Electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition

Abstract

The invention provides an electrochemical method for detecting B-type brain natriuretic peptide or an N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition, which comprises the following steps: S1, electrode surface function modification: soaking an electrode in a modification solution containing a functional reagent, and reacting in a dark place at room temperature, or electrochemically depositing the functional reagent in the modification solution on the surface of the electrode to obtain a modified electrode surface; a functionally modified electrode is obtained; s2, antibody immobilization; s3, carrying out antigen-antibody mutual reaction to obtain antigens / antibodies / electrodes with different concentrations; and S4, electrochemical detection. Through the interaction of the functional reagent and the surface of the electrode, an electrode sensing interface which can be combined with an antibody and can resist non-specific adsorption of non-target molecules in a complex sample is constructed; the immobilized antibody and the target BNP or NT-proBNP are subjected to a specific recognition effect, and the target BNP or NT-proBNP of a sample is captured, so that the BNP or NT-proBNP is rapidly and sensitively detected without a label.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensing, and particularly relates to an electrochemical method for detecting B-type natriuretic peptide or N-terminal pro-B-type natriuretic peptide based on antigen-antibody recognition. Background Art

[0002] Heart failure (HF) is an acute disease and an important cause of morbidity and mortality in cardiovascular diseases. B-type natriuretic peptide (BNP) or N-terminal pro-B-type natriuretic peptide (NT-proBNP) are two reliable and promising biomarkers for heart failure. Their contents in the blood of healthy humans are relatively low (BNP is about 20 pg / mL; NT-proBNP is less than 125 pg / mL). In patients with acute heart failure, BNP rises to about 2 ng / mL. The cut-off value for clinical diagnosis of BNP is 100 pg / ml, and its half-life in vivo is about 18 min and can be stable for 4 h in vitro. The cut-off value for clinical diagnosis of NT-proBNP is 300 pg / ml, and its half-life in vivo is about 1 - 2 h and can be stable for 72 h at room temperature in vitro. Detection of BNP and NT-proBNP can be used for auxiliary diagnosis and prognosis evaluation of HF.

[0003] Most of the reported methods for detecting BNP and NT-proBNP based on antigen-antibody recognition require labeling. Labeled immunoassays usually use enzyme, fluorescence or chemiluminescence methods to detect target analytes. Although labeled immunoassays have high sensitivity, they require expensive and bulky instruments and need multi-step detection to complete the experiment, which is time-consuming. A biosensor is an analytical device that uses a biological specific recognition process to detect a specific analyte. A typical biosensor mainly consists of two basic elements: one is the "recognition element" that can selectively respond to a specific analyte (such as DNA, antibody or cell), thus minimizing interference from other sample components and being responsible for the selective recognition of the target analyte; the other is the "transduction element", a sensor or detection device that converts a biological recognition event into a recognizable signal. An electrochemical biosensor refers to a type of biosensor that converts the response event occurring on the electrode surface (recognition element) into an electrical signal (current, resistance, potential, etc.) through the transduction element. Electrochemical biosensors are very popular point-of-care testing devices in the detection field due to their advantages such as high sensitivity, simple instrument and miniaturization.

[0004] In the prior art, by utilizing the high specific surface area and electrical conductivity of inorganic nanomaterials, bovine serum albumin (BSA), glutaraldehyde, and multi-walled carbon nanotubes are organically combined together to construct a nano-porous membrane interface on the surface of a screen-printed carbon electrode (SPCE). A glycated hemoglobin antibody is immobilized on the porous membrane interface. Through the recognition of antigen and antibody, a label-free electrochemical detection method is used to achieve the rapid detection of glycated hemoglobin.

[0005] However, due to the low content of BNP and NT-proBNP in blood samples and their short half-life, there is an urgent need to develop a highly sensitive detection method to determine them. Summary of the Invention

[0006] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide an electrochemical method for detecting B-type natriuretic peptide or N-terminal pro-brain natriuretic peptide based on antigen-antibody recognition, so as to achieve label-free, rapid, and sensitive detection of the target BNP or NT-proBNP.

[0007] To achieve the above purpose and other related purposes, the present invention provides an electrochemical method for detecting B-type natriuretic peptide or N-terminal pro-brain natriuretic peptide based on antigen-antibody recognition. The electrochemical method includes the following steps:

[0008] S1. Functional modification of the electrode surface: Immerse the electrode in a modification solution containing a functionalizing reagent and react in the dark at room temperature, or electrochemically deposit the functionalizing reagent in the modification solution onto the surface of the electrode. After rinsing and drying, a functionally modified electrode is obtained.

[0009] S2. Antibody immobilization: First, activate the surface of the functionally modified electrode, then drop an antibody solution onto the activated electrode surface. After reacting in the dark, wash and blow dry, and block the remaining carboxyl binding sites on the electrode surface to obtain an antibody / electrode.

[0010] S3. Antigen-antibody interaction: Drop different concentrations of antigens onto the surfaces of different antibody / electrodes respectively, react at 37°C for 10 minutes. After the reaction is completed, wash and blow dry to obtain antigen / antibody / electrodes with different concentrations.

[0011] S4. Electrochemical detection: Drop a mixed solution of potassium ferricyanide and potassium ferrocyanide onto the surfaces of the antigen / antibody / electrode at different concentrations respectively, and conduct electrochemical detection using an electrochemical workstation. Preferably, the modification solution in step S1 includes solution A, or a combination of solution A and solution B; wherein, the functional reagent in solution A includes one of 4-mercaptobenzoic acid, 4-aminobenzoic acid, 4-diazo-benzoic acid; the functional reagent in solution B includes one or a combination of 4-mercaptophenol, mercaptopropionylcholine, mercaptohexanol, 4-aminophenol, aminopropionylcholine, aminohexanol.

[0012] Preferably, the electrode in step S1 includes one of a gold electrode, a carbon electrode, and an indium tin oxide electrode.

[0013] Preferably, when the modification solution in step S1 is solution A, the concentration of solution A is 1 - 10 mmol / L.

[0014] Preferably, when the modification solution in step S1 is a mixed solution of solution A and solution B, the molar ratio between solution A and solution B is 1:1. In the modification solution, the concentration of solution A is 1 - 10 mmol / L, and the concentration of solution B is 1 - 10 mmol / L.

[0015] Preferably, when the electrode in step S1 is a gold electrode, before surface functional modification of the gold electrode, the surface of the gold electrode needs to be pretreated. The specific steps of the pretreatment include:

[0016] Drop 20 μL of 0.5 mol / L sodium hydroxide, 0.5 mol / L sulfuric acid, 0.1 mol / L sulfuric acid prepared from 0.01 mol / L potassium chloride, and 0.05 mol / L sulfuric acid onto the surface of the gold electrode in sequence, and conduct electrochemical cleaning respectively using cyclic voltammetry to remove impurities on the surface of the gold electrode, then rinse it with ultrapure water until clean, and finally dry it with nitrogen.

[0017] Preferably, the specific steps for activating the surface of the functionally modified electrode in step S2 include:

[0018] Mix 0.4 mol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.1 mol / L N-hydroxysuccinimide according to a volume ratio of 1:1 to obtain an activation mixture;

[0019] Take 20 - 80 μL of the activation mixture and drop it onto the surface of the functionally modified electrode, react for 8 - 20 min to activate the carboxyl group. After activation, rinse it with a 4-morpholineethanesulfonic acid buffer solution with a pH of 6.0, and then dry it with nitrogen.

[0020] Preferably, the antibody solution in step S2 includes a BNP antibody solution, an NT-proBNP antibody solution, or a BNP nucleic acid aptamer solution composed of single-stranded oligonucleotides; the concentrations of the BNP antibody solution and the NT-proBNP antibody solution are both 100-1000 μg / mL; the concentration of the BNP nucleic acid aptamer solution is 10-100 μmol / L.

[0021] Preferably, the amount of the antibody solution dropped on the surface of the activated electrode in step S2 is 20-80 μL.

[0022] Preferably, the temperature of the light-shielded reaction in step S2 is 25°C to 37°C, and the time of the light-shielded reaction is 2 h to 12 h.

[0023] Preferably, the washing in both step S2 and step S3 includes: washing 3-6 times with a phosphate buffer solution containing 0.05% Tween-20, and then washing 3-5 times with a phosphate buffer solution.

[0024] Preferably, the step of blocking the remaining carboxyl binding sites on the electrode surface in step S2 includes: taking 30 μL of a 5% casein blocking solution and dropping it on the electrode surface, and reacting for 30 min to block the remaining carboxyl groups on the electrode surface. Preferably, the antigen in step S3 is BNP or NT-proBNP, and the concentration range of the antigen is 0-200000 pg / mL.

[0025] Preferably, the concentration of the mixed solution of potassium ferricyanide and potassium ferrocyanide in step S4 is 1-5 mmol / L, and the amount of the mixed solution of potassium ferricyanide and potassium ferrocyanide to be dropped is 25-100 μL.

[0026] Preferably, the electrochemical detection in step S4 is differential pulse voltammetry and / or alternating current impedance detection.

[0027] Preferably, the conditions for the differential pulse voltammetry detection are: the voltage range is -0.2 V to 0.8 V, the amplitude is 5 mV, and the pulse period is 0.5 s.

[0028] Preferably, the frequency of the alternating current impedance detection is 0.1 MHz to 0.1 Hz, and the amplitude is 5 mV.

[0029] As described above, the electrochemical method for detecting BNP or NT-proBNP based on antigen-antibody recognition of the present invention has the following beneficial effects:

[0030] By modifying the interaction between the functional reagent in the solution and the electrode surface, the present invention performs functional modification on the electrode surface to construct an electrode sensing interface that can both bind antibodies and resist the non-specific adsorption of non-target molecules in complex samples. Then, the immobilized antibody specifically recognizes the target BNP or NT-proBNP antigen, captures the target BNP or NT-proBNP in the sample, and performs electrochemical detection by label-free electrochemical methods (differential pulse voltammetry or alternating current impedance), realizing label-free, rapid, and sensitive detection of BNP or NT-proBNP. It does not require expensive and bulky large-scale instruments, and label-free reduces the experimental steps, achieving rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It shows the electrochemical process flow chart of the present invention for detecting B-type natriuretic peptide or N-terminal pro-B-type natriuretic peptide based on antigen-antibody recognition.

[0032] Figure 2 It shows a schematic diagram of the present invention for detecting different concentrations of B-type natriuretic peptide (BNP) or N-terminal pro-B-type natriuretic peptide (NT-proBNP) by electrochemical method.

[0033] Figure 3 It shows the differential pulse voltammogram of detecting different concentrations of BNP by differential pulse voltammetry in Example 1 of the present invention.

[0034] Figure 4 It shows the standard curve graph of detecting different concentrations of BNP by differential pulse voltammetry in Example 1 of the present invention.

[0035] Figure 5 It shows the impedance graph of detecting different concentrations of BNP by alternating current impedance in Example 1 of the present invention.

[0036] Figure 6 It shows the specific investigation of detecting different targets by alternating current impedance in Example 2 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The following specific examples illustrate the embodiments 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 also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0038] Please refer to the attached drawings. It should be noted that the illustrations provided in this embodiment only illustrate the basic concept of the present invention in a schematic manner. Therefore, only the components related to the present invention are shown in the drawings, rather than being drawn according to the number, shape, and size of the components in actual implementation. The type, quantity, and proportion of each component in actual implementation can be arbitrarily changed, and the component layout type may also be more complex.

[0039] Due to the relatively complex composition of the actual sample, non-specific adsorption is likely to occur on the electrode surface. When identifying the target analyte, it is necessary to reduce the non-specific adsorption of non-target substances in the sample. This requires constructing an electrode sensing surface that can both identify the target analyte and reduce the adsorption of non-target substances. The present invention utilizes the interaction between the functionalized reagent in the modification solution and the electrode surface to perform functional modification on the electrode surface, and then immobilize the antibody. Through the specific recognition between the antibody and the antigen, BNP or NT-proBNP in the sample is captured, and through electrochemical detection, a method for rapidly and sensitively detecting BNP or NT-proBNP is provided.

[0040] Refer to Figure 1 、 Figure 2 , the present invention provides an electrochemical method for identifying the heart failure protein markers B-type natriuretic peptide (BNP) or N-terminal pro-brain natriuretic peptide (NT-proBNP) based on antigen-antibody recognition, including the following steps:

[0041] S1. Functional modification of the electrode surface: Immerse the electrode in a modification solution containing a functionalized reagent and react in the dark at room temperature, or deposit the functionalized reagent in the modification solution onto the electrode surface by electrochemical deposition. After rinsing and drying, a functionally modified electrode is obtained;

[0042] S2. Antibody immobilization: First, activate the surface of the functionally modified electrode, then drop the antibody solution onto the activated electrode surface. After reacting in the dark, wash and blow dry, and block the remaining carboxyl binding sites on the electrode surface to obtain an antibody / electrode;

[0043] S3. Antigen-antibody interaction: Drop different concentrations of antigens onto the surfaces of different antibody / electrodes respectively, react at 37 °C for 10 min, and after the reaction is completed, wash and blow dry to obtain antigen / antibody / electrodes with different concentrations;

[0044] S4. Electrochemical detection: Drop a mixed solution of potassium ferricyanide and potassium ferrocyanide onto the surfaces of antigen / antibody / electrodes with different concentrations respectively, and perform electrochemical detection using an electrochemical workstation.

[0045] Specifically, in steps S2 and S3, a fixed antibody specifically recognizes and interacts with the target BNP or NT-proBNP to capture the target BNP or NT-proBNP in the sample; then, label-free electrochemical detection (differential pulse voltammetry or alternating current impedance) is performed to achieve label-free, rapid, and sensitive detection of BNP or NT-proBNP.

[0046] Preferably, when performing functional modification on the electrode surface, the electrode is immersed in a modification solution containing a functionalizing reagent and reacted at room temperature in the dark for 12 h.

[0047] As an example, in step S1, the modification solution includes solution A, or a combination of solution A and solution B; wherein, the functionalizing reagent in solution A includes one of 4-mercaptobenzoic acid, 4-aminobenzoic acid, and 4-diazo-benzoic acid; the functionalizing reagent in solution B includes one or a combination of 4-mercaptophenol, mercaptopropionylcholine, mercaptohexanol, 4-aminophenol, aminopropionylcholine, and aminohexanol.

[0048] Specifically, the modification solution can be solution A alone or a mixed solution of solution A and solution B.

[0049] As an example, the electrode in step S1 includes one of a gold electrode, a carbon electrode, and an indium tin oxide electrode.

[0050] As an example, when the modification solution is solution A, the concentration of solution A is 1 - 10 mmol / L.

[0051] Specifically, the concentration of solution A can include any value within the range such as 1 mmol / L, 3 mmol / L, 5 mmol / L, 7 mmol / L, 9 mmol / L, 10 mmol / L, etc.

[0052] Preferably, the modification solution is a 4-mercaptobenzoic acid solution, and the concentration of the 4-mercaptobenzoic acid solution is 5 mmol / L.

[0053] As an example, when the modification solution in step S1 is a mixed solution of solution A and solution B, the molar ratio between solution A and solution B is 1:1. In the modification solution, the concentration of solution A is 1 - 10 mmol / L, and the concentration of solution B is 1 - 10 mmol / L.

[0054] Specifically, when the modification solution is a mixed solution of solution A and solution B, the molar ratio of the two is 1:1, and the concentration of solution A can include any value within the range of 1 mmol / L, 3 mmol / L, 5 mmol / L, 7 mmol / L, 9 mmol / L, 10 mmol / L, etc., and the concentration of solution B can include any value within the range of 1 mmol / L, 3 mmol / L, 5 mmol / L, 7 mmol / L, 9 mmol / L, 10 mmol / L, etc.

[0055] Preferably, in step S1, the modification solution is a mixed solution of 4-mercaptobenzoic acid and 4-mercaptophenol, and the molar ratio between 4-mercaptobenzoic acid and 4-mercaptophenol is 1:1. In the mixed solution, the concentration of 4-mercaptobenzoic acid is 5 mmol / L, and the concentration of 4-mercaptophenol is 5 mmol / L.

[0056] Specifically, in step S1, the functional groups such as mercapto, amino or diazo groups of the functionalizing reagent in the modification solution interact with the electrode surface to perform functional modification on the electrode surface, fix the functional molecules on the electrode surface, and perform rinsing after the reaction is completed. In the specific embodiments of the present invention, anhydrous ethanol is used for rinsing 5 times; thereby constructing an electrode sensing interface that can both bind antibodies and resist non-specific adsorption of non-target molecules in complex samples.

[0057] As an example, when the electrode in step S1 is a gold electrode, before the surface of the gold electrode is functionally modified, the surface of the gold electrode needs to be pretreated. The specific steps of the pretreatment include:

[0058] 20 μL of 0.5 mol / L sodium hydroxide, 0.5 mol / L sulfuric acid, 0.1 mol / L sulfuric acid prepared from 0.01 mol / L potassium chloride, and 0.05 mol / L sulfuric acid are successively dropped onto the surface of the gold electrode, and cyclic voltammetry is used for electrochemical cleaning respectively to remove impurities on the surface of the gold electrode, then rinsed with ultrapure water until clean, and finally dried with nitrogen.

[0059] Specifically, first drop 20 μL of 0.5 mol / L sodium hydroxide onto the surface of the gold electrode and perform electrochemical cleaning using cyclic voltammetry; then drop 20 μL of 0.5 mol / L sulfuric acid onto the surface of the gold electrode for one electrochemical cleaning, then drop 20 μL of 0.1 mol / L sulfuric acid (containing 0.01 mol / L potassium chloride) onto the surface of the gold electrode for one electrochemical cleaning, and finally drop 0.05 mol / L sulfuric acid onto the surface of the gold electrode for one electrochemical cleaning to remove impurities on the surface of the gold electrode, then rinse with ultrapure water until clean, and finally dried with nitrogen.

[0060] As an example, the specific steps for activating the surface of the functionally modified electrode in step S2 include:

[0061] Take 0.4 mol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.1 mol / L N-hydroxysuccinimide (NHS) and mix them according to a volume ratio of 1:1 to obtain an activation mixture.

[0062] Take 20 - 80 μL of the activation mixture and drop it onto the surface of the functionally modified electrode, and react for 8 - 20 min to activate the carboxyl group. After the activation is completed, rinse it with a 4-morpholineethanesulfonic acid buffer solution with a pH of 6.0, and then dry it with nitrogen.

[0063] Specifically, the amount of the activation mixture dropped onto the surface of the functionally modified electrode may include any value within the range such as 20 μL, 40 μL, 60 μL, 80 μL, etc., and can be specifically adjusted according to the actual situation; the reaction time may include any value within the range such as 8 min, 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, etc. Preferably, take 20 μL of the activation mixture and drop it onto the surface of the functionally modified electrode, and react for 10 min to activate the carboxyl group.

[0064] As an example, in step S2, the antibody solution includes a BNP antibody solution, an NT-proBNP antibody solution, or a BNP nucleic acid aptamer solution composed of single-stranded oligonucleotides; the concentrations of both the BNP antibody solution and the NT-proBNP antibody solution are 100 - 1000 μg / mL; the concentration of the BNP nucleic acid aptamer is 10 - 100 μmol / L.

[0065] Specifically, the nucleic acid aptamer can achieve high-affinity and high-specificity recognition with the target, similar to an antibody, and is also known as a "chemical antibody"; the concentrations of the BNP antibody solution and the NT-proBNP antibody solution may include any value within the range such as 100 μg / mL, 300 μg / mL, 500 μg / mL, 700 μg / mL, 900 μg / mL, 1000 μg / mL, etc.; the concentration of the BNP nucleic acid aptamer may include any value within the range such as 10 μmol / L, 30 μmol / L, 50 μmol / L, 70 μmol / L, 90 μmol / L, 100 μmol / L, etc.

[0066] As an example, in step S2, the amount of the BNP antibody or NT-proBNP antibody or BNP nucleic acid aptamer dropped onto the surface of the activated electrode is 20 - 80 μL.

[0067] Specifically, the amount of the BNP antibody or NT-proBNP antibody or BNP nucleic acid aptamer dropped onto the surface of the activated electrode in step S2 may include any value within the range such as 20 μL, 40 μL, 60 μL, 80 μL, etc.

[0068] As an example, the temperature of the light-shielded reaction in step S2 is 25°C to 37°C, and the time of the light-shielded reaction is 2 h to 12 h.

[0069] Specifically, the temperature of the light-shielded reaction may include any value within the range such as 25°C, 30°C, 32°C, 34°C, 36°C, 37°C, etc., and the time of the light-shielded reaction may include any value within the range such as 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, etc.

[0070] Preferably, 20 μL of an antibody solution with a concentration of 100 μg / mL is dropped onto the surface of the activated electrode, and a light-shielded reaction is carried out at 37°C for 2 h, or a reaction is carried out at 25°C for 12 h.

[0071] As an example, the washing in step S2 is carried out by washing 3 to 6 times with a phosphate buffer solution (PBST) containing 0.05% Tween-20, and then washing 3 to 5 times with a phosphate buffer solution (PBS).

[0072] Specifically, the number of times of washing with PBST can be 3, 4, 5, 6 times, etc., and the number of times of washing with PBS can be 3, 4, 5 times, and is specifically adjusted according to the actual situation.

[0073] As an example, the step of blocking the remaining carboxyl binding sites on the electrode surface in step S2 includes: taking 30 μL of a 5% casein blocking solution and dropping it onto the electrode surface, reacting for 30 min to block the remaining carboxyl groups on the electrode surface, and after the reaction is completed, washing and drying.

[0074] Specifically, the purpose of blocking the remaining carboxyl binding sites on the electrode surface is to reduce the binding of other substances to the electrode surface during the detection of BNP or NT-proBNP, resulting in false positive signals.

[0075] After the reaction of blocking the remaining carboxyl binding sites on the electrode surface is completed, it is washed 3 times with a phosphate buffer solution (PBST) containing 0.05% Tween-20, washed 2 times with a phosphate buffer solution (PBS), and then dried with nitrogen.

[0076] As an example, the antigen in step S3 is BNP or NT-proBNP, and the concentration range of the antigen is 0 to 200000 pg / mL.

[0077] Specifically, the concentration of BNP or NT-proBNP in step S3 may include any value within the range such as 0, 20 pg / mL, 200 pg / mL, 2000 pg / mL, 20000 pg / mL, 200000 pg / mL, etc.; the target BNP or NT-proBNP with different concentrations is prepared with 5% casein.

[0078] In addition, after the antigen-antibody interaction in step S3, washing is required. The washing is adjusted to be performed 3 to 6 times with a phosphate buffer solution (PBST) containing 0.05% Tween-20, and then 3 to 5 times with a phosphate buffer solution (PBS); preferably, it is washed 3 times with PBST, 3 times with PBS, and then dried with nitrogen.

[0079] As an example, the concentration of the potassium ferricyanide and potassium ferrocyanide mixed solution is 1 to 5 mmol / L, and the amount of the potassium ferricyanide and potassium ferrocyanide mixed solution to be added dropwise is 25 to 100 μL.

[0080] Specifically, the concentration of the potassium ferricyanide and potassium ferrocyanide mixed solution may include any value within the range such as 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, etc., and the amount of the potassium ferricyanide and potassium ferrocyanide mixed solution to be added dropwise may include any value within the range such as 25 μL, 50 μL, 75 μL, 100 μL, etc. Preferably, the concentration of the potassium ferricyanide and potassium ferrocyanide mixed solution is 5 mmol / L, and the amount of the potassium ferricyanide and potassium ferrocyanide mixed solution to be added dropwise is 30 μL.

[0081] As an example, the electrochemical detection in step S4 is differential pulse voltammetry (DPV) detection and / or alternating current impedance (EIS) detection.

[0082] As an example, the conditions for differential pulse voltammetry detection are: the voltage is -0.2V to 0.8V, the amplitude is 5 mV, and the pulse period is 0.5 s.

[0083] Specifically, the voltage may include any value within the range such as -0.2V, 0V, 0.2V, 0.4V, 0.6V, 0.8V, etc.

[0084] As an example, the frequency of the alternating current impedance is 0.1 MHz to 0.1 Hz, and the amplitude is 5 mV.

[0085] Specifically, the frequency of the alternating current impedance may include any value within the range such as 0.1 Hz, 1 Hz, 10 Hz, 100 Hz, 1000 Hz, 10 kHz, 0.1 MHz, etc., and can be specifically adjusted according to the actual situation.

[0086] To better understand the electrochemical method for detecting B-type natriuretic peptide or N-terminal pro-B-type natriuretic peptide based on antigen-antibody recognition in the present invention, the following describes the electrochemical method for detecting B-type natriuretic peptide or N-terminal pro-B-type natriuretic peptide based on antigen-antibody recognition in the present invention by referring to specific examples. It should be noted that these examples are merely descriptive and do not limit the present invention in any way.

[0087] Example 1

[0088] This embodiment provides an electrochemical method for detecting B-type natriuretic peptide based on antigen-antibody recognition, which includes the following steps:

[0089] S1. Functional modification of the gold electrode surface:

[0090] 20 μL of 0.5 mol / L sodium hydroxide, 0.5 mol / L sulfuric acid, 0.1 mol / L sulfuric acid (containing 0.01 mol / L potassium chloride), and 0.05 mol / L sulfuric acid are successively dropped onto the surface of the gold electrode, and cyclic voltammetry is used for electrochemical cleaning respectively to remove impurities on the surface of the gold electrode. Then, it is rinsed clean with ultrapure water, and finally dried with nitrogen;

[0091] The gold electrode is immersed in a 5 mmol / L 4-mercaptobenzoic acid solution and reacted in the dark at room temperature for 12 h. Gold-sulfur bonds are formed through the interaction between the mercapto group and gold, and 4-mercaptobenzoic acid is fixed on the surface of the gold electrode. After rinsing 5 times with absolute ethanol and drying, a functionally modified gold electrode is obtained;

[0092] S2. Fixation of BNP antibody: 0.4 mol / L EDC and 0.1 mol / L NHS are mixed according to a volume ratio of 1:1 to obtain an activation mixture; 20 μL of the activation mixture is taken with a pipette and dropped onto the surface of the functionally modified gold electrode, and the carboxyl group is activated by reacting for 10 min. After the activation is completed, it is rinsed 5 times with a 4-morpholineethanesulfonic acid buffer solution with a pH of 6.0, and then dried with nitrogen;

[0093] Then, 20 μL of a BNP monoclonal antibody solution with a concentration of 100 μg / mL is taken and dropped onto the surface of the activated gold electrode, and reacted in the dark at 37 °C for 2 h. It is washed 3 times with PBST and 3 times with PBS to wash away the BNP monoclonal antibody that is not bound to the electrode surface, and then dried with nitrogen; Then, 30 μL of a 5% mass concentration casein blocking solution is taken and dropped onto the surface of the gold electrode, and reacted for 30 min to block the remaining carboxyl binding sites on 4-mercaptobenzoic acid. After the reaction is completed, it is washed 3 times with PBST and 2 times with PBS, and then dried with nitrogen to obtain a BNP antibody / gold electrode;

[0094] S3. Antigen-antibody interaction reaction: 20 μL of BNP antigens with concentrations of 0, 20, 200, 2000, 20000, and 200000 pg / mL respectively, prepared from 5% mass concentration casein, are successively dropped onto the surfaces of different BNP antibody / gold electrodes, and reacted at 37 °C for 10 min. After the reaction is completed, it is washed 3 times with PBST and 3 times with PBS, and dried with nitrogen to obtain BNP / BNP antibody / gold electrodes with different concentrations;

[0095] S4. Electrochemical Detection: Drop 30 μL of a mixed solution of 5 mmol / L potassium ferricyanide and potassium ferrocyanide onto the surfaces of the gold electrodes with different concentrations of BNP / BNP antibody, and perform differential pulse voltammetry (voltage range: -0.2 V to 0.8 V, amplitude: 5 mV, pulse period: 0.5 s) and alternating current impedance (frequency: 0.1 MHz to 0.1 Hz, amplitude: 5 mV) detection using an electrochemical workstation.

[0096] See Figure 3 Figure is the differential pulse voltammogram for detecting BNP at different concentrations, obtained from the Figure 3 results. As the concentration of BNP increases, the peak current in the differential pulse voltammogram decreases; by plotting the change in the peak current in the differential pulse voltammogram in Figure 3 against the concentration of BNP, a standard curve graph is obtained (see Figure 4 ), achieving quantitative detection of BNP.

[0097] See Figure 5 Figure shows the detection results of detecting BNP at different concentrations by alternating current impedance, obtained from the Figure 5 results. As the concentration of BNP increases, the resistance in the alternating current impedance graph increases.

[0098] Example 2

[0099] This example provides an electrochemical detection method based on antigen-antibody recognition. The difference between this method and that in Example 1 is as follows: In the step of antigen-antibody interaction in step S3, replace the BNP with concentrations of 0, 20, 200, 2000, 20000, 200000 pg / mL prepared from 5% (mass concentration) casein with blank control (5% casein), 2000 pg / ml BNP (prepared from 5% casein), 2 mg / ml BSA (prepared from 5% casein), and 10 U / mL carbohydrate antigen 19-9 (CA19-9) calibration product (serum sample). Other steps and methods are the same as those in Example 1 and will not be elaborated here.

[0100] See Figure 6 Figure shows the specific investigation results of detecting different targets by alternating current impedance. It can be seen from the Figure 6 results that the impedance curves of high-concentration BSA (bovine serum albumin) and CA19-9 serum sample almost overlap with the impedance curve of the blank control, and only the impedance of 2000 pg / ml BNP has changed significantly, indicating that the electrochemical detection method based on antigen-antibody recognition in this invention has strong specificity and can specifically detect BNP.

[0101] In summary, the present invention constructs an electrode sensing interface that can both bind antibodies and resist non-specific adsorption of non-target molecules in complex samples by modifying the interaction between the functional reagent in the solution and the electrode surface; then, the immobilized antibody specifically recognizes the target BNP or NT-proBNP, captures the target BNP or NT-proBNP in the sample, and performs electrochemical detection by label-free electrochemical methods (differential pulse voltammetry or alternating current impedance), achieving label-free, rapid, and sensitive detection of BNP or NT-proBNP. Expensive and bulky large instruments are not required, and label-free reduces experimental steps and enables rapid detection. Therefore, the present invention effectively overcomes various drawbacks in the prior art and has high industrial utilization value.

[0102] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. An electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition, characterized in that: The electrochemical method comprises the following steps: S1. Functional modification of the electrode surface: immersing the electrode in a modification solution containing a functionalizing agent and reacting at room temperature in the dark, or electrochemically depositing the functionalizing agent in the modification solution onto the electrode surface, followed by rinsing and drying to obtain a functionally modified electrode; S2. Antibody immobilization: Activate the surface of the functionally modified electrode first, then drop the antibody solution onto the activated electrode surface, protect from light, and then wash and dry to block the remaining carboxyl binding sites on the electrode surface to obtain an antibody / electrode; S3. Antigen-antibody interaction: Different concentrations of antigens were added dropwise to the surface of different antibodies / electrodes, and the reaction was carried out at 37°C for 10 minutes. After the reaction was completed, the mixture was washed and dried to obtain antigens / antibodies / electrodes of different concentrations. S4. Electrochemical detection: A mixed solution of potassium ferrocyanide and potassium ferrocyanide was added dropwise to the antigen / antibody / electrode surface at different concentrations, and electrochemical detection was performed using an electrochemical workstation.

2. The electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition according to claim 1, characterized in that: Step S1 includes one or a combination of the following conditions: The modification solution includes solution A, or a combination of solution A and solution B; wherein the functionalizing agent in solution A includes one of 4-mercaptobenzoic acid, 4-aminobenzoic acid, and 4-diazonium-benzoic acid; and the functionalizing agent in solution B includes one of 4-mercaptophenol, mercaptophosphorylcholine, mercaptohexanol, 4-aminophenol, aminophosphorylcholine, and aminohexanol, or a combination thereof; The electrode includes one of a gold electrode, a carbon electrode, and an indium tin oxide electrode.

3. The electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition according to claim 2, characterized in that: Includes one or a combination of the following conditions: When the modification solution is solution A, the concentration of solution A is 1 to 10 mmol / L; When the modified solution is a mixed solution of solution A and solution B, the molar ratio of solution A to solution B is 1:

1. In the modified solution, the concentration of solution A is 1 to 10 mmol / L, and the concentration of solution B is 1 to 10 mmol / L.

4. The electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition according to claim 2, characterized in that: When the electrode in step S1 is a gold electrode, the surface of the gold electrode needs to be pretreated before functional modification of the surface of the gold electrode. The specific steps of the pretreatment include: 20 μL of 0.5 mol / L sodium hydroxide, 0.5 mol / L concentrated sulfuric acid, 0.1 mol / L concentrated sulfuric acid prepared from 0.01 mol / L potassium chloride, and 0.05 mol / L concentrated sulfuric acid were sequentially added dropwise to the surface of the gold electrode, and electrochemical cleaning was performed using cyclic voltammetry to remove impurities on the surface of the gold electrode. The electrode was then rinsed with ultrapure water and finally dried with nitrogen.

5. The electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition according to claim 1, characterized in that: The specific steps of activating the surface of the functionally modified electrode in step S2 include: According to a volume ratio of 1:1, 0.4 mol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.1 mol / L N-hydroxysuccinimide were mixed to obtain an activation mixture; 20 to 80 μL of the activation mixture was added dropwise to the surface of the functionally modified electrode and reacted for 8 to 20 minutes to activate the carboxyl groups. After activation, the electrode was rinsed with a pH 6.0 4-morpholineethanesulfonic acid buffer solution and then dried with nitrogen.

6. The electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition according to claim 1, characterized in that: Step S2 includes one or a combination of the following conditions: The antibody solution includes a BNP antibody solution, an NT-proBNP antibody solution, or a BNP nucleic acid aptamer solution composed of single-stranded oligonucleotides; the concentrations of the BNP antibody solution and the NT-proBNP antibody solution are both 100 to 1000 μg / mL; the concentration of the BNP nucleic acid aptamer solution is 10 to 100 μmol / L; The amount of the antibody solution added dropwise to the activated electrode surface is 20 to 80 μL; The temperature of the light-proof reaction is 25° C. to 37° C., and the time of the light-proof reaction is 2 h to 12 h.

7. The electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition according to claim 1, characterized in that: The washing in step S2 and step S3 both include: washing 3 to 6 times with a phosphate buffer solution containing 0.05% Tween-20, and then washing 3 to 5 times with a phosphate buffer solution.

8. The electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition according to claim 1, characterized in that: The step of blocking the remaining carboxyl binding sites on the electrode surface in step S2 includes: taking 30 μL of 5% casein blocking solution and adding it dropwise to the electrode surface, and reacting for 30 minutes to block the remaining carboxyl groups on the electrode surface.

9. The electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition according to claim 1, characterized in that: In step S3, the antigen is BNP or NT-proBNP, and the concentration range of the antigen is 0 to 200,000 pg / mL.

10. The electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition according to claim 1, characterized in that: Step S4 includes one or a combination of the following conditions: The concentration of the mixed solution of potassium ferrocyanide and potassium ferrocyanide is 1-5 mmol / L, and the amount of the mixed solution of potassium ferrocyanide and potassium ferrocyanide to be added dropwise is 25-100 μL; The electrochemical detection is differential pulse voltammetry and / or AC impedance detection.

11. The electrochemical method for detecting B-type brain natriuretic peptide or N-terminal B-type brain natriuretic peptide precursor based on antigen-antibody recognition according to claim 10, characterized in that: Includes one or a combination of the following conditions: The conditions for the differential pulse voltammetry detection are: voltage range of -0.2V to 0.8V, amplitude of 5mV, and pulse period of 0.5s; The frequency of the AC impedance detection is 0.1 MHz to 0.1 Hz, and the amplitude is 5 mV.

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