CN-116297733-B - Method for detecting steady-state damage of protein structure by nano-pore single molecules

Abstract

The invention relates to a method for detecting steady-state damage of a protein structure by using a nanopore single molecule, belonging to the technical field of electrochemical detection. The invention discloses a method for detecting stable damage of a protein structure by using nanopore single molecules, namely an electrochemical device with nanopores is adopted to collect electrochemical signals before and after natriuretic peptide proteins are acted by a reducing agent, and analysis is carried out through the amplitude value of the current signals and the current blocking time characteristics, so that the change information before and after the stable damage of the chemical structure on the single chemical bond breaking level of the natriuretic peptide proteins is detected, the advantages of the nanopores are fully exerted, the change information of tiny current caused by the stable damage change of the protein structure is accurately recorded, the repeatability is good, and the reliability is high.

Inventors

• ZHANG SHAOXIA
• WANG LIANG
• Wang Binjiao
• FANG SHAOXI
• YIN BOHUA
• SONG DANDAN

Assignees

• 重庆邮电大学
• 中国科学院重庆绿色智能技术研究院

Dates

Publication Date

20260505

Application Date

20230210

Claims (8)

1. 1. A method for detecting stable state damage of a protein structure by using nanopore single molecules is characterized in that a nanopore is arranged in an electrochemical detection device to form a nanopore electrochemical detection device, a protein sample is arranged in the nanopore electrochemical detection device, an electric signal of the protein sample is collected, the nanopore electrochemical detection device is started to detect the electric signal after a reducing agent in the nanopore electrochemical detection device breaks the stable state of the chemical structure of the protein sample, and the characteristics of amplitude, blocking time or signal quantity of the electric signal are analyzed to obtain the change information of the stable state damage of the protein sample structure on the single chemical bond level.
2. 2. The method according to claim 1, wherein the nanopore electrochemical detection device is assembled by placing a support film containing nanopores in a solution chamber containing electrolyte, placing two electrodes in the solution chambers containing the solution at two ends of the support film, and connecting a power supply and an ammeter in sequence between the two electrodes; the material of the supporting film is any one of silicon nitride, graphene, molybdenum disulfide or a Teflon film; The electrode material is a silver electrode material with the surface coated with silver chloride.
3. 3. The method of claim 2, wherein the nanopore electrochemical detection device further comprises a base buffer; The basic buffer solution is a mixed solution formed by dissolving alkali metal chloride and Tris (Tris) in water; The concentration of alkali metal chloride in the basic buffer solution is 0.1-2M, and the concentration of tris (hydroxymethyl) aminomethane is 1-50 mM.
4. 4. A method according to claim 3, wherein the pH of the base buffer is 7.4.
5. 5. A method according to claim 3, wherein the alkali chloride is any one or more of sodium chloride, potassium chloride and lithium chloride.
6. 6. The method of claim 1, wherein the protein sample comprises cardiac natriuretic peptide (atrial natriuretic peptide, ANP), brain natriuretic peptide (brain natriuretic peptide, BNP), or C-type natriuretic peptide (C-type natriuretic peptide, CNP).
7. 7. The method of claim 1, wherein the reducing agent in the nanopore electrochemical detection device is trichloroethyl phosphate flame retardant plasticizer (TCEP).
8. 8. The method of claim 1, wherein the molar ratio of the protein sample to the reducing agent during disruption of the steady state of the chemical structure of the protein sample is 1:3, provided that 2h is reacted in a 37 ℃ metal bath.

Description

Method for detecting steady-state damage of protein structure by nano-pore single molecules Technical Field The invention belongs to the technical field of electrochemical detection, and relates to a method for detecting steady-state damage of a protein structure by using a nanopore single molecule. Background Proteins are the material basis of life, are the basic organic substances that constitute cells, and are the main contributors to life activities. Protein structure refers to the spatial structure of a protein molecule. To perform a biological function, the protein needs to be properly folded into a specific configuration, primarily through a large number of non-covalent interactions. Furthermore, disulfide bonds play a key role in some protein folding, particularly secreted proteins. Disulfide bonds thus play an important role in the folding and stabilization of proteins or polypeptides, however, such chemical bonds are easily broken during biochemical reactions or microenvironment changes, so that the original structural homeostasis of the protein or polypeptide is broken and the original biological activity is lost. It is therefore necessary to study the chemical structure of proteins in order to further understand their physiological mechanism of action. There are a number of current methods for detecting protein structure. The most common methods are nuclear magnetic resonance (Nuclear Magnetic Resonance, NMR), X-ray crystallography (X-ray Crystallogra phy), freeze Electron microscopy (cryo-EM), mass spectrometry (Mass Spectrometry, MS), infrared spectroscopy (Infrared Spectroscopy, IR), ultraviolet spectroscopy (UV-visible Spectroscopy, UV), circular dichroism (Circular Dichroism, CD), and the like. The detection methods can detect and analyze the protein structure, but at present, the steady-state damage detection of the protein structure on the single chemical bond structure of the protein cannot be performed on the surface of the monomolecular layer, and in view of the characteristics of the detection methods, a simple method is urgently needed to study the steady-state damage of the protein chemical structure. Biosensors are electronic devices that use electrical, optical, chemical or mechanical devices to generate an electronic signal through biological interactions. Typically, for reliability, biosensors must have a high degree of sensitivity and selectivity to the desired molecules, and are therefore often designed for single species detection. Common biosensors include piezoelectric crystal immunosensors, fiber optic immunosensors, nanopore sensors, and the like. The nanopore sensor is used as an emerging platform, has the advantages of high flux, low cost, no mark, high sensitivity and the like, and is widely applied to the fields of nucleic acid, protein, biochemical reaction monitoring and the like. The nanopore sensing technology is an emerging technology with multiple advantages, can provide important information for the structure and function of nucleic acid and protein, can monitor biochemical reaction in real time, and solves the problems of traditional detection such as low selectivity and the like. Thus, attempts may be made to use nanopore-bound electrochemical detection methods for the detection and analysis of steady-state lesions in protein chemical structures. Therefore, further intensive research into methods for steady-state damage to protein chemical structures is necessary. Disclosure of Invention In view of the above, the present invention is directed to a method for detecting steady-state damage of protein structure by using a nanopore single molecule. In order to achieve the above purpose, the present invention provides the following technical solutions: 1. A method for detecting stable state damage of a protein structure by using nanopore single molecules comprises the steps of arranging a nanopore in an electrochemical detection device to form a nanopore electrochemical detection device, placing a protein sample in the nanopore electrochemical detection device, collecting an electric signal of the protein sample, starting the nanopore electrochemical detection device to a reducing agent in the nanopore electrochemical detection device to damage the stable state of the chemical structure of the protein sample, detecting the electric signal, and analyzing the characteristics of amplitude, blocking time or signal quantity of the electric signal before and after the electric signal, so as to obtain the change information of the stable state damage of the protein sample structure on a single chemical bond level. Preferably, the nanopore electrochemical detection device is assembled and formed according to the following method that a supporting film containing nanopores is placed in a solution chamber containing electrolyte, two electrodes are respectively placed in the solution chambers containing the solution at two ends of the supporting film, and a power supply and