Preparation method of a label-free electrochemical immunosensor based on copper-iron bimetallic organic framework nanozyme and immunoassay method

Abstract

The present invention relates to a preparation method and immunoassay method of a label-free electrochemical immunosensor based on a copper-iron bimetallic organic framework nanozyme. First, a spindle-shaped copper-iron bimetallic organic framework nanozyme is synthesized, and then the spindle-shaped copper-iron bimetallic organic framework nanozyme is used to modify the glassy carbon electrode interface to fix antibody molecules, so as to prepare a label-free electrochemical immunosensor. The copper-iron bimetallic organic framework nanozyme has a large specific surface area, and with the help of streptavidin, the antibody has a high selectivity for biotinylated antibodies, so that the antibody can be effectively fixed on the surface of the copper-iron bimetallic organic framework. The sensor can catalyze the oxidation of o-phenylenediamine in an o-phenylenediamine solution system to generate a strong electrochemical signal, so that the immune complex formed by the antigen-antibody specific reaction inhibits the catalysis of the copper-iron bimetallic organic framework nanozyme, and causes the electrochemical signal intensity to decrease. By reducing the linear relationship between the signal and the antigen concentration, the label-free and highly sensitive detection of IgG can be quickly achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical immunoassay, and in particular to a preparation method of a label-free electrochemical immunosensor based on a copper-iron bimetallic organic framework nanozyme and an immunoassay method. Background Art

[0002] Electrochemical immunosensors combine antigen-antibody recognition technology and have the advantages of high specificity, high sensitivity, simple operation, low operating cost, short detection time, and easy miniaturization. They are widely used in food safety, clinical diagnosis, and environmental analysis. Immunoglobulin G (IgG) is an important immunoglobulin that can neutralize toxins and viruses and activate complement aggregation. It is an important biomarker for clinical diagnosis of chronic infections, chronic liver diseases, lymphoma, systemic lupus erythematosus, multiple myeloma, and various immunodeficiency diseases, as well as leukemia. The concentration of IgG in blood and other body fluids is directly related to the standards of humoral immunity, and abnormal IgG concentrations often indicate the risk of disease or the degree of susceptibility to infection. As an important branch of electrochemical immunosensors, label-free immunosensors have become an attractive and promising method for direct detection of disease markers due to their simple operation, ease of use, rapidity, and the absence of secondary antibodies compared to sandwich-type immunosensors.

[0003] Enzyme-mimicking materials have stable peroxidase-like catalytic activity and are gradually replacing natural enzymes in electrochemical sensing. In particular, noble metal nanoparticles (such as gold and platinum) have been successfully used as electrode substrate modification materials or as signal probes in signal amplification strategies to construct electrochemical biosensors. However, metal-organic framework nanomaterials have rarely been reported to catalyze active substrates in electrochemical biosensors.

[0004] Metal-organic frameworks (MOFs) are a class of porous materials composed of metal nodes and organic ligands. They have large surface area, uniform and adjustable pores, controllable and diverse morphologies, and changeable surface properties. They have attracted widespread attention due to their potential applications.

[0005] Bimetallic MOF material is a new type of material proposed in recent years. It not only has the advantages of metal organic framework materials, but also has excellent properties that single metal MOFs do not have. Bimetallic MOF contains two metal active centers, which helps to improve the stability of the skeleton structure and enhance its catalytic performance. The application of CuFe-MOF in the field of electrochemical immunoassay has not been disclosed in the current technology. The present invention introduces it into electrochemical immunoassay for the first time, functionalizes it with streptavidin, and then combines it with biotinylated antibodies to construct a novel human IgG electrochemical immunosensor. CuFe-MOF has good biocompatibility and a large specific surface area. After functionalization with streptavidin, it can effectively fix a large number of biotinylated IgG antibodies, thereby improving the sensitivity of the immunosensor. Summary of the invention

[0006] One object of the present invention is to provide a method for preparing a label-free electrochemical immunosensor based on a copper-iron bimetallic organic framework nanozyme, so as to construct a novel IgG electrochemical label-free immunosensor, which utilizes the good biocompatibility and large specific surface area of ​​CuFe-MOF. After being functionalized with streptavidin, a large amount of biotinylated IgG antibodies can be effectively fixed, thereby improving the sensitivity of the immunosensor.

[0007] The object of the present invention is achieved by a method for preparing a label-free electrochemical immunosensor based on a copper-iron bimetallic organic framework nanozyme, characterized in that it comprises the following steps

[0008] The first step is to prepare spindle-shaped copper-iron bimetallic organic framework nanozyme microparticles: 2-aminoterephthalic acid is dissolved in N,N-dimethylformamide, and then NaOH, CuCl2.2H2O and FeCl3.6H2O are added dropwise, respectively, and stirred to obtain a mixed solution, and the mixed solution is transferred into a polytetrafluoroethylene-lined reactor, and then the reactor is placed in a constant temperature forced air drying oven at 90-110°C for 16-20 hours; then the high-pressure reactor is cooled naturally to room temperature, the precipitate after the reaction is separated and washed with distilled water and N,N-dimethylformamide for 3 times each, and finally vacuum dried at 80-90°C for 10-12 hours to obtain a dark brown powder, which is the spindle-shaped copper-iron bimetallic organic framework nanozyme microparticles with a particle size of 500-600 nm;

[0009] In the second step, the spindle-shaped copper-iron bimetallic organic framework nanozyme particles prepared in the first step are biofunctionalized with streptavidin and then used to modify the glassy carbon electrode interface to fix biotinylated antibody molecules to prepare a label-free electrochemical immunosensor.

[0010] Furthermore, in the first step, the concentration of 2-aminoterephthalic acid dissolved in N,N-dimethylformamide is 0.1-0.2 mol / L, the ratio of the amount of 2-aminoterephthalic acid, CuCl2.2H2O and FeCl3.6H2O is 1:1:1, the concentration of NaOH is 4 mol / L, and the amount added is 4-5% of the volume of the boiling liquid.

[0011] Furthermore, the second step specifically includes the following steps:

[0012] (1) ultrasonically dispersing the spindle-shaped copper-iron bimetallic organic framework nanozyme in secondary deionized water to form a uniformly dispersed suspension, and then mixing the suspension with an equal volume of chitosan solution and uniformly dispersing it by ultrasonication; and then uniformly mixing the above mixed solution with an equal volume of streptavidin solution to obtain a streptavidin biofunctionalized spindle-shaped copper-iron bimetallic organic framework nanozyme mixed solution;

[0013] (2) The glassy carbon electrode was first polished with alumina powder, and the residual alumina powder was rinsed off with secondary deionized water, and then the glassy carbon electrode was ultrasonically cleaned with ethanol and secondary distilled water in sequence, and finally dried under nitrogen;

[0014] (3) taking a mixed solution of the streptavidin biofunctionalized spindle-shaped copper-iron bimetallic organic framework nanozyme prepared in step (1), drop-coating it on the surface of the clean glassy carbon electrode obtained in step (2), and drying it in a low-temperature refrigeration environment;

[0015] (4) applying the biotinylated antibody to the surface of the glassy carbon electrode modified in step (3), reacting at room temperature, and then washing with a phosphate buffer solution;

[0016] (5) applying a bovine serum albumin solution dropwise onto the surface of the glassy carbon electrode obtained in step (4), sealing the electrode at room temperature, and then rinsing the electrode with a phosphate buffer solution to obtain the label-free electrochemical immunosensor.

[0017] Furthermore, in step (1), the dispersion concentration of the spindle-shaped copper-iron bimetallic organic framework nanozyme suspension is 1-2.0 mg / mL, the concentration of the chitosan solution is 0.5-1.0wt%, and the concentration of streptavidin is 200-300 μg / mL; the mixed volume ratio of the spindle-shaped copper-iron bimetallic organic framework nanozyme suspension, chitosan solution and streptavidin is 1:1:1; in step (2), the particle size of the alumina powder is 0.5 μm.

[0018] Furthermore, in step (3), the refrigeration temperature is 0 to 4° C.; in step (4), the concentration of the biotinylated antibody is 100 to 200 μg / mL.

[0019] Furthermore, in step (5), the concentration of the bovine serum albumin solution is 1.0 to 2.0 wt%.

[0020] The label-free electrochemical immunosensor based on the copper-iron bimetallic organic framework nanozyme prepared by the present invention has the following advantages:

[0021] The spindle-shaped copper-iron bimetallic organic framework nanozyme (CuFe-MOF) nanozyme material used in the sensor has a large specific surface area and abundant amino groups, can effectively adsorb streptavidin, and increase the fixed amount of biotinylated antibodies. On the other hand, the biotin-streptavidin system has high specificity and one molecule of streptavidin can bind to four molecules of biotin. A large amount of biotinylated antibodies can be fixed on the interface of the spindle-shaped copper-iron bimetallic organic framework nanozyme functionalized with streptavidin, thereby constructing a more efficient sensing platform and further improving the sensitivity of the sensor; In addition, MOFs are recognized as a new type of nanozyme by virtue of their excellent catalytic properties, and have been used as signal probes for labeling electrochemical immunosensors. However, the analysis process of labeling electrochemical immunosensors is complicated and cumbersome. The spindle-shaped copper-iron bimetallic organic framework nanozyme synthesized by the method of the present invention has a large specific surface area, good biocompatibility, and exhibits excellent peroxidase-like catalytic activity. It can be used to directly catalyze reaction substrates and is used to construct label-free electrochemical immunosensors, which not only avoids the cumbersome labeling process, but also reduces the use of natural enzymes.

[0022] Another object of the present invention is to provide a label-free electrochemical immunoassay method based on the copper-iron bimetallic organic framework nanozyme, characterized in that it comprises the following analysis steps:

[0023] a. The label-free electrochemical immunosensor of the hammer-shaped copper-iron bimetallic organic framework nanozyme prepared as claimed in claim 1 is incubated in an IgG antigen incubation solution and then rinsed with a phosphate buffer solution;

[0024] b. Using the label-free electrochemical immunosensor after antigen incubation in step a as the working electrode, a saturated calomel electrode as the auxiliary electrode, and a platinum electrode as the counter electrode, in an o-phenylenediamine test solution, the electrochemical signal is detected by differential pulse voltammetry, and the current signal curves of the antigens of different known concentrations are output respectively;

[0025] c. The concentration of each known antigen is marked horizontally, the peak value of the current signal curve corresponding to each concentration output in step b is marked vertically, and the points are fitted to the antigen immunoassay standard curve;

[0026] d. Perform electrochemical immunoassay of unknown antigen concentration according to the process of step a and step b, output the current signal curve, use the peak value of the current signal curve in this step as the vertical mark, plot the antigen immunoassay standard curve in step c, and determine the concentration of the unknown antigen.

[0027] In step a, the incubation time is 30 to 40 minutes.

[0028] In step b, the o-phenylenediamine test solution is composed of a mixture of 20.0 mM o-phenylenediamine and 6.0 mM hydrogen peroxide, the pH of the solution is 7.0, and the volume of the solution is 10 to 20 mL.

[0029] The label-free electrochemical immunoassay method of the present invention uses a CuFe MOF nanozyme with peroxidase-like activity to catalyze the oxidation of o-phenylenediamine (o-PD) to produce a significantly increased reduction current. After incubating the IgG antigen, the specific immune complex formed inhibits the CuFe-MOF enzyme catalysis, causing the electrochemical signal intensity to decrease. According to the linear relationship between the change of the electrochemical signal and the antigen concentration, the quantitative detection of IgG is achieved, and the detection sensitivity is high and the detection range is large. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the SEM image of the spindle-shaped copper-iron bimetallic organic framework nanozyme material prepared in Example 1.

[0031] Figure 2 This is the TEM image of the spindle-shaped copper-iron bimetallic organic framework nanozyme material prepared in Example 1.

[0032] Figure 3 This is a schematic diagram of the preparation and immunoassay of the electrochemical immunosensor of the present invention.

[0033] Figure 4 This is the linear curve of the IgG standard sample detection obtained in Example 2. DETAILED DESCRIPTION

[0034] In order to better illustrate the technical process and purpose of the present invention, the present invention is described in detail below in conjunction with the accompanying drawings and specific implementation methods. Example

[0035] The present invention provides a method for preparing an IgG label-free electrochemical immunosensor based on a spindle-shaped copper-iron bimetallic organic framework nanozyme. The method for preparing the IgG label-free immunosensor comprises the following steps:

[0036] (1) Preparation of spindle-shaped copper-iron bimetallic organic framework nanozyme materials

[0037] In a 50 mL beaker, 0.7246 g of 2-aminoterephthalic acid was dissolved in 20 mL of N,N-dimethylformamide (0.2 mol / L), and then 0.8 mL of NaOH (4M), 0.682 g of CuCl2.2H2O and FeCl3.6H2O were added dropwise, respectively, and stirred for 30 minutes. After mixing evenly, the solution was transferred into the lining of the reactor made of polytetrafluoroethylene, the lining was placed into the reactor made of steel material and the upper cover of the reactor was tightened, and then the reactor was placed in a constant temperature blast drying oven with the temperature set to 100 °C and the reaction time set to 18 h. After the reaction was completed, the high-pressure reactor was naturally cooled to room temperature, the precipitate was separated and washed with distilled water and N,N-dimethylformamide 3 times each, and finally vacuum dried at 80 °C for 12 h to obtain a dark brown powder as a spindle-shaped copper-iron bimetallic organic framework nanozyme material. Figure 1 Shown Figure 1 This is the SEM image of the spindle-shaped copper-iron bimetallic organic framework nanozyme material prepared in this example. Figure 1 Scanning photos show that the copper-iron bimetallic organic framework has a spindle-shaped structure and a smooth surface, with relatively uniform morphology and size, and a particle size of about 600 nm; Figure 2 is a TEM image of the spindle-shaped copper-iron bimetallic organic framework nanozyme material prepared in this embodiment, Figure 2 It can be seen that the spindle-shaped structure of the nanoscale copper-iron bimetallic organic framework is clearly visible and has a regular shape.

[0038] (2) mixing the spindle-shaped copper-iron bimetallic organic framework nanozyme material prepared in step (1) above with deionized water to prepare a spindle-shaped copper-iron bimetallic organic framework nanozyme material suspension with a concentration of 2.0 mg / mL, and mixing it with a chitosan solution with a concentration of 0.5 wt % in equal volumes and then ultrasonically dispersing it;

[0039] (3) Take 20 μL of the mixture from step (2) and 20 μL of 200 μg / mL streptavidin solution and mix them in equal volumes;

[0040] (4) Take 5 μL of the streptavidin functionalized mixed solution from step (3) and evenly apply it on the surface of a clean glassy carbon electrode, then place it at 4°C to dry;

[0041] (5) Take 10 μL of 200 μg / mL biotinylated IgG antibody and evenly apply it on the surface of the modified material, incubate at room temperature for 12 h, and rinse with phosphate buffer solution;

[0042] (6) Take 5 μL of 0.5 wt% bovine serum albumin and evenly apply it on the surface layer of the electrode modified in step (5), block it at room temperature for 60 min, and rinse it with phosphate buffer solution to obtain the IgG label-free electrochemical immunosensor of this example. Example

[0043] As Figure 3 The process shown in the figure, the immunoassay method for the IgG label-free electrochemical immunosensor based on the spindle-shaped copper-iron bimetallic organic framework nanozyme prepared in Example 1 includes:

[0044] (a) taking a number of the IgG label-free immunosensors prepared above, incubating them in incubation solutions with different known concentrations of IgG antigens for 40 min, and then washing them with phosphate buffer solution;

[0045] (b) The prepared IgG label-free immunosensor was used as the working electrode, the saturated calomel electrode as the auxiliary electrode, and the platinum electrode as the counter electrode. The electrochemical signal was detected by differential pulse voltammetry in a test solution containing 20.0 mM o-phenylenediamine and 6.0 mM hydrogen peroxide. The output current signal curves of the electrodes incubated with different known concentrations of antigens were shown in Figure 2. Figure 4 As shown in A;

[0046] (c) Using the concentration values ​​of each known antigen as horizontal markers and the valley values ​​of the current signal curves corresponding to each known concentration output in step b as vertical markers, a point-fitting antigen immunoassay standard curve is drawn, such as Figure 4 As shown in B.

[0047] by Figure 4 In order to investigate the reliability of the practical application of the label-free electrochemical sensor, the IgG content in the clinical blood samples provided by the CDC was subjected to a spike recovery experiment according to the process of steps (a) and (b) of this embodiment. The results are shown in Table 1, which are basically consistent with the standard addition value, and the recovery rate is 94.40%-106.3%.

[0048] Table 1.

[0049] sample Standard concentration (ng / mL) Detection concentration of this example (ng / mL) Recovery rate (%) 1 0.050 0.049 98.00 2 5.00 4.72 94.40 3 15.00 15.94 106.3 4 20.00 20.73 103.7 5 25.00 26.11 104.4

Claims

1. A method for preparing a label-free electrochemical immunosensor based on a copper-iron bimetallic organic framework nanozyme, characterized in that: The following steps are included The first step is to prepare spindle-shaped copper-iron bimetallic organic framework nanozyme microparticles: 2-aminoterephthalic acid is dissolved in N,N-dimethylformamide, and then NaOH, CuCl2.2H2O and FeCl3.6H2O are added dropwise, respectively, and stirred to obtain a mixed solution, and the mixed solution is transferred to a polytetrafluoroethylene-lined reactor, and then the reactor is placed in a constant temperature forced air drying oven at 90-110°C for 16-20 hours; then the high-pressure reactor is naturally cooled to room temperature, the precipitate after the reaction is separated and washed with distilled water and N,N-dimethylformamide for 3 times each, and finally vacuum dried at 80-90°C for 10-12 hours to obtain a dark brown powder, which is the spindle-shaped copper-iron bimetallic organic framework nanozyme microparticles with a particle size of 500-600 nm; wherein, the concentration of 2-aminoterephthalic acid dissolved in N,N-dimethylformamide is: 0.1-0.2 mol / L, The ratio of the amount of 2-aminoterephthalic acid, CuCl2.2H2O and FeCl3.6H2O is: 1:1:1; In the second step, the spindle-shaped copper-iron bimetallic organic framework nanozyme particles prepared in the first step are functionalized with streptavidin and then used to modify the glassy carbon electrode interface to fix biotinylated antibody molecules to prepare a label-free electrochemical immunosensor.

2. The method for preparing a label-free electrochemical immunosensor based on a copper-iron bimetallic organic framework nanozyme according to claim 1, characterized in that: In the first step, the concentration of NaOH is 4 mol / L, and the amount added is 4-5% of the volume of the mixed solution.

3. The method for preparing a label-free electrochemical immunosensor based on a copper-iron bimetallic organic framework nanozyme according to claim 1, characterized in that: The second step specifically includes the following steps: (1) ultrasonically dispersing the spindle-shaped copper-iron bimetallic organic framework nanozyme in secondary deionized water to form a uniformly dispersed suspension, and then mixing the suspension with an equal volume of chitosan solution and uniformly dispersing it by ultrasonication; and then uniformly mixing the above mixed solution with an equal volume of streptavidin solution to obtain a streptavidin biofunctionalized spindle-shaped copper-iron bimetallic organic framework nanozyme mixed solution; (2) The glassy carbon electrode was first polished with alumina powder, and the residual alumina powder was rinsed off with secondary deionized water, and then the glassy carbon electrode was ultrasonically cleaned with ethanol and secondary distilled water in sequence, and finally dried under nitrogen; (3) taking the mixed solution of the streptavidin biofunctionalized spindle-shaped copper-iron bimetallic organic framework nanozyme prepared in step (1), drop-coating it on the surface of the clean glassy carbon electrode obtained in step (2), and drying it in a low-temperature refrigeration environment; (4) applying the biotinylated antibody to the surface of the glassy carbon electrode modified in step (3), reacting at room temperature, and then washing with a phosphate buffer solution; (5) applying a bovine serum albumin solution dropwise onto the surface of the glassy carbon electrode obtained in step (4), sealing the electrode at room temperature, and then washing the electrode with a phosphate buffer solution to obtain the label-free electrochemical immunosensor.

4. The method for preparing a label-free electrochemical immunosensor based on a copper-iron bimetallic organic framework nanozyme according to claim 3, characterized in that: In step (1), the dispersion concentration of the spindle-shaped copper-iron bimetallic organic framework nanozyme suspension is 1-2.0 mg / mL, the concentration of the chitosan solution is 0.5-1.0wt%, and the concentration of streptavidin is 200-300μg / mL; the mixing volume ratio of the spindle-shaped copper-iron bimetallic organic framework nanozyme suspension, chitosan solution and streptavidin is 1:1:1; in step (2), the particle size of the alumina powder is 0.5μm.

5. The method for preparing a label-free electrochemical immunosensor based on a copper-iron bimetallic organic framework nanozyme according to claim 3, characterized in that: In step (3), the refrigeration low temperature is 0 to 4° C.; in step (4), the biotinylated antibody concentration is 100 to 200 μg / mL.

6. The method for preparing a label-free electrochemical immunosensor based on a copper-iron bimetallic organic framework nanozyme according to claim 3, characterized in that: In step (5), the concentration of the bovine serum albumin solution is 1.0 to 2.0 wt %.

7. A label-free electrochemical immunoassay method based on copper-iron bimetallic organic framework nanozyme, characterized in that: The analysis steps include: a. incubating the spindle-shaped copper-iron bimetallic organic framework nanozyme label-free electrochemical immunosensor prepared according to any one of claims 1 to 6 in an IgG antigen incubation solution, and then washing with a phosphate buffer solution; b. Using the label-free electrochemical immunosensor after antigen incubation in step a as the working electrode, a saturated calomel electrode as the auxiliary electrode, and a platinum electrode as the counter electrode, in an o-phenylenediamine test solution, the electrochemical signal is detected by differential pulse voltammetry, and the current signal curves of the antigens of different known concentrations are output respectively; c. The concentration of each known antigen is marked horizontally, the peak value of the current signal curve corresponding to each known concentration output in step b is marked vertically, and the points are fitted to the antigen immunoassay standard curve; d. Perform electrochemical immunoassay of unknown antigen concentration according to the process of step a and step b, output the current signal curve, use the peak value of the current signal curve in this step as the vertical mark, plot the antigen immunoassay standard curve in step c, and determine the concentration of the unknown antigen.

8. The label-free electrochemical immunoassay method according to claim 7, characterized in that: In step a, the incubation time is 30 to 40 minutes.

9. The label-free electrochemical immunoassay method according to claim 7, characterized in that: In step b, the o-phenylenediamine test solution is composed of a mixture of 20.0 mM o-phenylenediamine and 6.0 mM hydrogen peroxide, the pH of the solution is 7.0, and the volume of the solution is 10 to 20 mL.

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