Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Digital Bioanalysis Laboratory, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
ACS Sens. 2023 Sep 22;8(9):3400-3407. doi: 10.1021/acssensors.3c00587. Epub 2023 Aug 17.
Digital enzyme assays are emerging biosensing methods for highly sensitive quantitative analysis of biomolecules with single-molecule detection sensitivity. However, current digital enzyme assays require a fluorogenic substrate for detection, which limits the applicability of this method to certain enzymes. ATPases and kinases are representative enzymes for which fluorogenic substrates are not available; however, these enzymes form large domains and play a central role in biology. In this study, we implemented a fluorogenic cascade reaction in a femtoliter reactor array device to develop a digital bioassay platform for ATPases and kinases. The digital cascade assay enabled quantitative measurement of the single-molecule activity of F-ATPase, the catalytic portion of ATP synthase. We also demonstrated a digital assay for human choline kinase α. Furthermore, we developed a digital cascade assay for ATP-synthesizing enzymes and demonstrated a digital assay for pyruvate kinase. These results show the high versatility of this assay platform. Thus, the digital cascade assay has great potential for the highly sensitive detection and accurate characterization of various ADP- and ATP-producing enzymes, such as kinases, which may serve as disease biomarkers.
数字酶分析是一种新兴的生物传感方法，可实现具有单分子检测灵敏度的生物分子的高灵敏度定量分析。然而，目前的数字酶分析需要荧光底物进行检测，这限制了该方法在某些酶中的适用性。ATP 酶和激酶是没有荧光底物的代表性酶；然而，这些酶形成大的结构域，在生物学中起着核心作用。在这项研究中，我们在皮升反应器阵列装置中实现了荧光级联反应，开发了用于 ATP 酶和激酶的数字生物分析平台。数字级联分析能够定量测量 ATP 合酶催化部分 F-ATP 酶的单分子活性。我们还展示了用于人胆碱激酶 α 的数字分析。此外，我们开发了用于合成酶的数字级联分析，并展示了用于丙酮酸激酶的数字分析。这些结果表明该分析平台具有很高的多功能性。因此，数字级联分析在高度灵敏的检测和准确表征各种 ADP 和 ATP 产生酶方面具有巨大潜力，例如激酶，激酶可能作为疾病生物标志物。
