A method of nanopore single molecule detection of protein structure homeostasis impairment

Abstract

The present application relates to a kind of nanometer hole single molecule detection protein structure steady state damage method, belong to electrochemical detection technical field.The present application discloses a kind of nanometer hole single molecule detection protein structure steady state damage method, i.e. using electrochemical device with nanometer hole, gathers the electrochemical signal before and after sodium peptide protein via the action of reducing agent, the amplitude of current signal and current block time characteristics are analyzed, to detect the change information before and after the chemical structure steady state damage of sodium peptide protein single chemical bond rupture level, give full play to nanometer hole advantage, accurately record the change information of small current caused by protein structure steady state damage change, repeatability is good, and reliability is high.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection technology and relates to a method for detecting steady-state damage to protein structures using nanopore single molecules. Background Technology

[0002] Proteins are the material basis of life, the basic organic components of cells, and the main carriers of life activities. Protein structure refers to the spatial structure of protein molecules. To perform their biological functions, proteins need to fold correctly into a specific conformation, mainly through numerous non-covalent interactions. Furthermore, disulfide bonds play a crucial role in the folding of some proteins, especially secretory proteins. Therefore, disulfide bonds play an important role in the folding and stability of proteins or polypeptides. However, these chemical bonds are easily broken during biochemical reactions or changes in the microenvironment, disrupting the original structural homeostasis of the protein or polypeptide and causing it to lose its original biological activity. Therefore, it is essential to study the chemical structure of proteins to further understand their physiological mechanisms of action.

[0003] Currently, there are numerous methods for detecting protein structures. The most common methods include nuclear magnetic resonance (NMR), X-ray crystallography, cryo-electron microscopy (cryo-EM), mass spectrometry (MS), infrared spectroscopy (IR), ultraviolet-visible spectroscopy (UV), and circular dichroism (CD). While all these methods can detect and analyze protein structures, they currently cannot detect steady-state damage to individual chemical bonds at the single-molecule level. Given the limitations of these methods, there is an urgent need for a simpler method to study steady-state damage in protein chemical structures.

[0004] Biosensors are electronic devices that utilize electrical, optical, chemical, or mechanical means to generate electronic signals through biological interactions. For reliability, biosensors typically require high sensitivity and selectivity for specific molecules, and are therefore often designed for single-species detection. Common biosensors include piezoelectric crystal immunosensors, fiber optic immunobiosensors, and nanopore sensors. Nanopore sensors, as an emerging platform, offer advantages such as high throughput, low cost, label-free operation, and high sensitivity, and are widely used in nucleic acid, protein, and biochemical reaction monitoring. Nanopore sensing technology, as an emerging technology, provides crucial information on the structure and function of nucleic acids and proteins, and can monitor biochemical reactions in real time, solving traditional detection challenges such as low selectivity. Therefore, combining nanopores with electrochemical detection methods can be explored for the detection and analysis of steady-state damage to protein chemical structures.

[0005] Therefore, it is necessary to conduct further in-depth research on methods for damaging the steady-state chemical structure of proteins. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a method for detecting steady-state damage to protein structures using nanopore single molecules.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] 1. A method for detecting protein structural steady-state damage at the nanopore single-molecule level, the method comprising: setting a nanopore in an electrochemical detection device to form a nanopore electrochemical detection device; placing a protein sample in the nanopore electrochemical detection device; acquiring the electrical signal of the protein sample; activating the nanopore electrochemical detection device until the reducing agent in the nanopore electrochemical detection device disrupts the chemical structural steady-state of the protein sample, and then detecting the electrical signal again; analyzing the amplitude, retardation time, or signal quantity characteristics of the two electrical signals to detect the changes in the protein sample structural steady-state damage at the single chemical bond level.

[0009] Preferably, the nanopore electrochemical detection device is assembled as follows: a supporting film containing nanopores is placed in a solution chamber containing electrolyte, two electrodes are placed in solution chambers containing solution at both ends of the supporting film, and a power supply and an ammeter are connected in sequence between the two electrodes to form the nanopore electrochemical detection device.

[0010] The material of the supporting film is any one of silicon nitride, graphene, molybdenum disulfide, or Teflon film;

[0011] The electrode material is a silver electrode material with silver chloride coated on its surface.

[0012] More preferably, the nanopore electrochemical detection device also includes a basic buffer solution;

[0013] The basic buffer solution is a mixed solution of alkali metal chloride and tris(hydroxymethyl)aminomethane (Tris) dissolved in water;

[0014] The concentration of alkali metal chloride in the basic buffer solution is 0.1–2 M, and the concentration of tris(hydroxymethyl)aminomethane is 1–50 mM.

[0015] More preferably, the pH of the base buffer solution is 7.4.

[0016] More preferably, the alkali metal chloride is any one or more of sodium chloride, potassium chloride, or lithium chloride.

[0017] Preferably, the protein sample includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or c-type natriuretic peptide (CNP).

[0018] Preferably, the reducing agent in the nanoporous electrochemical detection device is trichloroethyl phosphate flame retardant plasticizer (TCEP).

[0019] Preferably, during the process of the reducing agent disrupting the chemical structural stability of the protein sample, the molar ratio of the protein sample to the reducing agent is 1:3, and the condition is: reaction in a metal bath at 37°C for 2 hours.

[0020] The beneficial effects of this invention are as follows: This invention discloses a method for detecting steady-state damage to protein structures using nanopore single-molecule detection. By setting up nanopores in an electrochemical detection and analysis device, electrical signals are collected before and after the protein sample interacts with the nanopores. The amplitude, retardation time, and signal intensity of the electrical signals are analyzed to detect information such as protein content, charged properties, protein length, or molecular weight. The method of this invention can detect three natriuretic peptides (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or c-type natriuretic peptide (CNP)) and their corresponding linear structures. Electrochemical signals are collected in the electrochemical analysis device to distinguish different structures of the same protein, thus realizing the cross-application of nanopores and bioinformatics. It can detect three natriuretic peptides in the natriuretic peptide family and their corresponding linear structures. This application has the advantages of low cost, label-free operation, and convenient detection. In addition, the method of the present invention fully utilizes the advantages of low cost, label-free and rapid operation of nanopore electrochemical devices, accurately records the minute current changes caused by damage to the chemical structure of proteins, and has good repeatability and high reliability.

[0021] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0023] Figure 1 The results of the detection and analysis in Example 1 are shown in the following figures: a is the current graph of the test solution containing atrial natriuretic peptide (ANP), b is the current graph of the linear atrial natriuretic peptide (ANP), c is the current graph of the atrial natriuretic peptide (ANP) after being reduced by the reducing agent TCEP, and df is a scatter plot of the blocking time and blocking amplitude of atrial natriuretic peptide proteins with three different structures.

[0024] Figure 2 The results of the detection and analysis in Example 2 are shown in the following figures: a is the current graph of the test solution containing brain natriuretic peptide (BNP), b is the current graph of the linear brain natriuretic peptide (BNP), c is the current graph of the brain natriuretic peptide (BNP) after reduction by the reducing agent TCEP, and df is a scatter plot of the blocking time and blocking amplitude of brain natriuretic peptide (BNP) protein corresponding to three different structures.

[0025] Figure 3 The results of the detection and analysis in Example 3 are shown in the following figures: a is the current graph of the test solution containing C-type natriuretic peptide (CNP), b is the current graph of the solution containing linear C-type natriuretic peptide (CNP), c is the current graph of the solution containing C-type natriuretic peptide (CNP) reduced by the reducing agent TCEP, and df is a scatter plot of the blocking time and blocking amplitude of C-type natriuretic peptide (CNP) protein corresponding to three different structures.

[0026] Figure 4 This is a schematic diagram illustrating the principle of using TCEP as a reducing agent to reduce disulfide bonds in the chemical structure of natriuretic peptide protein. Detailed Implementation

[0027] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0028] The proteins involved in the following examples include atrial natriuretic peptide (ANNP), the sequence of which is shown in SEQ ID NO: 1; brain natriuretic peptide (BNP), the sequence of which is shown in SEQ ID NO: 2; and C-type natriuretic peptide (CNP), the sequence of which is shown in SEQ ID NO: 3, wherein:

[0029] SEQ ID NO: 1 is: NH2-Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met AspArg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr-COOH.

[0030] SEQ ID NO: 2 is: NH2-Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly ArgLys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His-COOH.

[0031] SEQ ID NO: 3 is: NH2-Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp ArgIle Gly Ser Met Ser Gly Leu Gly Cys-COOH.

[0032] Example 1

[0033] The following are common methods for nanopore electrochemical detection of atrial natriuretic peptide (ANP) protein chemical structure steady-state damage in solution studies:

[0034] 1. Prepare the solution:

[0035] (1) Preparation of basic buffer solution: Dissolve sodium chloride and tris(hydroxymethyl)aminomethane (Tris) in deionized water and adjust the pH of the solution to 7.4 to form a basic buffer solution containing sodium chloride and tris(hydroxymethyl)aminomethane (Tris) (the concentration of sodium chloride is 1M and the concentration of tris(hydroxymethyl)aminomethane (Tris) is 10mM). Finally, filter the buffer solution using a 0.22um filter before use.

[0036] (2) Preparation of protein samples: Dissolve the atrial natriuretic peptide (ANP) and its linear protein (L-ANP) in enzyme-free water to obtain solutions with a concentration of 2 mM, and store them in a refrigerator at -20℃ for later use.

[0037] (3) Preparation of reduction reaction protein sample: Add 6mM of reducing agent TCEP to the ANP protein sample solution prepared in step (2), mix well and place it in a metal bath at 37°C for 2 hours to fully destroy the disulfide bond between the two cysteine ​​residues in the ANP protein structure.

[0038] 2. Constructing a nanoporous electrochemical detection and analysis device: A supporting film containing nanopores (the supporting film is made of silicon nitride) is placed in a solution chamber containing an electrolyte. Two electrodes (both of which are silver electrode materials with silver chloride coating) are placed in solution chambers at both ends of the supporting film, and a power supply and an ammeter are connected in sequence between the two electrodes to form a nanoporous electrochemical detection and analysis device.

[0039] 3. Protein detection:

[0040] (1) The above-prepared atrial natriuretic peptide ANP, linear ANP and the prepared atrial natriuretic peptide ANP protein sample solution (5.0 μM) after reduction reaction were added separately to the above-prepared nanopore electrochemical analysis device. The above-prepared basic buffer solution was used as the electrolyte. After applying an external electric field, the peptide sample in the protein sample solution was driven to interact with the nanopore under the action of the electric field force, and the changing current signal was collected.

[0041] (2) The current signal collected in step (1) was amplified using a low-noise current amplifier (Axon Axopatch 200B), and then the amplitude and current lag time characteristics of the current signal were analyzed (the collected electrical signal file was selected by the software clamfit to select the characteristic electrical signal, and then the signal was statistically analyzed by Origin) to obtain the corresponding protein sample feature map.

[0042] Proteins can be analyzed and compared, enabling the analysis and detection of proteins using nanopores, such as... Figure 1 As shown, a is the current graph of the test solution containing atrial natriuretic peptide (ANP), b is the current graph of the solution containing linear atrial natriuretic peptide (ANP), c is the current graph of the solution containing atrial natriuretic peptide (ANP) reduced by the reducing agent TCEP, and df is a scatter plot of atrial natriuretic peptide proteins with three different structures. Figure 1 As can be seen from the comparison of the proportion of blocking signal amplitude and the blocking current time, there are significant differences among the three types of ANP: linear ANP, and ANP whose disulfide bonds in the original protein structure were disrupted by the reducing agent TCEP. Analysis of their scatter plots also shows that the current generated by the interaction of the three ANP structures with the nanopores is different. The ANP ANP reduced by TCEP did not form a protein structure completely consistent with the linear structure, indicating that some kind of protein structural steady-state damage occurred during the reduction process, preventing it from being reduced to the same structure as the linear structure. This further demonstrates that the nanopore has excellent resolution and can distinguish different steady-state structures of proteins, identifying chemical structural steady-state damage at the level of single chemical bond breakage.

[0043] Example 2

[0044] The following are common methods for nanopore electrochemical detection of brain natriuretic peptide (BNP) protein chemical structure steady-state damage in solutions:

[0045] 1. Prepare the solution:

[0046] (1) Preparation of basic buffer solution: Dissolve sodium chloride and tris(hydroxymethyl)aminomethane (Tris) in deionized water and adjust the pH of the solution to 7.4 to form a basic buffer solution containing sodium chloride and tris(hydroxymethyl)aminomethane (Tris) (the concentration of sodium chloride is 1M and the concentration of tris(hydroxymethyl)aminomethane (Tris) is 10mM). Finally, filter the buffer solution using a 0.22um filter before use.

[0047] (2) Preparation of protein samples: Dissolve brain natriuretic peptide BNP and its linear protein in enzyme-free water to obtain solutions with a concentration of 2mM, and store them in a refrigerator at -20℃ for later use.

[0048] (3) Preparation of reduction reaction protein sample: Add 6mM of reducing agent TCEP to the brain natriuretic peptide (BNP) protein sample solution prepared in step (2), mix well and place it in a metal bath at 37°C for 2 hours to fully destroy the disulfide bond between the two cysteine ​​residues in the brain natriuretic peptide protein structure.

[0049] 2. Constructing a nanoporous electrochemical detection and analysis device: A supporting film containing nanopores (the supporting film is made of graphene) is placed in a solution chamber containing an electrolyte. Two electrodes (both of which are silver electrode materials with silver chloride coating) are placed in solution chambers at both ends of the supporting film, and a power supply and an ammeter are connected in sequence between the two electrodes to form a nanoporous electrochemical detection and analysis device.

[0050] 3. Protein detection:

[0051] (1) The prepared brain natriuretic peptide BNP, linear BNP and the prepared reduced brain natriuretic peptide BNP protein sample solution (5.0 μM) were added separately to the above-mentioned nanopore electrochemical analysis device. The above-mentioned basic buffer solution was used as the electrolyte. After applying an external electric field, the protein sample in the protein sample solution was driven to interact with the nanopore under the action of the electric field force, and the changing current signal was collected.

[0052] (2) The current signal collected in step (1) was amplified using a low-noise current amplifier (Axon Axopatch 200B), and then the amplitude and current lag time characteristics of the current signal were analyzed (the collected electrical signal file was selected by the software clamfit to select the characteristic electrical signal, and then the signal was statistically analyzed by Origin) to obtain the corresponding protein sample feature map.

[0053] Proteins can be analyzed and compared, enabling the analysis and detection of proteins using nanopores, such as... Figure 2 As shown, a is the current graph of the test solution containing brain natriuretic peptide (BNP), b is the current graph of the solution containing linear BNP, c is the current graph of the solution containing BNP reduced by the reducing agent TCEP, and df is a scatter plot of BNP proteins corresponding to three different structures. Figure 2As can be seen from the comparison of the proportion of blocking signal amplitude and the blocking current time, there are significant differences between BNP, linear BNP, and BNP whose original disulfide bonds in the protein structure were disrupted by the reducing agent TCEP. Analysis of their scatter plots also shows that the current generated by the interaction of the three BNP structures with the nanopores is different. The BNP reduced by TCEP did not form a protein structure completely consistent with the linear structure, indicating that some kind of protein structural homeostasis damage occurred during the reduction process, preventing it from being reduced to the same structure as the linear structure. This further demonstrates that the nanopore has excellent resolution and can distinguish different protein homeostasis structures, identifying chemical structural homeostasis damage at the level of single chemical bond breakage.

[0054] Example 3

[0055] The following is a common method for using nanopore electrochemical detection to study the steady-state damage of the chemical structure of C-type natriuretic peptide (CNP) protein in solution:

[0056] 1. Prepare the solution:

[0057] (1) Preparation of basic buffer solution: Dissolve sodium chloride and tris(hydroxymethyl)aminomethane (Tris) in deionized water and adjust the pH of the solution to 7.4 to form a basic buffer solution containing sodium chloride and tris(hydroxymethyl)aminomethane (Tris) (the concentration of sodium chloride is 1M and the concentration of tris(hydroxymethyl)aminomethane (Tris) is 10mM). Finally, filter the buffer solution using a 0.22um filter before use.

[0058] (2) Preparation of protein samples: C-type natriuretic peptide (CNP) and its linear protein were dissolved in enzyme-free water to obtain solutions with a concentration of 2 mM, and then stored in a refrigerator at -20°C for later use.

[0059] (3) Preparation of reduction reaction protein sample: Add 6mM of reducing agent TCEP to the C-type natriuretic peptide (CNP) protein sample solution prepared in step (2), mix well and place it in a metal bath at 37°C for 2 hours to fully destroy the disulfide bond between the two cysteine ​​residues in the C-type natriuretic peptide protein structure.

[0060] 2. Constructing a nanoporous electrochemical detection and analysis device: A supporting film containing nanopores (the material of the supporting film is molybdenum disulfide) is placed in a solution chamber containing an electrolyte. Two electrodes (both of which are silver electrode materials with silver chloride coating) are placed in the solution chambers at both ends of the supporting film, and a power supply and an ammeter are connected in sequence between the two electrodes to form a nanoporous electrochemical detection and analysis device.

[0061] 3. Protein detection:

[0062] (1) The prepared C-type natriuretic peptide CNP, linear CNP and the C-type natriuretic peptide CNP protein sample solution (5.0 μM) after reduction reaction were added separately to the above-mentioned nanopore electrochemical analysis device. The above-mentioned basic buffer solution was used as the electrolyte. After applying an external electric field, the protein sample in the protein sample solution was driven to interact with the nanopore under the action of the electric field force, and the changing current signal was collected at this time.

[0063] (2) The current signal collected in step (1) was amplified using a low-noise current amplifier (Axon Axopatch 200B), and then the amplitude and current lag time characteristics of the current signal were analyzed (the collected electrical signal file was selected by the software clamfit to select the characteristic electrical signal, and then the signal was statistically analyzed by Origin) to obtain the corresponding protein sample feature map.

[0064] Proteins can be analyzed and compared, enabling the analysis and detection of proteins using nanopores, such as... Figure 3 As shown, a is the current graph of the test solution containing C-type natriuretic peptide (CNP), b is the current graph of the solution containing linear C-type natriuretic peptide (CNP), c is the current graph of the solution containing C-type natriuretic peptide (CNP) reduced by the reducing agent TCEP, and df is a scatter plot of brain natriuretic peptide proteins corresponding to three different structures. Figure 3 It can be seen from the comparison of the proportion of blocking signal amplitude and the blocking current time that there are obvious differences between C-type natriuretic peptide (CNP), linear CNP, and CNP whose original disulfide bonds in the protein structure were destroyed by the reducing agent TCEP. Analysis of their scatter plots also shows that the current generated by the interaction of the three CNP structures with the nanopores is different. The CNP (CNP) reduced by the reducing agent TCEP did not form a protein structure completely consistent with the linear structure, indicating that some kind of protein structural homeostasis damage occurred during the reduction of CNP, preventing it from being reduced to the same structure as the linear structure. This further demonstrates that the nanopore has excellent resolution and can distinguish different protein homeostasis structures, identifying chemical structural homeostasis damage at the level of single chemical bond breakage.

[0065] Figure 4This is a schematic diagram illustrating the principle of reducing disulfide bonds in the chemical structure of natriuretic peptides using the reducing agent TCEP. As can be seen, the three natriuretic peptides of this invention (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or c-type natriuretic peptide (CNP)) form a relatively obvious linear structure after being reduced by the reducing agent TCEP in a nanopore electrochemical detection device. This makes them easier to pass through the nanopores in the nanopore electrochemical detection device, thereby generating electrical signal variations to test their structural steady-state damage.

[0066] In summary, this invention discloses a method for single-molecule detection of protein structural steady-state damage using nanopores. By setting up nanopores in an electrochemical detection and analysis device, electrical signals are collected before and after the protein sample interacts with the nanopores. The amplitude, retardation time, and signal intensity of the electrical signals are analyzed to detect information such as protein content, charged properties, length, or molecular weight. This method can detect three natriuretic peptides (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or c-type natriuretic peptide (CNP)) and their corresponding linear structures. Electrochemical signals are collected in the electrochemical analysis device to distinguish different structures of the same protein, thus realizing the cross-application of nanopores and bioinformatics. It can detect three natriuretic peptides in the natriuretic peptide family and their corresponding linear structures. This application has the advantages of low cost, label-free operation, and convenient detection. In addition, the method of the present invention fully utilizes the advantages of low cost, label-free and rapid operation of nanopore electrochemical devices, accurately records the minute current changes caused by damage to the chemical structure of proteins, and has good repeatability and high reliability.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting steady-state damage to protein structures using nanopore single molecules, characterized in that, The method specifically involves: setting 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 the electrical signal of the protein sample; activating the nanopore electrochemical detection device until the reducing agent in the nanopore electrochemical detection device disrupts the chemical structural steady state of the protein sample; and then detecting the electrical signal again. The amplitude, retardation time, or signal quantity characteristics of the two electrical signals are analyzed to detect changes in the structural steady-state damage of the protein sample at the single chemical bond level.

2. The method according to claim 1, characterized in that, The nanopore electrochemical detection device is assembled as follows: a supporting film containing nanopores is placed in a solution chamber containing electrolyte, two electrodes are placed in solution chambers containing solution at both ends of the supporting film, and a power supply and an ammeter are connected in sequence between the two electrodes to form the nanopore electrochemical detection device. The material of the supporting film is any one of silicon nitride, graphene, molybdenum disulfide, or Teflon film; The electrode material is a silver electrode material with silver chloride coated on its surface.

3. The method according to claim 2, characterized in that, The nanopore electrochemical detection device also includes a basic buffer solution; The basic buffer solution is a mixed solution of alkali metal chloride and tris(hydroxymethyl)aminomethane (Tris) dissolved in water; The concentration of alkali metal chloride in the basal buffer is 0.1–2 M, and the concentration of tris(hydroxymethyl)aminomethane is 1–50 mM.

4. The method according to claim 3, characterized in that, The pH of the base buffer solution is 7.

4.

5. The method according to claim 3, characterized in that, The alkali metal chloride is any one or more of sodium chloride, potassium chloride, or lithium chloride.

6. The method according to claim 1, characterized in that, The protein samples include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or c-type natriuretic peptide (CNP).

7. The method according to claim 1, characterized in that, The reducing agent in the nanoporous electrochemical detection device is trichloroethyl phosphate flame retardant plasticizer (TCEP).

8. The method according to claim 1, characterized in that, During the process of the reducing agent disrupting the chemical structural stability of the protein sample, the molar ratio of the protein sample to the reducing agent is 1:3, and the condition is: reaction in a metal bath at 37°C for 2 h.

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