separate chambers, which was monitored by fluorescence microscopy. Photooxidation of Amplex Red and photobleaching of resorufin have previously limited the analysis of HRP in femtoliter arrays. We have strongly reduced these effects by optimizing the fluorescence excitation and detection scheme to yield accurate single molecule substrate turnover rates. We demonstrate the presence of long-lived kinetic states of single HRP molecules that are individually different for each molecule in the array. The large number of molecules investigated in parallel provides excellent statistics on the activity distribution in the enzyme population, which is similar to that reported for other enzymes such as β-galactosidase. We have further confirmed that the product formation of HRP in femtoliter chambers is 10-fold lower than that in the bulk solution due to the particular two-step redox reaction mechanism of HRP.
大量纳升级别的微腔被刻蚀在熔融石英载玻片的表面，以封闭并平行观测数百个单个辣根过氧化物酶（HRP）分子。HRP 分子在单独的微腔中氧化荧光底物 Amplex Red 生成荧光产物 Resorufin，通过荧光显微镜进行监测。Amplex Red 的光氧化和 Resorufin 的光漂白以前限制了纳升级别 HRP 的分析。我们通过优化荧光激发和检测方案，极大地降低了这些影响，从而获得了准确的单个分子底物转化率。我们证明了单个 HRP 分子存在长寿命的动力学状态，对于阵列中的每个分子而言都是不同的。平行研究的大量分子为酶群体中的活性分布提供了极好的统计数据，与其他酶（如β-半乳糖苷酶）的报道相似。我们还进一步证实，由于 HRP 的特殊两步氧化还原反应机制，HRP 在纳升级别的腔室中的产物形成比在体相溶液中低 10 倍。
