Registration of activity of a single molecule of horseradish peroxidase using a detector based on a solid-state nanopore.

Q3 Biochemistry, Genetics and Molecular Biology Biomeditsinskaya khimiya Pub Date : 2024-09-01 DOI:10.18097/PBMC20247005349
Yu D Ivanov, A N Ableev, A V Vinogradova, E D Nevedrova, I D Shumov, V S Ziborov, A F Kozlov, I A Ivanova, N V Vaulin, D V Lebedev, A S Bukatin, I S Mukhin, E A Ponomarenko, A I Archakov
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Abstract

This work demonstrates the use of a solid-state nanopore detector to monitor the activity of a single molecule of a model enzyme, horseradish peroxidase (HRP). This detector includes a measuring cell, which is divided into cis- and trans- chambers by a silicon nitride chip (SiN structure) with a nanopore of 5 nm in diameter. To entrap a single HRP molecule into the nanopore, an electrode had been placed into the cis-chamber; HRP solution was added into this chamber after application of a negative voltage. The reaction of the HRP substrate, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), oxidation by the enzyme molecule was performed in the presence of hydrogen peroxide. During this reaction, the functioning of a single HRP molecule, entrapped in the nanopore, was monitored by recording the time dependence of the ion current flowing through the nanopore. The approach proposed in our work is applicable for further studies of functioning of various enzymes at the level of single molecules, and this is an important step in the development of single-molecule enzymology.

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使用基于固态纳米孔的检测器登记单分子辣根过氧化物酶的活性。
这项研究展示了如何利用固态纳米孔检测器来监测单分子辣根过氧化物酶(HRP)的活性。该检测器包括一个测量池,测量池被氮化硅芯片(SiN 结构)分为顺式和反式腔室,氮化硅芯片上有一个直径为 5 纳米的纳米孔。为了将单个 HRP 分子捕获到纳米孔中,在顺式腔中放置了一个电极;在施加负电压后,将 HRP 溶液加入该腔中。在过氧化氢的作用下,酶分子与 HRP 底物 2,2-叠氮双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)发生氧化反应。在这一反应过程中,通过记录流经纳米孔的离子电流的时间依赖性来监测纳米孔中夹带的单个 HRP 分子的功能。我们工作中提出的方法适用于在单分子水平上进一步研究各种酶的功能,这是发展单分子酶学的重要一步。
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来源期刊
Biomeditsinskaya khimiya
Biomeditsinskaya khimiya Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)
CiteScore
1.30
自引率
0.00%
发文量
49
期刊介绍: The aim of the Russian-language journal "Biomeditsinskaya Khimiya" (Biomedical Chemistry) is to introduce the latest results obtained by scientists from Russia and other Republics of the Former Soviet Union. The Journal will cover all major areas of Biomedical chemistry, including neurochemistry, clinical chemistry, molecular biology of pathological processes, gene therapy, development of new drugs and their biochemical pharmacology, introduction and advertisement of new (biochemical) methods into experimental and clinical medicine etc. The Journal also publish review articles. All issues of journal usually contain invited reviews. Papers written in Russian contain abstract (in English).
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Fundamentals of protein chemistry at the Institute of Biomedical Chemistry. In silico and in cellulo approaches for functional annotation of human protein splice variants. Nanowire-based biosensors for solving biomedical problems. Proteome of plasma extracellular vesicles as a source of colorectal cancer biomarkers. Registration of activity of a single molecule of horseradish peroxidase using a detector based on a solid-state nanopore.
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