A kit and method for detecting a marker of heart failure

Abstract

The present application relates to a kind of kit and detection method for detecting heart failure marker, belong to heart failure marker detection technical field.The kit includes basic buffer A (pH=7.4, containing 0.5mol / L NaCl and 10mmol / L Tris aqueous solution), basic buffer B (pH=7.4, containing 3mol / L NaCl and 10mmol / L Tris aqueous solution) and washing liquid (0.5~3mol / L alkali metal chloride).The detection method disclosed in the present application mainly forms ion gradient direction on the two sides of the electrochemical device with nano-pore respectively by basic buffer A and basic buffer B, releases the electrochemical signal characteristics of large sodium peptide, compares the sample characteristic map of serum sample with the sample characteristic map of standard B-type natriuretic peptide by the amplitude of current signal and current block time characteristic analysis, detects and quantifies B-type natriuretic peptide in serum to-be-detected object, and its detection limit is as low as 767.92fmol / L.

Description

Technical Field

[0001] This invention belongs to the field of heart failure biomarker detection technology, and relates to a reagent kit and detection method for detecting heart failure biomarkers. Background Technology

[0002] Heart failure is a complex clinical syndrome, encompassing acute and critical stages of various cardiovascular diseases, such as coronary artery disease, hypertension, and valvular heart disease. Various cardiac structural or diseased conditions can impair ventricular filling or ejection capacity, leading to systolic or diastolic dysfunction. Heart failure occurs in the terminal stages of these diseases and is one of the leading causes of death in adults worldwide. Current global medical strategies focus on early detection and treatment to provide proactive, specific, and aggressive interventions. However, early diagnosis of heart failure is difficult. B-type natriuretic peptide (BNP) has gained international recognition as a biomarker for the diagnosis and prognosis of heart failure and is considered one of the gold standards for its diagnosis. In early heart failure, left ventricular cardiomyocytes secrete large amounts of BNP due to disease. This secretion status has extremely high accuracy and reliability in subsequent assessments, greatly facilitating the diagnosis and treatment of early heart failure. BNP is an important member of the natriuretic peptide family (NPs), sharing high structural homology with atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP). Natriuretic peptides have diuretic, natriuretic, and vasodilatory effects. Therefore, BNP can dilate blood vessels, reduce vascular resistance, promote higher stroke volume, increase renal sodium secretion, increase urine output, and decrease blood volume, thereby lowering blood pressure. Studies have shown that in addition to BNP, ANP and CNP are also associated with heart failure.

[0003] Due to the important role of BNP biomarkers in the diagnosis of heart failure, analytical immunosensors for detecting BNP in human serum and whole blood samples have been developed. Currently, BNP detection methods can be broadly classified into three categories. The first is radioimmunoassay, a competitive immunoassay using radiolabeled tracers. However, due to the presence of several BNP-related peptides in plasma, the specificity of this method varies greatly; for example, NT-proBNP can interfere with the detection values. To overcome the problem of specificity differences, a non-competitive immunoassay based on a two-site sandwich immunoassay has been proposed. However, the radiation from this method may cause harm to humans and the environment. The second method is electrochemiluminescent enzyme immunoassay. While this method has high specificity, it is time-consuming and very expensive. The third method is enzyme-linked immunosorbent assay (ELISA). This method is selective for BNP and is unaffected by natriuretic peptides, but it is also costly. Although the accuracy of the above methods meets the requirements, they are time-consuming, labor-intensive, and require professionally trained personnel. Currently, there is no method that can simultaneously detect the natriuretic peptide family; therefore, this invention proposes an electrochemical rapid quantitative kit for heart failure biomarkers and its detection method.

[0004] Biosensors are electronic devices that generate electronic signals through biological interactions using electrical, optical, chemical, or mechanical means. 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 important information on the structure and function of nucleic acids and proteins, and can monitor biochemical reactions in real time, solving the problems of traditional detection methods, such as low selectivity. Therefore, kits using nanopore sensors can achieve low-cost, simple, and efficient detection of heart failure biomarkers.

[0005] Therefore, it is necessary to conduct further in-depth research on methods for quantitative detection of heart failure biomarkers. Summary of the Invention

[0006] In view of this, one objective of the present invention is to provide a reagent kit for detecting heart failure biomarkers; another objective of the present invention is to provide a method for detecting heart failure biomarkers.

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

[0008] 1. A kit for detecting heart failure biomarkers, the kit comprising basal buffer A, basal buffer B, and washing buffer;

[0009] The heart failure marker is B-type natriuretic peptide;

[0010] The pH of the basal buffer A is 7.4, and the basal buffer A contains 0.5 mol / L NaCl and 10 mmol / L tris(hydroxymethyl)aminomethane (Tris) aqueous solution.

[0011] The pH of the basal buffer B is 7.4, and the basal buffer B contains 3 mol / L NaCl and 10 mmol / L tris(hydroxymethyl)aminomethane (Tris) aqueous solution.

[0012] The washing solution is an alkali metal chloride at a concentration of 0.5–3 mol / L.

[0013] Preferably, the alkali metal chloride is any one or more of sodium chloride, potassium chloride, or lithium chloride.

[0014] 2. A method for detecting a heart failure biomarker, wherein the heart failure biomarker is B-type natriuretic peptide, and the detection method includes the following steps:

[0015] (1) Dissolve the analyte in the blood sample and then dissolve it in enzyme-free water to form solution I with a concentration of 3-20000 μg / mL;

[0016] (2) Mix the above-mentioned kit for detecting heart failure markers with solution I to form a mixed solution;

[0017] (3) Place the mixed solution and solution I described in step (2) into an electrochemical device with nanopores, use the basic buffer A contained in the above kit as the electrolyte at the ground end and the basic buffer B as the electrolyte at the anode end, and collect the change in current signal after adding electric field strength;

[0018] (4) After amplifying the change in the current signal, perform amplitude and current hysteresis time characteristic analysis of the current signal to obtain the sample feature map;

[0019] (5) Compare the sample characteristic map of solution I with the sample characteristic map of standard B-type natriuretic peptide. If the current amplitude ratios of the sample characteristic map of solution I and the sample characteristic map of standard B-type natriuretic peptide are the same, it indicates that the analyte contains B-type natriuretic peptide; otherwise, it does not.

[0020] Comparing the sample characteristic map of the mixed solution with that of the standard B-type natriuretic peptide, the concentration curve of B-type natriuretic peptide is: C BNP =10 0.083*N-1.995 ,

[0021] Where N represents the number of electrochemical signaling events of B-type natriuretic peptide in the blood sample, C BNPThe concentration of B-type natriuretic peptide in the blood sample is expressed in pmol / L.

[0022] The beneficial effects of this invention are as follows:

[0023] 1. This invention discloses a kit for detecting heart failure biomarkers. The kit comprises basal buffer A (pH 7.4 containing 0.5 mol / L NaCl and 10 mmol / L Tris aqueous solution), basal buffer B (pH 7.4 containing 3 mol / L NaCl and 10 mmol / L Tris aqueous solution), and washing buffer (0.5–3 mol / L alkali metal chloride). It can detect three natriuretic peptides in the natriuretic peptide family and quantify the heart failure biomarker B-type natriuretic peptide. This kit has the advantages of high selectivity, ultrasensitivity, low cost, label-free operation, and convenient detection.

[0024] 2. This invention also discloses a method for detecting heart failure biomarkers. The method primarily involves detecting the electrical signal of solution I (formed by dissolving the analyte in enzyme-free water and mixing it with blood) in an electrochemical device with nanopores. The sample characteristic map is obtained by analyzing the amplitude and retardation time of the current signal and compared with the sample characteristic map of a standard B-type natriuretic peptide (BNP), thereby detecting BNP in the analyte. This detection method has the advantages of being unaffected by the complex matrix in blood and exhibiting high selectivity and ultrasensitivity for BNP.

[0025] 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

[0026] 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:

[0027] Figure 1 Figure 2 shows the detection and analysis results, where a is the current graph and blocking signal amplitude histogram of the test solution containing atrial natriuretic peptide (ANP), b is the current graph and blocking signal amplitude histogram of the test solution containing B-type natriuretic peptide (BNP), c is the current graph and blocking signal amplitude histogram of the test solution containing C-type natriuretic peptide (CNP), d is the characteristic signal of BNP, mixed solution I and mixed solution II, e is the scatter plot of blocking current and blocking time of mixed solution II (mixture of three natriuretic peptides), and f is the blocking time histogram of the three natriuretic peptides.

[0028] Figure 2The results of the detection and analysis in Example 3 are shown in Figure a, where a is the electrophoresis and histogram of the blocking signal amplitude of serum, and b is the electrophoresis and histogram of the blocking signal amplitude of the mixed solution of serum and natriuretic peptides.

[0029] Figure 3 This is the concentration curve detected and analyzed in Example 3. Detailed Implementation

[0030] 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.

[0031] The following examples involve a heart failure biomarker, B-type natriuretic peptide, the sequence of which is shown in SEQ ID NO: 1. Another heart failure-related natriuretic peptide is shown in SEQ ID NO: 2. A third heart failure-related natriuretic peptide is C-type natriuretic peptide, the sequence of which is shown in SEQ ID NO: 3.

[0032] SEQ ID NO: 1 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.

[0033] SEQ ID NO: 2 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.

[0034] 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.

[0035] Example 1

[0036] The components of a reagent kit for detecting heart failure biomarkers are as follows:

[0037] 1. Composition and distribution system:

[0038] (1) Preparation of basic buffer: Mix the aqueous solution of tris(hydroxymethylaminomethane) and the aqueous solution of sodium chloride, and adjust the pH with hydrochloric acid to obtain basic buffer A with pH = 7.4 containing 10 mmol / L of aqueous solution of tris(hydroxymethylaminomethane) and 0.5 mol / L of aqueous solution of chloride. Also obtain basic buffer B with pH = 7.4 containing 10 mmol / L of aqueous solution of tris(hydroxymethylaminomethane) and 3.0 mol / L of aqueous solution of chloride. Filter the buffers using a 0.22 μm filter before use.

[0039] (2) Preparation of washing solution: Mix the aqueous solution of tris(hydroxymethylaminomethane) and the aqueous solution of sodium chloride, and adjust the pH with hydrochloric acid to obtain a washing solution with pH=7.4 containing an aqueous solution of tris(hydroxymethylaminomethane) at a concentration of 10 mmol / L and an aqueous solution of alkali metal chloride at a concentration of 0.5-3 mol / L.

[0040] 2. Kit components: 50mL basic buffer A, 50mL basic buffer A, 50mL washing buffer.

[0041] Example 2

[0042] The detection principle of the electrochemical rapid quantitative kit for the heart failure biomarker B-type natriuretic peptide (BNP):

[0043] 1. Prepare the solution to be tested:

[0044] (1) Prepare the test solution containing B-type natriuretic peptide (BNP): Dissolve B-type natriuretic peptide (BNP) in enzyme-free water to obtain a solution with a concentration of 20 mg / ml as the test solution. Dissolve atrial natriuretic peptide (ANP) and C-type natriuretic peptide in enzyme-free water to obtain solutions with a concentration of 20 mg / ml as interference solution 1 and interference solution 2, respectively. Store in a refrigerator at -20℃ for later use.

[0045] (2) Preparation of mixed solution: Mix the test solution containing B-type natriuretic peptide (BNP) with interference solution 1 to obtain mixed solution I, then mix mixed solution I with interference solution 2 to obtain mixed solution II, and store in a refrigerator at -20℃ for later use;

[0046] (3) 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 alkali metal chloride in basic buffer solution A is 0.5 mol / L, the concentration of alkali metal chloride in basic buffer solution B is 3 mol / L, and the concentration of tris(hydroxymethyl)aminomethane (Tris) in basic buffer solution A and basic buffer solution B is 10 mM). Finally, filter the solution using a 0.22 μm filter before use.

[0047] 2. Constructing a nanoporous electrochemical detection and analysis device: A supporting film containing nanopores is placed in a solution chamber containing electrolyte. Two electrodes 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.

[0048] 3. Detection of the test solution, mixed solution I, and mixed solution II containing B-type natriuretic peptide (BNP):

[0049] (1) The prepared 1.0 μM B-type natriuretic peptide (BNP) test solution, mixed solution I (a mixture of 1.0 μM B-type natriuretic peptide (BNP) test solution and 5.0 μM atrial natriuretic peptide (ANP)) and mixed solution II (a mixture of mixed solution I and 5.0 μM C-type natriuretic peptide (CNP)) were added separately to the above-mentioned electrochemical analysis device with nanopores. The prepared basic buffer solution was used as the electrolyte. After applying an external electric field, the sample in the solution was pushed to interact with the nanopores under the action of the electric field force, and the changing current signal during the interaction was collected.

[0050] (2) The current signal collected in step (1) is amplified by a low-noise current amplifier (Axon Axopatch 200B), and then the amplitude and current hysteresis time characteristics of the current signal are analyzed (the characteristic electrical signals are selected by the software clamfit from the collected electrical signal files, and then the signals are statistically analyzed by Origin) to obtain the corresponding sample feature map.

[0051] The test solution containing B-type natriuretic peptide (BNP) and mixed solutions I and II were analyzed and compared to demonstrate the selective analysis and detection of BNP using nanopores. The results are as follows: Figure 1As shown, figure a is the current graph of the test solution containing B-type natriuretic peptide (BNP), figure b is the current graph of mixed solution I, figure c is the current graph of mixed solution II, figure d is the characteristic signal of BNP, mixed solution I and mixed solution II, figure e is the scatter plot of blocking current and blocking time of mixed solution II (mixture of three natriuretic peptides), and figure f is the bar chart of blocking time of the three natriuretic peptides. Figure 1 It can be seen that BNP has the smallest blocking current. After adding ANP (mixed solution I), the signals of the two peptides do not interfere with each other. Finally, adding CNP (mixed solution II) introduces a new characteristic signal, namely the blocking signal of CNP. The experimental results show that the characteristic signals of the three natriuretic peptides related to heart failure do not interfere with each other and can all be detected. Analysis of their scatter plots also shows that the three natriuretic peptides can be distinguished by the blocking current amplitude and blocking time. Finally, the analysis shows that the blocking current to open current ratios of the three natriuretic peptides are 94.65±2.29% (ANP), 44.84±1.1% (BNP), and 86.21±4.45% (CNP), respectively, and the corresponding blocking times are 88.56±9.59ms, 0.64±0.16ms, and 8.73±0.73ms, respectively. To demonstrate the high selectivity of the detection method, the three natriuretic peptides were detected in serum samples, and the results are as follows. Figure 2 As shown, a) is the electrophoresis diagram, histogram of the percentage of blocking current, and scatter plot over time for serum; b) is the electrophoresis diagram, histogram of the percentage of blocking current, and scatter plot over time for a simulated blood sample prepared by mixing three natriuretic peptides with serum. The experimental results show that the complex matrix in serum does not interfere with the high selectivity of the nanopores, demonstrating the high selectivity and practical applicability of the detection method. Finally, based on the above experiments, different concentrations of BNP were detected in serum (results shown in Figure 1). Figure 3 As shown), i.e., C BNP =10 0.083*N-1.995 Where N represents the number of electrochemical signaling events of B-type natriuretic peptide in the blood sample, and C BNP The concentration of B-type natriuretic peptide in the blood sample is expressed in pmol / L.

[0052] For example, a serum sample containing approximately 1 nmol / L of B-type natriuretic peptide (BNP) was added to an electrolytic cell. Nanopore electrochemical experiments were then performed, detecting 60 BNP signal events (N = 60). Substituting these values ​​into the concentration calculation formula, the BNP concentration was found to be 9.66 nmol / L, with a BNP recovery rate of 96.6%, i.e., C0.05. BNP =10 0.083*60-1.995 = 9.66 nmol / L.

[0053] Example 3

[0054] The steps for detecting B-type natriuretic peptide (BNP) content using the kit of the present invention are as follows:

[0055] 1. Read the instruction manual in its entirety before testing.

[0056] 2. Remove the sample to be tested from the storage environment and mix it with the basic buffer A.

[0057] 2. Construction of a nanoporous electrochemical detection and analysis device: A supporting membrane containing nanopores is placed in a solution chamber containing electrolyte (basic buffer B is added to the CIS end, and basic buffer A is added to the trans end). Two electrodes are placed in the solution chambers at both ends of the supporting membrane, and a power supply and an ammeter are connected sequentially between the two electrodes to form a nanoporous electrochemical detection and analysis device.

[0058] 3. Quantitative detection of B-type natriuretic peptide (BNP) was achieved based on the electrochemical signal of nanopores.

[0059] In summary, this invention discloses a rapid electrochemical quantitative reagent kit for heart failure biomarkers. By incorporating nanopores in an electrochemical detection and analysis device, the electrical signals generated by the interaction between heart failure biomarkers and the nanopores are collected. The amplitude, blocking time, and signal intensity of these signals are analyzed to achieve quantitative detection of heart failure biomarkers in the complex matrix of serum. This invention enables the detection of heart failure biomarkers in serum, with the electrochemical analysis device collecting electrochemical signals and distinguishing three homologous natriuretic peptides, thus realizing the cross-application of nanopores with clinical medicine. Furthermore, this invention also discloses a method for electrochemically detecting heart failure biomarkers, fully leveraging the advantages of high selectivity, ultrasensitivity, speed, label-free operation, and accuracy of the nanopore electrochemical device. It precisely records minute current changes caused by similar peptides and various complex matrices in serum, exhibiting good repeatability and high reliability.

[0060] 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 heart failure biomarkers, characterized in that, The detection method steps are as follows: S1. Prepare B-type natriuretic peptide (BNP) solution, mixed solution I, and mixed solution II: Prepare an enzyme-free aqueous solution of 20 mg / ml B-type natriuretic peptide (BNP) as B-type natriuretic peptide (BNP) solution; prepare an enzyme-free aqueous solution of 20 mg / ml atrial natriuretic peptide (ANP) solution as interference solution 1; prepare an enzyme-free aqueous solution of 20 mg / ml C-type natriuretic peptide (CNP) solution as interference solution 2. Mixing B-type natriuretic peptide (BNP) solution with interference solution 1 yields mixed solution I; mixing mixed solution I with interference solution 2 yields mixed solution II. S2. Prepare basic buffer A, basic buffer B, and washing buffer: Prepare a basic buffer A containing 0.5 mol / L NaCl and 10 mmol / L tris(hydroxymethyl)aminomethane in an aqueous solution with pH=7.4; Prepare a basic buffer B containing an aqueous solution of 3 mol / L NaCl and 10 mmol / L tris(hydroxymethyl)aminomethane, pH=7.4; The washing solution is prepared as an alkali metal chloride at a concentration of 0.5–3 mol / L, wherein the alkali metal chloride is any one or more of sodium chloride, potassium chloride, or lithium chloride; S3. Constructing a nanoporous electrochemical detection and analysis device: A nanoporous support film is placed in a solution chamber containing an electrolyte. Two electrodes are placed in the solution chambers at both ends of the support 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. S4. Detection of B-type natriuretic peptide (BNP) solution, mixed solution I, and mixed solution II: B-type natriuretic peptide (BNP) solution, mixed solution I and mixed solution II were added separately to the nanopore electrochemical detection and analysis device. Basic buffer A was used as the electrolyte at the ground terminal and basic buffer B was used as the electrolyte at the anode terminal. After applying an external electric field, the sample in the solution was pushed to interact with the nanopore under the action of the electric field force, and the changing current signal during the interaction was collected. The acquired current signal was amplified using a low-noise current amplifier. Then, the amplitude and current retardation time characteristics of the current signal were analyzed for B-type natriuretic peptide (BNP) solution, mixed solution I, and mixed solution II, respectively, yielding corresponding sample characteristic maps. Comparison of the characteristic maps of BNP solution, mixed solution I, and mixed solution II showed that the characteristic signals of the three natriuretic peptides (BNP, ANP, and CNP) did not interfere with each other. Based on the above experiments, different concentrations of B-type natriuretic peptide were detected in serum, obtaining the concentration curve of B-type natriuretic peptide as shown in Figure C. BNP =10 0.083*N-1.995 Where N represents the number of electrochemical signaling events of B-type natriuretic peptide in the blood sample, and C BNP The concentration of B-type natriuretic peptide in the blood sample is expressed in pmol / L.

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