Preparation method and application of copper-doped NiFe PBA nano-enzyme probe

Abstract

The invention discloses a preparation method and application of a copper-doped NiFe PBA nano-enzyme probe. The preparation method comprises the following steps: (1) preparing a copper-doped NiFe PBA nano-enzyme, and connecting a secondary antibody Ab2 to prepare a Cu-NiFe PBA-Ab2 nano-enzyme probe; and (2) the antigen molecules and a Cu-NiFe PBA-Ab2 nano-enzyme probe are dropwise added to the surface of the primary antibody Ab1 and streptavidin modified epoxy activated carrier sheet, and the chemiluminescence imaging immunosensor with a double-antibody sandwich is formed through incubation, so that high-sensitivity detection of the antigen molecules is realized, and the detection limit can reach a femtogram level.

Description

Technical Field

[0001] The present invention relates to immunological analysis and detection technology, and specifically relates to a preparation method and application of a copper-doped NiFe PBA nanozyme probe for detecting protein molecules in body fluids such as blood, urine, saliva, pleural effusion, and ascites. Background Art

[0002] Chemiluminescent imaging immunoassay (CLIIA) combines chemiluminescent immunoassay and imaging technology, and has the advantages of chemiluminescent immunoassay and high throughput of imaging analysis. It has a wide linear range, fast analysis speed, and can be used to detect trace medical substances such as proteins, hormones, and bacteria, and also plays an important role in the diagnosis of diseases, the monitoring of treatment effects, and the monitoring of disease conditions. In the chemiluminescent system, the catalyst plays an important role in enhancing the chemiluminescent intensity. Although natural enzymes, as commonly used catalysts, can improve the detection sensitivity of chemiluminescent analysis, they are easily inactivated, have high preparation costs, and are not easy to store, which seriously hinders the development and practical application of chemiluminescent immunoassay methods. Compared with natural enzymes, nanozymes have the advantages of low cost, high stability, and good biocompatibility, and have been widely used in fields such as food, chemical engineering, and medicine.

[0003] Prussian blue analogues (PBAs) are a class of nanomaterials composed of cyanide ligands and transition metal nodes. Due to their three-dimensional open framework, high surface area, unique enzyme-like catalytic activity, and biocompatibility, they have been applied in fields such as energy and environment, including energy conversion and storage, adsorption, electrochemical sensors, and biosensors.

[0004] Compared with single-metal nanozymes, bimetallic nanozymes have better catalytic performance and stability. On the one hand, the effective charge transfer ability between different elements of the bimetal helps to improve the catalytic activity; on the other hand, the construction of the bimetal center can increase the number of catalytic centers. High chemical surface area, more exposed active sites, and appropriate lattice strain are identified as factors affecting the enhancement of intrinsic activity and the promotion of kinetic reaction rate. Therefore, it is urgent to develop a nanozyme probe with high catalytic activity for highly sensitive detection of the antigen to be measured. Summary of the Invention

[0005] The object of the present invention is to provide a preparation method of a copper-doped NiFe PBA nanozyme probe and then prepare a chemiluminescent imaging immunosensor, which combines the double-antibody sandwich method to achieve high-throughput, high-specificity, and high-sensitivity detection of target antigen molecules, and the detection limit can reach the femtogram level.

[0006] To achieve the above object of the invention, the present invention provides the following technical solutions:

[0007] A preparation method of a copper-doped NiFe PBA nanozyme probe includes the following steps:

[0008] S1: Prepare copper-doped NiFe PBA nanozyme;

[0009] S2: Modify the surface of the copper-doped NiFe PBA nanozyme with carboxyl groups and connect the secondary antibody Ab2 to obtain the Cu@NiFePBA-Ab2 nanozyme probe.

[0010] Preferably, S1 includes the following steps:

[0011] Weigh Ni(NO3)2·6H2O, C6H5Na3O7 and Cu(NO3)2·3H2O, disperse them in 50 mL of deionized water, and use ultrasonic treatment to completely dissolve them to form solution A, which is blue-green. Then dissolve K3[Fe(CN)6] in 50 mL of deionized water to form solution B, which is yellow. Dropwise add solution B to solution A, stir at room temperature for 10 min, age for 24 h, collect by centrifugation at 8000 r / min for 5 min in a centrifuge, wash three times alternately with ethanol and water, and then dry in vacuum at 60 °C for 12 h to obtain copper-doped NiFe PBA nanozyme.

[0012] Preferably, the concentrations of Ni(NO3)2·6H2O, C6H5Na3O7 and Cu(NO3)2·3H2O in solution A are 30 mmol / L, 45 mmol / L and 6 mmol / L respectively, and the concentration of K3[Fe(CN)6] in solution B is 20 mmol / L.

[0013] Preferably, S2 includes the following steps:

[0014] Dissolve the copper-doped NiFe PBA nanozyme in a phosphate buffer solution, add a 11-mercaptoundecanoic acid (MUA) solution to modify the carboxyl groups on the surface of the copper-doped NiFe PBA, then activate the carboxyl groups with a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), stir at room temperature for 1 h, add the secondary antibody (Ab2), stir at 4 °C for 3 h, and then continue to centrifuge and wash at 4 °C to obtain the Cu@NiFe PBA-Ab2 nanozyme probe, which is dispersed in a phosphate buffer solution and stored at low temperature.

[0015] Preferably, the concentration of the copper-doped NiFe PBA solution is 2 mg / mL, the concentration of 11-mercaptoundecanoic acid is 0.01 mol / L, the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are both 10 mg / mL, and the concentration of the secondary antibody is 500 μg / mL.

[0016] As another object of the present invention, the present invention also provides a method for detecting an antigen by a chemiluminescent imaging immunosensor based on a copper-doped NiFe PBA nanozyme probe, comprising the following steps:

[0017] (a) Fabricate a reaction array on the surface of an epoxy-silanized glass slide using screen printing technology with the aid of a template;

[0018] (b) Mix equal volumes of a chitosan solution and a streptavidin solution. Each time, take 5 μL of the mixed solution and drop it into the microholes of the reaction array, wait for natural drying, then add 5 μL of the primary antibody solution to each well, dry it, incubate at 4 °C in a refrigerator for 12 hours;

[0019] (c) Drop a bovine serum albumin solution on the surface of the microholes, seal and incubate at 4 °C for 6 hours to block unbound sites, then rinse with a phosphate buffer solution and dry at room temperature;

[0020] (d) Drop an antigen solution on the surface of the microholes, incubate at room temperature for 25 min, then rinse with a phosphate buffer solution and dry at room temperature;

[0021] (e) Drop a Cu@NiFe PBA-Ab2 nanozyme probe on the surface of the microholes to react with the antigen, incubate at room temperature for 30 min, then rinse with a phosphate buffer solution and dry at room temperature;

[0022] (f) Prepare a luminescent substrate, drop it on the surface of the microholes, and quickly place it in a full-automatic chemiluminescent imaging system to detect the luminescence intensity.

[0023] Further, the sensing array prepared in step (a) has a 4×12 array of microholes, the diameter of the microholes is 4 mm, the edge-to-edge distance between the holes is 1.5 mm, and the outside of the microholes is green insulating paint with a hydrophobic effect.

[0024] Further, in step (b), the mass concentration of chitosan is 0.5%, the concentration of the streptavidin solution is 50 μg / mL, and the concentration of the primary antibody is 5 μg / mL.

[0025] Further, in step (c), the mass concentration of the bovine serum albumin solution is 2%, and in step (e), the concentration of the probe is 2 mg / mL.

[0026] Further, in step (f), the luminescent substrate is prepared from a 0.1 mol / L Tris-HCL buffer solution, 0.01 mol / L luminol, 0.01 mol / L p-iodophenol, and 0.01 mol / L hydrogen peroxide, and the phosphate buffer solution in steps (a)-(f) is 0.01 mol / L.

[0027] In the copper-doped NiFe PBA nanozyme of the present invention, Ni and Fe as dual metal centers synergistically enhance the catalytic activity of the nanozyme. At the same time, the incorporation of Cu changes the electronic structure and further enhances the catalytic activity of the nanozyme. In addition, the core-shell and hollow structures can provide a higher cavity volume and a higher surface area, exposing more active sites.

[0028] Using the chemiluminescence system based on copper-doped NiFe PBA nanozyme as a probe to label antibodies can effectively overcome the defects of natural enzymes such as easy inactivation, high preparation cost, and difficult storage. At the same time, the copper-doped NiFe PBA nanozyme has peroxidase-like activity, which can catalyze hydrogen peroxide to generate strongly oxidizing hydroxyl radicals to trigger the chemiluminescence signal of luminol. The chemiluminescence signal is collected by a charge-coupled device CCD. The detection of the antigen to be measured can be realized by using the linear relationship between the chemiluminescence signal intensity and the antigen concentration. The addition of copper ions in the copper-doped NiFe PBA nanozyme enhances the peroxidase-like catalytic activity and stability of NiFe PBA. The addition of streptavidin in the reaction system amplifies the chemiluminescence signal. Combined with the double antibody sandwich method, the sensitivity and specificity of protein molecule detection are improved. Brief Description of the Drawings

[0029] Figure 1 It is a schematic flow chart for preparing a chemiluminescent imaging immunosensor based on copper-doped NiFe PBA nanozyme;

[0030] Figure 2 It is a scanning electron microscope image and a transmission electron microscope image for preparing NiFe PBA and copper-doped NiFe PBA nanozymes;

[0031] Figure 3 It is a graph for evaluating the peroxidase-like performance of NiFe PBA doped with different amounts of copper using a UV spectrophotometer;

[0032] Figure 4 It is a chemiluminescent imaging graph and its linear regression curve for detecting BTN3A2 standard by the chemiluminescent imaging immunosensor based on copper-doped NiFe PBA nanozyme of the present invention;

[0033] Figure 5 It is a correlation analysis for measuring the concentration of BTN3A2 in the serum of gastric cancer patients in Yizheng People's Hospital by the chemiluminescent imaging immunosensor based on copper-doped NiFe PBA nanozyme of the present invention and an ELISA kit;

[0034] Figure 6 It is a correlation analysis for measuring the concentration of BTN3A2 in the serum of gastric cancer patients in Northern Jiangsu People's Hospital by the chemiluminescent imaging immunosensor based on copper-doped NiFe PBA nanozyme of the present invention and an ELISA kit;

[0035] Figure 7 Correlation analysis of the chemiluminescence imaging immunosensor based on copper-doped NiFe PBA nanozyme and ELISA kit for the determination of serum BTN3A2 concentration in gastric cancer patients in Anhui Provincial Hospital in the present invention. Detailed implementation mode

[0036] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments. The present invention can be implemented in various forms and should not be limited by the embodiments described herein.

[0037] Example 1

[0038] As Figure 1 shown in the process, a chemiluminescence imaging immunosensor based on a copper-doped NiFe PBA nanozyme probe is prepared.

[0039] First, synthesize the Cu@NiFe PBA-Ab2 nanozyme probe:

[0040] (1) Weigh Ni(NO3)2·6H2O, C6H5Na3O7 and Cu(NO3)2·3H2O, disperse them in 50 mL of deionized water, and use ultrasonic treatment to completely dissolve them to form solution A, and the solution is blue-green. Then dissolve K3[Fe(CN)6] in 50 mL of deionized water to form solution B, and the solution is yellow. Dropwise add solution B to solution A, stir at room temperature for 10 min, age for 24 h, collect by centrifugation at 8000 r / min for 5 min in a centrifuge, wash three times alternately with ethanol and water, and then dry in vacuum at 60 °C for 12 h to obtain copper-doped NiFe PBA nanozyme;

[0041] (2) Dissolve the copper-doped NiFe PBA nanozyme in a phosphate buffer solution, add 11-mercaptoundecanoic acid (MUA) solution to modify carboxyl groups on the surface of the copper-doped NiFe PBA, and then activate the carboxyl groups with a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), stir at room temperature for 1 h, add the secondary antibody (Ab2), stir at 4 °C for 3 h, and then continue to centrifuge and wash at 4 °C to obtain the Cu@NiFe PBA-Ab2 nanozyme probe, which is dispersed in a phosphate buffer solution and stored at low temperature.

[0042] Then, prepare a chemiluminescence imaging immunosensor based on the copper-doped NiFe PBA nanozyme probe for antigen detection, which specifically includes the following steps:

[0043] (a) Use screen printing technology and with the aid of a template to fabricate a reaction array on the surface of an epoxy-silanized glass slide;

[0044] (b) Mix the same volume of chitosan solution and streptavidin solution. Each time, take 5 μL of this mixed solution and drop it into the micro-wells of the reaction array. Wait for it to dry naturally, and then add 5 μL of the primary antibody solution to each well. Let it dry for 30 min and incubate in a refrigerator at 4 °C for 12 hours;

[0045] (c) Drop the bovine serum albumin solution on the surface of the micro-wells, seal and incubate in an environment at 4 °C for 6 hours to block the unbound sites, and then rinse with phosphate buffer solution and dry at room temperature;

[0046] (d) Drop the antigen solution on the surface of the micro-wells, incubate at room temperature for 25 min, and then rinse with phosphate buffer solution and dry at room temperature;

[0047] (e) Drop the probe to react with the antigen on the surface of the micro-wells, incubate at room temperature for 30 min, and then rinse with phosphate buffer solution and dry at room temperature;

[0048] (f) Prepare the luminescent substrate, drop it on the surface of the micro-wells, and quickly put it into a full-automatic luminescence imaging system to detect the luminescence intensity.

[0049] Perform electron microscopy scanning analysis on the prepared NiFe PBA and copper-doped NiFe PBA as Figure 2 shown, Figure 2 A, Figure 2 C are the scanning and transmission electron microscopy images of NiFe PBA. It can be seen that it is in the shape of a cube with a particle size of about 150 nm. Figure 2 B, Figure 2 D are the scanning and transmission electron microscopy images of copper-doped NiFe PBA. It can be observed that its shape remains unchanged, still in the shape of a cube, and its size becomes smaller, with a particle size of about 100 nm.

[0050] Perform peroxidase-like performance analysis on NiFe PBA and NiFe PBA doped with different amounts of copper by ultraviolet-visible spectrophotometer, as Figure 3 shown. The solution systems are respectively (a) 3.0 mM Cu-NiFe PBA + (b) 1.5 mM Cu-NiFe PBA + (c) NiFe PBA + (d) 6.0 mM Cu-NiFe PBA + (e) 4.5 mM Cu-NiFe PBA. The inset shows the color change of the solution. NiFe PBA and NiFe PBA doped with different amounts of copper have strong absorption at 652 nm, and the signal of 3.0 mM Cu-NiFe PBA is stronger, confirming that 3.0 mM Cu-NiFe PBA has strong peroxidase-like catalytic performance.

[0051] Figure 4This is the chemiluminescence imaging diagram and its linear regression curve for detecting the BTN3A2 standard sample by the chemiluminescence imaging immunosensor based on the copper-doped NiFe PBA nanozyme probe of the present invention. From Figure 4 it can be clearly seen that the chemiluminescence signal increases with the increase of the BTN3A2 concentration. The linear range is 0.5 - 5000 pg / mL, and the linear regression equation is I = 33103.73 + 4500.13 log C BTN3A2 (pg / mL), and the linear correlation coefficient is 0.9942. Based on the above data, the chemiluminescence imaging immunosensor based on the copper-doped NiFe PBA nanozyme probe has high sensitivity and low detection limit for detecting BTN3A2.

[0052] Example 2

[0053] As Figures 5-7 shown, the chemiluminescence imaging immunosensor prepared in Example 1 was used in this example to detect BTN3A2 in the serum samples of gastric cancer patients from three hospitals, namely Yizheng People's Hospital (n = 20), Northern Jiangsu People's Hospital (n = 20), and Anhui Provincial Hospital (n = 20). Before detection, the serum was diluted 10 times with PBS (10 mM, pH 7.4) without further pretreatment. The obtained results were compared with the test values of the ELISA kit, and the correlation coefficients were 0.9964, 0.9988, and 0.9943 respectively.

[0054] Obviously, the examples listed in this application are only for more clearly explaining the present invention and are not a limitation on the implementation modes of this application. For those skilled in the art, various changes and modifications can be made to this application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the protection scope of this application.

Claims

1. A method for preparing a copper-doped NiFe PBA nanozyme probe, characterized in that: The following steps are involved: S1: Preparation of copper-doped NiFe PBA nanozymes; S2: The surface of copper-doped NiFe PBA nanozyme was modified with carboxyl groups and connected to the secondary antibody Ab2 to obtain the Cu@NiFe PBA-Ab2 nanozyme probe.

2. The preparation method according to claim 1, characterized in that: S1 includes the following steps: Weigh Ni(NO3)2·6H2O, C6H5Na3O7 and Cu(NO3)2·3H2O, disperse them in 50mL deionized water, and dissolve them completely under ultrasonication to form liquid A, which is blue-green in color. Then dissolve K3[Fe(CN)6] in 50mL deionized water to form liquid B, which is yellow in color. Add liquid B drop by drop into liquid A, stir at room temperature for 10min, age for 24h, collect by centrifugation at 8000r / min for 5min, wash three times with ethanol and water alternately, and then dry under vacuum at 60℃ for 12h to obtain copper-doped NiFe PBA nanozyme.

3. The preparation method according to claim 2, characterized in that: The concentrations of Ni(NO3)2·6H2O, C6H5Na3O7 and Cu(NO3)2·3H2O in liquid A are 30mmol / L, 45mmol / L and 6mmol / L respectively, and the concentration of K3[Fe(CN)6] in liquid B is 20mmol / L.

4. The preparation method according to claim 1, characterized in that: S2 includes the following steps: The copper-doped NiFe PBA nanozyme was dissolved in a phosphate buffer solution, and 11-mercaptoundecanoic acid (MUA) solution was added to modify the carboxyl groups on the surface of the copper-doped NiFe PBA. The carboxyl groups were then activated with a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The mixture was stirred for 1 h at room temperature, and then the secondary antibody (Ab2) was added. The mixture was stirred for 3 h at 4°C, and then centrifuged and washed at 4°C to obtain the Cu@NiFe PBA-Ab2 nanozyme probe, which was dispersed in a phosphate buffer solution and stored at low temperature.

5. The preparation method according to claim 4, characterized in that: The concentration of the copper-doped NiFe PBA solution was 2 mg / mL, the concentration of 11-mercaptoundecanoic acid was 0.01 mol / L, the concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were both 10 mg / mL, and the concentration of the secondary antibody was 500 μg / mL.

6. A method for detecting antigens using a chemiluminescent imaging immunosensor based on a copper-doped NiFe PBA nanozyme probe, comprising the following steps: (a) The reaction array was fabricated on the surface of epoxy-silanized glass slide using screen printing technology and templates; (b) Mix the chitosan solution and streptavidin solution of the same volume, and drop 5 μL of the mixed solution into the microwells of the reaction array each time, wait for it to dry naturally, then add 5 μL of the primary antibody solution to each well, dry it for 30 minutes, and incubate it in a refrigerator at 4°C for 12 hours; (c) adding bovine serum albumin solution to the micropore surface, sealing and incubating at 4°C for 6 hours to block the unbound sites, then rinsing with phosphate buffer solution and drying at room temperature; (d) dropping antigen solution onto the micropore surface, incubating at room temperature for 25 min, then rinsing with phosphate buffer solution and drying at room temperature; (e) adding the Cu@NiFe PBA-Ab2 nanozyme probe prepared according to any one of claims 1 to 5 to react with the antigen on the micropore surface, incubating at room temperature for 30 minutes, then rinsing with phosphate buffer solution and drying at room temperature; (f) Prepare the luminescent substrate, drop it onto the surface of the microwell, and quickly place it into the fully automatic luminescent imaging system to detect the luminescent intensity.

7. The method according to claim 6, characterized in that The sensor array prepared in step (a) has a 4×12 array of micropores, a micropore diameter of 4 mm, a spacing of 1.5 mm between the pore edges, and a green insulating paint with a hydrophobic effect outside the micropores.

8. The method according to claim 6, characterized in that In step (b), the mass concentration of chitosan is 0.5%, the concentration of streptavidin solution is 50 μg / mL, and the concentration of the primary antibody is 5 μg / mL.

9. The method according to claim 6, characterized in that The mass concentration of the bovine serum albumin solution in step (c) is 2%, and the probe concentration in step (e) is 2 mg / mL.

10. The method according to claim 6, characterized in that The luminescent substrate in step (f) is prepared from 0.1 mol / L Tris-HCL buffer solution, 0.01 mol / L luminol, 0.01 mol / L p-iodophenol and 0.01 mol / L hydrogen peroxide, and the phosphate buffer solution in steps (a)-(f) is 0.01 mol / L.

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