南京大学江德臣团队开发了单个活细胞中低丰度酶基于电化学分子陷阱的传感方法。相关研究成果发表在2022年9月16日出版的《美国化学会杂志》。

测量低丰度酶的活性，即一个活细胞中的几个分子的活性，对于阐明其生物学功能是重要的，但具有挑战性。

该文中，在带有电化学检测器的纳米移液管尖端建立电化学分子陷阱，其中通过电渗流（EOF）阻止分子远离电化学检测器的扩散。因此，捕获有限数量的酶，以持续催化底物的转化，生成足够数量的副产物过氧化氢，用于电化学测量。单个脂质体中酶的电阻脉冲传感验证了低至15个分子的检测灵敏度。使用这种超灵敏的电化学策略，研究人员测量了单个未经刺激的活J774细胞内60个鞘氨醇酶分子的活性，这是以前的方法很难检测到的。建立的基于电化学分子陷阱的传感方法为在一个活细胞中进行单分子电化学检测打开了大门。

这一成功将解决长期以来关于在细胞天然生理状态下研究低丰度蛋白质活性的问题，并极大地增强对蛋白质在细胞行为中作用的理解。

附：英文原文

Title: Electrochemical Molecule Trap-Based Sensing of Low-Abundance Enzymes in One Living Cell

Author: Rongrong Pan, Dengchao Wang, Kang Liu, Hong-Yuan Chen, Dechen Jiang

Issue&Volume: September 16, 2022

Abstract: Measuring the activity of low-abundance enzymes, down to a few molecules in one living cell, is important but challenging to elucidate their biological function. Here, an electrochemical molecule trap is established at the tip of a nanopipette with an electrochemical detector, in which the diffusion of the molecules away from the electrochemical detector is prevented by electro-osmotic flow (EOF). Accordingly, a limited amount of enzymes is trapped to continuously catalyze the conversion of the substrate to generate a sufficient amount of the byproduct hydrogen peroxide for electrochemical measurements. The resistive pulse sensing of the enzymes in single liposomes validates the detection sensitivity down to 15 molecules. Using this ultrasensitive electrochemical strategy, the activity of 60 sphingomyelinase molecules inside single unstimulated living J774 cells is measured, which was hardly detected by previous methods. The established electrochemical molecule trap-based sensing approach opens the door toward single-molecule electrochemical detection in one living cell. This success will solve the long-standing problem regarding the study of the activity of low-abundance proteins in cells in their native physiological state and greatly enhance the understanding of the roles of proteins in cellular behavior.

DOI: 10.1021/jacs.2c06962

Source: https://pubs.acs.org/doi/10.1021/jacs.2c06962