Preparation method of rapid detection sensor for early-warning marker BNP (Brain Natriuretic Peptide) of acute heart failure

Abstract

The invention discloses a preparation method of an acute heart failure early warning marker BNP rapid detection sensor. The preparation method comprises the following steps: preparation of an M-NTO-CNT composite material, preparation of M-NTO-CNT / SPCE and preparation of an electrochemical immunosensor. The electrochemical immunosensor based on the MXene composite material is used for detecting the BNP, the real-time sensitive monitoring of trace BNP is realized by simplifying the detection steps, reducing the detection cost and shortening the detection period, and a novel and convenient detection method is established for the early warning diagnosis of acute heart failure. The invention not only provides a novel and convenient detection method for process screening, early warning diagnosis and prognosis risk assessment of AHF, but also provides a scientific basis for efficient and convenient new technology development.

Description

Technical Field

[0001] The invention belongs to the field of cardiovascular disease monitoring, and in particular relates to a method for preparing a BNP rapid detection sensor which is an early warning marker for acute heart failure. Background Art

[0002] Cardiovascular disease (CVD) is the leading cause of death from chronic diseases worldwide, and acute heart failure (AHF) is a severe manifestation and terminal stage of CVD, with extremely high disability and mortality rates. Therefore, early warning, diagnosis, monitoring and prognosis of AHF patients are of great significance. B-type natriuretic peptide (BNP) has a high sensitivity to the occurrence of AHF, and its level is closely related to the condition and prognosis of AHF patients. It can be used as a biomarker for early warning, diagnosis, monitoring and prognosis evaluation of AHF. BNP is mainly expressed in the ventricles. It is a natural hormone with biological activity synthesized by myocardial cells. It can inhibit the renin-angiotensin-aldosterone system and the sympathetic nervous system, dilate blood vessels, and regulate blood sodium, blood pressure and fluid balance. The determination of BNP has been recommended for the diagnosis and risk stratification of heart failure. When BNP ≥ 4.00 × 10 2 pg mL -1 When BNP is 1.00 × 10 2 -4.00 × 10 2 pg mL -1 It can be confirmed by combining with other indicators.

[0003] There are many methods reported for BNP detection. The more common detection methods are radioimmunoassay (IRA), immunoradiometric measurement (IRMA), and electrochemiluminescence (ECLA). However, these conventional methods have radiation, demanding operation requirements, and are difficult to be portable and popularized for instant detection. Electrochemical immunosensor technology has attracted widespread attention from researchers due to its advantages such as high sensitivity, wide linear range, strong specificity, and suitability for point-of-care testing (POCT) diagnosis. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a BNP rapid detection sensor for early warning marker of acute heart failure, so as to realize real-time sensitive monitoring of trace BNP, which has the advantages of simple operation, short detection time, good stability and can be applied to on-site rapid detection.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A method for preparing a BNP rapid detection sensor, which is an early warning marker for acute heart failure, comprises the following steps: S1. Preparation of M-NTO-CNT composite materials: 80-100 mg of MXene powder was added to 20-40 mL of water, and ultrasonic dispersion was performed for 30 min to obtain dispersion liquid I. 1-3 M NaOH and 30% H2O2 were mixed, and the obtained mixture was added to dispersion liquid I. The mixture was washed by centrifugation with deionized water and then centrifuged and dried to obtain M-NTO composite material powder. 90-110 mg MXene powder and 20-40 mg CNT powder were added to water, and ultrasonic dispersion was performed for 30 min to obtain dispersion II. 1-3 M NaOH and 30% H2O2 were mixed, and the obtained mixture was added to dispersion II. The mixture was washed by centrifugation with deionized water and then centrifuged and dried to obtain M-NTO-CNT composite material powder. S2. Preparation of M-NTO-CNT / SPCE: 10-30 mg of M-NTO-CNT powder was dissolved in 5-15 mL of deionized water and ultrasonicated for 10 min to make it evenly dispersed. -1 20 μL of M-NTO-CNT dispersion was applied to the surface of the working electrode of SPCE and dried naturally to prepare M-NTO-CNT modified M-NTO-CNT@SPCE; the SPCE electrode (screen-printed carbon electrode) integrated a three-electrode system, including a carbon-based working electrode, a carbon-based counter electrode and an Ag / AgCl reference electrode; S3. Preparation of electrochemical immunosensor: 30-50 μL of 100× dilution of biotinylated BNP antibody was added dropwise to the working electrode surface of M-NTO-CNT@SPCE, and M-NTO-CNT@SPCE was placed in a sealed environment at room temperature of 20 °C for 4 hours, then taken out, and the electrode was rinsed with PBST solution and immersed in BSA blocking solution for 1 hour to obtain BSA@BNP-Ab@M-NTO-CNT@SPCE, that is, an electrochemical immunosensor for BNP detection was constructed. The electrode was rinsed with PBST solution to remove physically adsorbed antibodies, and immersed in BSA blocking solution to block nonspecific adsorption sites.

[0006] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses a composite material composed of carbon nanotubes (CNT) with good chemical stability, thermal conductivity and electrical conductivity and a nanomaterial MXene with a large specific surface area and numerous surface groups that can contact the analyte more quickly as an electrode substrate material. The M-NTO-CNT composite material formed by mixing in a one-pot method combines the advantages of both to become an excellent electrode modification material with a large specific surface area, high stability, high conductivity and numerous surface groups, so that it can effectively bind various functional groups or biomolecules and can contact the analyte more quickly. The performance of the electrochemical sensor can be significantly improved by using M-NTO-CNT as a combination of electrode substrate materials. In order to further improve the conductivity of the electrochemical sensor, a functionalized composite material (M-NTO-CNT) is prepared by combining CNT with MXene and modified on the electrode. The synergistic effect between the two can not only utilize its open macroporous structure to load materials with good conductivity, which can well improve the conductivity of the electrode, but also reduce material aggregation, increase the specific surface area and thus increase the catalytic current response to the target molecule.

[0007] Therefore, in the process of functional modification of MXene, the synergistic effect between the two can not only utilize its open macroporous structure to load materials with good conductivity, which can greatly improve the conductivity of the electrode, but also reduce material aggregation, increase the specific surface area and thus increase the catalytic current response to the target molecule. The modified composite material (M-NTO-CNT) was modified on the electrode surface to construct an electrochemical immunosensor with high sensitivity and high selectivity for rapid detection, which was used to achieve real-time sensitive monitoring of trace BNP. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to optimize the experimental conditions affecting the electrochemical immunosensor. Under the optimized experimental conditions, the sensor was 3.13 × 10 1 -8.00 ×10 3 pg mL -1 The detection limit was as low as 1.748 × 10 -3 pg mL -1 When used for actual sample detection, the sensor exhibits good specificity and stability, and can achieve highly sensitive and rapid detection of trace BNP in actual samples. The present invention not only provides an effective means for convenient, efficient and accurate detection of BNP, a marker of acute heart failure, but also establishes a novel and convenient detection method for early warning diagnosis of acute heart failure.

[0008] Compared with the authorized patent CN201521139284.9, an immunofluorescence detector for detecting heart failure, the present invention has the following advantages: Sensitivity: Electrochemical immunosensors can significantly improve sensitivity, amplify electrochemical detection signals, and reduce the detection limit of BNP by using new nanomaterials such as MXene and CNT carbon nanotube modified electrodes.

[0009] Specificity: The highly specific binding between antigen and antibody can accurately identify BNP and reduce nonspecific interference. BNP can be specifically detected in blood samples without being affected by other similar substances.

[0010] Adaptability to clinical applications: Due to its high sensitivity, specificity and stability, as well as good ease of operation and detection speed, it is very suitable for rapid and accurate detection of BNP in clinical diagnosis, providing timely basis for the diagnosis and treatment of diseases such as heart failure.

[0011] The present invention has a wider detection range and a lower detection limit, and has greatly improved sensitivity compared with the prior art.

[0012] The electrochemical immunosensor based on MXene composite materials constructed by the present invention detects BNP, which simplifies the detection steps, reduces the detection cost, shortens the detection cycle, realizes real-time sensitive monitoring of trace BNP, and also establishes a novel and convenient detection method for early warning diagnosis of acute heart failure. It not only provides a novel and convenient detection method for process screening, early warning diagnosis and prognosis risk assessment of AHF, but also provides a scientific basis for the development of efficient and convenient new technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The figure is a flow chart of the preparation of the sensor of the present invention; Figure 2 The images of SEM electron microscopy and Raman spectroscopy characterization of the M-NTO-CNT@SPCE modified electrode in the experimental example of the present invention, wherein A is the SEM image of CNT; B is the SEM image of MXene; C is the SEM image of M-NTO; D is the SEM image of M-NTO-CNT; E is the Raman image, a is MXene, b is M-NTO, c is CNT, d is M-NTO-CNT; Figure 3 The electrochemical behavior characterization diagram in the experimental example of the present invention, wherein A is the CV curve; B is the EIS curve; C is the chrono-Coulomb curve; D is the Qt 1 / 2 Curve; E is the CV curve of M-NTO-CNT@SPCE at different scan rates; F is the linear relationship between the logarithm of the redox peak current and the logarithm of the scan rate; G is the linear relationship between the logarithm of the redox peak potential and the scan rate; a in each figure of AG is SPCE, b is M-NTO@SPCE, c is CNT@SPCE, and d is M-NTO-CNT@SPCE; Figure 4 The results of the gradient concentration sample detection in the experimental example of the present invention, where A is the DPV response of the BSA@BNP-Ab@M-NTO-CNT@SPCE sensor to different concentrations of BNP (3.13 × 10 1 -8.00 × 10 3 pg mL -1 ), B is the linear relationship between the DPV response value of the BSA@BNP-Ab@M-NTO-CNT@SPCE sensor and the BNP concentration; Figure 5 These are the test results of the specificity and stability of the BSA@BNP-Ab@M-NTO-CNT@SPCE sensor in the experimental example of the present invention, where A is the test result of the specificity and B is the test result of the stability. DETAILED DESCRIPTION

[0014] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0015] like Figure 1 As shown, a method for preparing a BNP rapid detection sensor, which is an early warning marker for acute heart failure, comprises the following steps: S1. Preparation of M-NTO-CNT composite materials: 80-100 mg of MXene powder was added to 20-40 mL of water, and ultrasonic dispersion was performed for 30 min to obtain dispersion liquid I. 1-3 M NaOH and 30% H2O2 were mixed, and the obtained mixture was added to dispersion liquid I. The mixture was washed by centrifugation with deionized water and then centrifuged and dried to obtain M-NTO composite material powder. 90-110 mg MXene powder and 20-40 mg CNT powder were added to water, and ultrasonic dispersion was performed for 30 min to obtain dispersion II. 1-3 M NaOH and 30% H2O2 were mixed, and the obtained mixture was added to dispersion II. The mixture was washed by centrifugation with deionized water and then centrifuged and dried to obtain M-NTO-CNT composite material powder. S2. Preparation of M-NTO-CNT / SPCE: 10-30 mg of M-NTO-CNT powder was dissolved in 5-15 mL of deionized water and ultrasonicated for 10 min to make it evenly dispersed. -1 20 μL of M-NTO-CNT dispersion was applied to the surface of the working electrode of SPCE and dried naturally to prepare M-NTO-CNT modified M-NTO-CNT@SPCE; the SPCE electrode (screen-printed carbon electrode) integrated a three-electrode system, including a carbon-based working electrode, a carbon-based counter electrode and an Ag / AgCl reference electrode; S3. Preparation of electrochemical immunosensor: 30-50 μL of 100× dilution of biotinylated BNP antibody was added dropwise to the working electrode surface of M-NTO-CNT@SPCE, and M-NTO-CNT@SPCE was placed in a sealed environment at room temperature of 20 °C for 4 h, then taken out, and the electrode was rinsed with PBST solution and immersed in BSA blocking solution for 1 h to obtain BSA@BNP-Ab@M-NTO-CNT@SPCE, that is, an electrochemical immunosensor for BNP detection was constructed. The electrode was rinsed with PBST solution to remove physically adsorbed antibodies, and immersed in BSA blocking solution to block nonspecific adsorption sites.

[0016] Embodiment 1: 1. Preparation of M-NTO-CNT composites Take 80 mg of MXene powder and add it to 20 mL of water, ultrasonically disperse it for 30 min to make it evenly dispersed, then add a mixed solution of 1 M NaOH and 30% H2O2, centrifuge and wash it with deionized water, and centrifuge and dry it to obtain M-NTO composite material powder. At the same time, take 90 mg of MXene powder and 20 mg of CNT powder, and prepare them in the same way as above to obtain M-NTO-CNT composite material powder; 2. Preparation of M-NTO-CNT / SPCE Using screen-printed carbon electrode (SPCE), 10 mg M-NTO-CNT powder was dissolved in 5 mL deionized water and ultrasonicated for 10 min to make it evenly dispersed, and 2 mg mL -1 20 μL of M-NTO-CNT dispersion was applied to the surface of SPCE working electrode and dried naturally to prepare M-NTO-CNT modified SPCE electrode (M-NTO-CNT@SPCE). 3. Preparation of electrochemical immunosensor 30 μL of 100× dilution of biotinylated BNP antibody (BNP-Ab) was added dropwise to the surface of the M-NTO-CNT@SPCE working electrode. The modified electrode was incubated in a sealed environment at room temperature of 20 ℃ for 4 h and then taken out. The electrode was then rinsed with a pre-prepared PBST solution to remove the physically adsorbed antibody and then immersed in a BSA blocking solution for 1 h to block the nonspecific adsorption sites to obtain BSA@BNP-Ab@M-NTO-CNT@SPCE. An electrochemical immunosensor for BNP detection was constructed.

[0017] Embodiment 2: 1. Preparation of M-NTO-CNT composites Take 90 mg of MXene powder and add it to 30 mL of water, ultrasonically disperse it for 30 min to make it evenly dispersed, then add a mixed solution of 2 MNaOH and 30% H2O2, centrifuge and wash it with deionized water, and centrifuge and dry it to obtain M-NTO composite material powder. At the same time, take 100 mg of MXene powder and 30 mg of CNT powder, and prepare them in the same way as above to obtain M-NTO-CNT composite material powder; 2. Preparation of M-NTO-CNT / SPCE Using screen-printed carbon electrode (SPCE), 20 mg M-NTO-CNT powder was dissolved in 10 mL deionized water and ultrasonicated for 10 min to make it evenly dispersed, and 2 mg mL -1 20 μL of M-NTO-CNT dispersion was applied to the surface of SPCE working electrode and dried naturally to prepare M-NTO-CNT modified SPCE electrode (M-NTO-CNT@SPCE). 3. Preparation of electrochemical immunosensor 40 μL of 100× dilution of biotinylated BNP antibody (BNP-Ab) was added dropwise to the surface of the M-NTO-CNT@SPCE working electrode. The modified electrode was incubated in a sealed environment at room temperature of 20 ℃ for 4 h and then taken out. The electrode was then rinsed with a pre-prepared PBST solution to remove the physically adsorbed antibody and then immersed in a BSA blocking solution for 1 h to block the nonspecific adsorption sites to obtain BSA@BNP-Ab@M-NTO-CNT@SPCE. An electrochemical immunosensor for BNP detection was constructed.

[0018] Embodiment 3: 1. Preparation of M-NTO-CNT composites Take 100 mg of MXene powder and add it to 40 mL of water, ultrasonically disperse it for 30 min to make it evenly dispersed, then add a mixed solution of 3 MNaOH and 30% H2O2, centrifuge and wash it with deionized water, and centrifuge and dry it to obtain M-NTO composite material powder. At the same time, take 110 mg of MXene powder and 40 mg of CNT powder, and prepare them in the same way as above to obtain M-NTO-CNT composite material powder; 2. Preparation of M-NTO-CNT / SPCE Using screen-printed carbon electrode (SPCE), 30 mg of M-NTO-CNT powder was dissolved in 15 mL of deionized water and ultrasonicated for 10 min to make it evenly dispersed, and 2 mg mL -120 μL of M-NTO-CNT dispersion was applied to the surface of SPCE working electrode and dried naturally to prepare M-NTO-CNT modified SPCE electrode (M-NTO-CNT@SPCE). 3. Preparation of electrochemical immunosensor 50 μL of 100× dilution of biotinylated BNP antibody (BNP-Ab) was added dropwise to the surface of the M-NTO-CNT@SPCE working electrode. The modified electrode was incubated in a sealed environment at room temperature of 20 ℃ for 4 h and then taken out. The electrode was then rinsed with a pre-prepared PBST solution to remove the physically adsorbed antibody and then immersed in a BSA blocking solution for 1 h to block the nonspecific adsorption sites to obtain BSA@BNP-Ab@M-NTO-CNT@SPCE. An electrochemical immunosensor for BNP detection was constructed.

[0019] Experimental examples, electrochemical measurements and reaction processes: 1. Electrochemical characterization of basic electrodes like Figure 2 As shown in the figure, after the M-NTO-CNT@SPCE modified electrode was constructed, it was characterized by Raman spectroscopy and SEM electron microscopy. The test results show that the composite material and modified electrode were successfully prepared.

[0020] Electrochemical measurements include cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). CV measurements were performed at a sensitivity of 10 -4 The potential range was -0.4 V-0.8 V and the scan rate was 0.1 V / s. The EIS measurement frequency parameters were 0.1 Hz-10 Hz, the rest time was 2 s, and the voltage amplitude was 0.005 V. The sensitivity of DPV was 10 -5 , potential range 0.6 V- -0.2 V, rest time 2 s, voltage amplitude 0.05 V, pulse period 0.5 s, sampling width 0.0167 s. The test base solution contains 5.0 mM [Fe(CN)6] 3- / 4- and a mixed solution of 0.01 M KCl.

[0021] like Figure 3 As shown in Figure 2, the composite materials and modified electrodes were successfully prepared, and the electroactive effective specific surface areas of SPCE, M-NTO@SPCE, CNT@SPCE, and M-NTO-CNT@SPCE were 0.632 cm 2 、0.889 cm 2 、2.447 cm 2 、3.361 cm 2 .

[0022] 2. Cyclic voltammetry characterization of electrochemical immunosensor The CV method was used in the voltage range of -0.4 V to 0.8 V at a rate of 100 mVs -1 Scan rate, CV characterization of M-NTO-CNT@SPCE electrochemical immunosensor was carried out in 5.0 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution of 0.01 M KCl.

[0023] 3. BNP measurement process The BSA@BNP-Ab@M-NTO-CNT@SPCE modified electrode was prepared by drop coating and self-assembly. The electrochemical response of the BSA@BNP-Ab@M-NTO-CNT@SPCE modified electrode was investigated by differential pulse voltammetry (DPV) in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] in 0.1 M PBS solution, and the current response was recorded. The sensitivity of DPV was 10 -5 , potential range 0.6 V - -0.2 V, rest time 2 s, voltage amplitude 0.05 V, pulse period 0.5 s, sampling width 0.0167 s. The test base solution contains 5.0 mM [Fe(CN)6] 3- / 4- and a mixed solution of 0.01 M KCl. Finally, the linear functional relationship between the DPV of BNP at different concentrations and the electrochemical response value was obtained by mathematical modeling.

[0024] Perform gradient concentration sample testing under optimized conditions, such as Figure 4 It can be seen that the logarithm of the oxidation peak current (Ipa, μA) of BNP and its concentration (pg / mL) is 3.13× 10 1 - 8.00 × 10 3 pg mL -1 The corresponding linear regression equation is: p = 257.65108-29.40923Lg[C BNP ] (R 2 = 0.99607), and the detection limit was 1.748 × 10 -3 pg mL -1 (S / N=3).

[0025] To verify the practicality of the sensor, the present invention conducted specificity and stability tests on the BSA@BNP-Ab@M-NTO-CNT@SPCE sensor. Figure 4 As shown, the BSA@BNP-Ab@M-NTO-CNT@SPCE sensor has excellent specificity and stability.

[0026] In order to further evaluate the applicability of the established immunosensor in clinical detection and analysis, the present invention used the standard addition method to test in real serum samples (n=3) and compared with the commercial ELISA method. The results in Table 1 show that the sensor has high sensitivity and the detection results are better than other detection methods. The sensor can be used for the detection of BNP in actual samples.

[0027] .

Claims

1. A method for preparing a BNP rapid detection sensor for early warning marker of acute heart failure, characterized in that The steps include: S1. Preparation of M-NTO-CNT composite materials: 80-100 mg of MXene powder was added to 20-40 mL of water, and ultrasonically dispersed for 30 min to obtain dispersion I. 1-3 M NaOH and 30% H2O2 were mixed, and the obtained mixture was added to dispersion I. The mixture was washed with deionized water and then centrifuged and dried to obtain M-NTO composite material powder. 90-110 mg MXene powder and 20-40 mg CNT powder were added to water, and ultrasonic dispersion was performed for 30 min to obtain dispersion II. 1-3 M NaOH and 30% H2O2 were mixed, and the obtained mixture was added to dispersion II. The mixture was washed by centrifugation with deionized water and then centrifuged and dried to obtain M-NTO-CNT composite material powder. S2. Preparation of M-NTO-CNT / SPCE: 10-30 mg of M-NTO-CNT powder was dissolved in 5-15 mL of deionized water and ultrasonicated for 10 min to make it evenly dispersed. -1 20 μL of M-NTO-CNT dispersion was taken and dropped on the working electrode surface of SPCE, and then dried naturally to prepare M-NTO-CNT modified M-NTO-CNT@SPCE. S3. Preparation of electrochemical immunosensor: 30-50 μL of 100× dilution of biotinylated BNP antibody was added dropwise to the working electrode surface of M-NTO-CNT@SPCE. M-NTO-CNT@SPCE was incubated in a closed environment at room temperature of 20 ℃ for 4 h and then taken out. The electrode was rinsed with PBST solution and immersed in BSA blocking solution for 1 h to obtain BSA@BNP-Ab@M-NTO-CNT@SPCE. Thus, an electrochemical immunosensor for BNP detection was constructed.

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