Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Molecular Physiology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Sci Adv. 2020 Mar 11;6(11):eaay0888. doi: 10.1126/sciadv.aay0888. eCollection 2020 Mar.
We established an ultrasensitive method for identifying multiple enzymes in biological samples by using a multiplexed microdevice-based single-molecule enzymatic assay. We used a paradigm in which we "count" the number of enzyme molecules by profiling their single enzyme activity characteristics toward multiple substrates. In this proof-of-concept study of the single enzyme activity-based protein profiling (SEAP), we were able to detect the activities of various phosphoric ester-hydrolyzing enzymes such as alkaline phosphatases, tyrosine phosphatases, and ectonucleotide pyrophosphatases in blood samples at the single-molecule level and in a subtype-discriminating manner, demonstrating its potential usefulness for the diagnosis of diseases based on ultrasensitive detection of enzymes.
我们建立了一种超灵敏的方法，用于通过基于多重微设备的单分子酶分析来鉴定生物样品中的多种酶。我们使用了一种范例，通过分析它们对多种底物的单一酶活性特征来“计数”酶分子的数量。在这个基于单酶活性的蛋白质分析（SEAP）的概念验证研究中，我们能够以单分子水平和亚型区分的方式检测血液样本中各种磷酸酯水解酶（如碱性磷酸酶、酪氨酸磷酸酶和核苷酸焦磷酸酶）的活性，证明了其在基于酶的超灵敏检测的疾病诊断方面的潜在用途。
