A chemiluminescence imaging immunoassay method based on Au@CuZn-ZIF nanozyme probe

Abstract

The present invention relates to a chemiluminescent imaging immunoassay method based on Au@CuZn‑ZIF nanozyme probe in the field of immunological analysis and detection technology. First, Au@CuZn‑ZIF nanozyme is prepared and carboxyl groups are modified on its surface. Secondary antibody Ab2 is connected to prepare Au@CuZn‑ZIF‑Ab2 nanozyme probe; then antigen molecule and Au@CuZn‑ZIF‑Ab2 nanozyme probe are added dropwise to the surface of epoxy-activated carrier sheet modified by primary antibody Ab1, and incubated to form a sandwich-shaped chemiluminescent imaging immunosensor to achieve high sensitivity inspection of antigen molecules. In the present invention, the peroxidase-like activity of Au@CuZn‑ZIF nanozyme can catalyze hydrogen peroxide to generate strong oxidizing hydroxyl radicals to trigger luminol to produce chemiluminescent signals, and the linear relationship between chemiluminescent signals and antigen concentration can be used to achieve antigen detection. At the same time, the introduction of copper ions in Au@CuZn‑ZIF nanozyme enhances the enzyme-like catalytic activity and stability of the composite material, and combined with the sandwich method, the sensitivity and specificity of protein molecule detection are improved.

Description

Technical Field

[0001] The present invention relates to immunological analysis and detection technology, and in particular to a chemiluminescence imaging immunoassay method based on Au@CuZn-ZIF nanozyme for detecting protein molecules. Background Art

[0002] Chemiluminescence is an analytical method that uses the energy provided by chemical reactions to excite substances and generate light radiation. Chemiluminescence imaging immunoassay combines the advantages of chemiluminescence immunoassay with imaging technology, offering a wide linear range and fast analysis speed. It can be used to detect trace substances such as proteins, hormones, and cytokines, and plays an important role in disease diagnosis, immune function monitoring, and treatment efficacy and condition monitoring.

[0003] Traditional chemiluminescent immunoassays typically use natural enzymes (HRP) to label antibodies. However, the development and application of chemiluminescent immunoassays are limited by the drawbacks of natural enzymes, such as their easy inactivation, difficulty in extraction, and poor tolerance to extreme environments. Compared to natural enzymes, nanozymes, as nanomaterials with excellent enzyme-like catalytic activity, have attracted widespread attention among researchers. Metal-organic frameworks (MOFs), formed by the self-assembly of metal ions and organic linkers, are being continuously developed due to their large surface area, high porosity, and excellent stability. Introducing MOF nanozymes into chemiluminescent systems as probes for antibody labeling can effectively overcome the limitations of natural enzymes. They catalyze hydrogen peroxide to generate highly oxidizing hydroxyl radicals, which trigger luminol to produce a chemiluminescent signal, which then specifically binds to the antigen for detection.

[0004] Compared with single-metal coordination structures, bimetallic MOF nanozymes have better catalytic performance and stability. On the one hand, the construction of bimetallic centers can increase the number of catalytic centers; on the other hand, the potential difference between the two metal centers can promote electron transfer, thereby helping to improve catalytic activity. However, biocompatibility and detection sensitivity need to be further improved. Summary of the Invention

[0005] The purpose of the present invention is to provide a chemiluminescent imaging immunosensor using Au@CuZn-ZIF nanozymes to improve the biocompatibility and catalytic activity of metal organic framework nanozymes, and combine it with the sandwich method to achieve high-throughput, high-specificity and high-sensitivity detection of target antigens.

[0006] The object of the present invention is achieved by a chemiluminescent imaging immunoassay method based on Au@CuZn-ZIF nanozyme probe, characterized in that it is prepared by the following steps:

[0007] Step 1: Prepare Au@CuZn-ZIF nanozyme and modify its surface with carboxyl groups, then connect it to the secondary antibody Ab2 to prepare the Au@CuZn-ZIF-Ab2 nanozyme probe;

[0008] In the second step, the antigen molecules and Au@CuZn-ZIF-Ab2 nanozyme probes are dropped onto the surface of the epoxy-activated carrier modified with the primary antibody Ab1, and incubated to form a sandwich chemiluminescence imaging immunosensor to detect the antigen molecules.

[0009] The present invention utilizes a chemiluminescent imaging immunosensor using an Au@CuZn-ZIF nanozyme. The Au@CuZn-ZIF nanozyme is first prepared and then applied to a chemiluminescent system. The introduction of copper ions, along with zinc as a bimetallic center, synergistically enhances the enzyme-like catalytic activity of the MOF. Simultaneously, the introduction of gold nanoparticles enhances the biocompatibility of the nanozyme, effectively immobilizing antibodies. The Au@CuZn-ZIF nanozyme can be used to replace a natural enzyme to label a secondary antibody to prepare a nanozyme probe. Combined with a sandwich method, the corresponding luminescent signal is collected and detected. The linear relationship between the chemiluminescent signal and the antigen concentration allows for accurate detection of target protein molecules, while also improving detection sensitivity and specificity.

[0010] To facilitate the preparation of the Au@CuZn-ZIF-Ab2 nanozyme probe, the first step includes the following sub-steps:

[0011] 1.1 First, Zn(NO3)2·6H2O and Cu(NO3)2·3H2O were dissolved in methanol to form solution A. Then, 2-methylimidazole was dissolved in methanol to form solution B. Solution A was added dropwise to solution B. The mixture was stirred at room temperature for 30-60 minutes. The solid phase product in the mixture was then collected and washed by centrifugation to obtain CuZn-ZIF nanoparticles.

[0012] 1.2 Dissolve CuZn-ZIF nanoparticles in water to form a solution, add it to the block polyether F-127 aqueous solution, stir at room temperature for 30-60 minutes, add HAuCl4 solution, stir at room temperature for another 30-60 minutes, then add NaBH4 solution, continue stirring for two hours, and then centrifuge to collect the solid phase product in the washing mixture to obtain Au@CuZn-ZIF nanozyme;

[0013] 1.3 The Au@CuZn-ZIF nanozyme was dissolved in phosphate buffer solution, and 11-mercaptoundecanoic acid (MUA) solution was added to modify the carboxyl group on the Au@CuZn-ZIF surface. The carboxyl group was then activated with a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The secondary antibody (Ab2) was added and stirred at 4°C for 3 hours. The mixture was then centrifuged and washed at 4°C to obtain the Au@CuZn-ZIF-Ab2 nanozyme probe, which was dispersed in phosphate buffer solution and stored at low temperature.

[0014] Furthermore, in step 1.1, the concentrations of Zn(NO3)2·6H2O and Cu(NO3)2·3H2O in solution A are 0.013 mol / L and 0.05 mol / L, respectively, the concentration of 2-methylimidazole in solution B is 0.55 mol / L, and the volume ratio of solution A to solution B is 1:2.

[0015] Furthermore, in step 1.2, the concentration of the CuZn-ZIF solution was 4 mg / mL, the concentration of the F-127 aqueous solution was 3 mg / mL, the concentration of the HAuCl4 solution was 10 mmol / L, the concentration of the NaBH4 solution was 0.1 mol / L, and the volume ratio of the CuZn-ZIF, F-127, HAuCl4, and NaBH4 solutions was 50:100:20:3.

[0016] Furthermore, in step 1.3, the concentration of the Au@CuZn-ZIF solution was 2 mg / mL, the concentration of the MUA solution was 0.01 mol / L, the concentrations of the EDC and NHS solutions were both 10 mg / mL, the concentration of the secondary antibody (Ab2) solution was 1 mg / mL, and the volume ratio of the Au@CuZn-ZIF, MUA, EDC, NHS, and Ab2 solutions was 100:20:10:7:4.

[0017] To facilitate luminescent immunoassay using the prepared Au@CuZn-ZIF-Ab2 nanozyme probe, step 2 includes the following sub-steps:

[0018] 2.1 Using screen printing technology and templates to fabricate reactive microwell arrays on epoxy-silanized glass slides;

[0019] 2.2 Mix the primary antibody (Ab1) solution with the chitosan solution, shake until evenly mixed, and then drip into the microwells of the reaction array. Store at 4°C for 12 hours.

[0020] 2.3 Add bovine serum albumin solution to the micropore surface, seal and incubate at 4°C for 12 hours to block unbound sites, then rinse with phosphate buffer solution and dry at room temperature;

[0021] 2.4 Add antigen solution to the microwell surface to react with primary antibody (Goat anti-HIgG, Ab1), incubate at room temperature for half an hour, then rinse with phosphate buffer solution and dry at room temperature;

[0022] 2.5 Add Au@CuZn-ZIF-Ab2 nanozyme probe to the micropore surface to react with the antigen, incubate at room temperature for half an hour, then rinse with phosphate buffer solution and dry at room temperature;

[0023] 2.6 Prepare the luminescent substrate and drip it onto the microwell surface. Immediately place it in the fully automated luminescence imaging system to detect the luminescence intensity.

[0024] 2.7 Based on the luminescence intensity measured in step 2.6 for the antigen solution of known concentration, a standard curve is constructed to show the linear relationship between the luminescence signal and the antigen concentration. The luminescence intensity corresponding to the unknown antigen concentration is then plotted against the coordinates corresponding to the standard curve to determine the antigen concentration.

[0025] Furthermore, in step 2.2, the concentration of the primary antibody Ab1 is 200 μg / mL, the mass concentration of the chitosan is 0.8%, and they are mixed in equal volumes.

[0026] Furthermore, in step 2.3, the mass concentration of the bovine serum albumin solution is 1%.

[0027] Furthermore, in step 2.5, the Au@CuZn-ZIF-Ab2 nanozyme probe solution was a PBS solution with a concentration of 2 mg / mL.

[0028] Furthermore, in step 2.6, the luminescent substrate is prepared from 5 mM luminol, 0.6 mM p-iodophenol, 4 mM hydrogen peroxide, and 0.1 mol / L Tris-HCl buffer solution, with the volume ratio of each solution being 50:60:1:889. In steps (a)-(f), the phosphate buffer solution is 0.01 mol / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of immunoassay of the Au@CuZn-ZIF nanozyme-based chemiluminescence imaging immunosensor of the present invention.

[0030] Figure 2 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of CuZn-ZIF and Au@CuZn-ZIF nanozymes prepared in Example 1.

[0031] Figure 3 This is a diagram showing the peroxidase-like performance of the Au@CuZn-ZIF prepared in Example 1 evaluated using an ultraviolet spectrophotometer.

[0032] Figure 4 The chemiluminescence imaging image and its linear regression curve of the chemiluminescence imaging immunosensor based on Au@CuZn-ZIF nanozyme of the present invention for detecting HIgG standard samples. DETAILED DESCRIPTION Example 1

[0033] like Figure 1 The process shown is to prepare the chemiluminescent imaging immunosensor based on Au@CuZn-ZIF nanozyme.

[0034] First, the Au@CuZn-ZIF-Ab2 nanozyme probe was synthesized

[0035] (1) Zn(NO3)2·6H2O and Cu(NO3)2·3H2O were dissolved in methanol to form liquid A, wherein the concentrations of Zn(NO3)2·6H2O and Cu(NO3)2·3H2O in liquid A were 0.013 mol / L and 0.05 mol / L, respectively. 2-Methylimidazole was then dissolved in methanol to form a solution with a concentration of 0.55 mol / LB. Liquid A and liquid B were mixed at a volume ratio of 1:2, stirred at room temperature for half an hour, and then the solid phase product in the washed mixture was collected by centrifugation to obtain CuZn-ZIF nanoparticles.

[0036] (2) Dissolve the CuZn-ZIF nanoparticles in water to form 10 mL of 4 mg / mL solution, add it to 20 mL of 3 mg / mL F-127 aqueous solution, stir at room temperature for half an hour, add 4 mL of 10 mM HAuCl4 solution, stir at room temperature for 1 hour, then add 600 μL of 0.1 mol / L NaBH4 solution, continue stirring for two hours, and then centrifuge to collect the solid phase product in the washing mixture to obtain Au@CuZn-ZIF nanoparticles;

[0037] (3) 2 mg of Au@CuZn-ZIF nanoparticles were dissolved in 1 mL of phosphate buffer solution, and 200 μL of 0.01 mol / L MUA solution was added. The mixture was stirred at room temperature for 2 h to modify the carboxyl groups on the surface of Au@CuZn-ZIF. The carboxyl groups were then activated with a mixed solution of 100 μL of EDC (10 mg / mL) and 70 μL of NHS (10 mg / mL). 40 μL of 1 mg / mL secondary antibody (Rabbit anti-HIgG, Ab2) was added and stirred at 4°C for 3 h. The mixture was then centrifuged and washed at 4°C to obtain the Au@CuZn-ZIF-Ab2 nanozyme probe, which was dispersed in 0.01 mol / L phosphate buffer solution and stored at low temperature.

[0038] The CuZn-ZIF and Au@CuZn-ZIF prepared above were analyzed by electron microscopy. Figure 2 As shown, Figures A and C are the scanning and transmission electron microscopy images of CuZn-ZIF, which can be seen to be dodecahedral in shape with a particle size of about 130 nm. Figures B and D are the scanning and transmission electron microscopy images of Au@CuZn-ZIF, which show no significant shape change and a large number of gold nanoparticles are loaded on the surface.

[0039] Then, the peroxidase-like performance of CuZn-ZIF and Au@CuZn-ZIF was analyzed by UV-visible spectrophotometer. Figure 3 As shown, the solution systems are (a) 2 mL of ABS (0.1 M); (b) 2 mL of ABS (0.1 M) + 50uL of TMB (30mM); (c) 2 mL of ABS (0.1 M) + 50uL of TMB (30mM) + 30uL of H2O2 (10M); (d) 2 mL of ABS (0.1 M) + 50uL of TMB (30mM) + 30uL of H2O2 (10M) + 1mg of CuZn-ZIF; (e) 2 mL of ABS (0.1M) + 50uL of TMB (30mM) + 30uL of H2O2 (10M) + 1mg of Au@CuZn-ZIF, Figure 3 (A) shows the color changes of each solution after being placed into transparent test tubes. As can be seen from the figure, the color of each solution changes from colorless in tubes a, b, and c to darker blue in tubes d and e. Figure 3 (B) is the UV absorption curve. It can be seen that curves a, b, and c coincide with the horizontal axis. CuZn-ZIF (curve d) and Au@CuZn-ZIF (curve e) have strong absorption at 652nm, and the signal of Au@CuZn-ZIF (curve e) is stronger, confirming that Au@CuZn-ZIF has strong peroxidase-like catalytic properties.

[0040] Then, the HigG protein molecules were detected by chemiluminescence imaging immunoassay based on the Au@CuZn-ZIF nanozyme probe, which specifically includes the following steps:

[0041] a. Using screen printing technology and with the help of a template to produce a reaction array on the surface of an epoxy-silanized glass slide, the reaction array used in this embodiment is a 4×12 array of microwells with a microwell diameter of 2 mm and an edge spacing of 4 mm;

[0042] b. Mix a 200 µg / mL primary antibody solution (Goat anti-HIgG) with an equal volume of a 0.8% chitosan solution, shake well, and drip into the microwells of the reaction array. Store at 4°C for 12 hours.

[0043] c. Add 1% bovine serum albumin solution to the micropore surface, seal and incubate at 4°C for 12 hours to block unbound sites, then rinse with 0.01 mol / L phosphate buffer solution and allow to dry at room temperature.

[0044] d. Different known concentrations of antigen solutions were added dropwise to different microwell surfaces to react with the primary antibody, incubated at room temperature for half an hour, then rinsed with 0.01 mol / L phosphate buffer solution and allowed to dry at room temperature. In this step, the known concentrations of antigen were HIgG, with concentrations of 1 ng / mL, 10 ng / mL, 100 ng / mL, 1000 ng / mL, and 10,000 ng / mL, respectively.

[0045] e. Add 2 mg / mL of Au@CuZn-ZIF-Ab2 nanozyme probe to the surface of each micropore to react with the antigen, incubate at room temperature for half an hour, then rinse with 0.01 mol / L phosphate buffer solution and dry at room temperature;

[0046] f. Mix 50µL of 5 mM luminol, 60µL of 0.6 mM p-iodophenol, 1µL of 4 mM hydrogen peroxide, and 889µL of 0.1 mol / L Tris-HCl buffer to prepare a luminescent substrate. This solution was then dripped onto the microwell surface obtained in the previous step. The luminescence intensity was then measured using an automated luminescence imaging system. The luminescence intensity values corresponding to different known concentrations of HigG antigen were recorded.

[0047] g. Prepare the chemiluminescence imaging immunosensor based on Au@CuZn-ZIF nanozyme to detect the chemiluminescence imaging graph and its linear regression curve of the HIgG standard sample. The logarithm of the HIgG antigen depth is used as the horizontal axis, and the corresponding luminescence intensity value obtained in step f is used as the vertical axis. The curve shown in the figure is the chemiluminescence imaging graph and its linear regression curve of the HIgG standard sample. It can be clearly seen from the figure that the chemiluminescence signal increases with the increase of HIgG concentration. The linear range is 1 ng / mL-10 μg / mL, and the linear regression equation is I=13167.18+708.36logC HIgG (ng / mL), with a linear correlation coefficient of 0.9957. Based on the above data, the chemiluminescence imaging immunosensor based on Au@CuZn-ZIF nanozyme has high sensitivity and low detection limit for detecting HIgG. Example 2

[0048] In this example, the Au@CuZn-ZIF-Ab2 nanozyme probe prepared in Example 1 was used. According to the implementation of steps a to e in Example 1, the spiked recovery experiment of HIgG in the untreated actual serum sample was performed, and the luminescence intensity value of the detection was compared with the Figure 4 The vertical coordinate of the standard regression curve was plotted to obtain the logarithmic value of the horizontal coordinate and then the detection concentration value of HIgG was obtained. The comparison of each test result with the standard addition value and the recovery rate obtained by detection are shown in Table 1.

[0049] Table 1. Determination of HIgG actual samples

[0050] .

[0051] It can be seen from Table 1 that the recovery rate ranges from 95.00% to 106.2%, indicating that the chemiluminescence imaging immunosensor can accurately detect HIgG in actual serum samples.

Claims

1. A chemiluminescence imaging immunoassay method based on Au@CuZn-ZIF nanozyme probe, characterized in that: Prepared by the following steps: Step 1: Prepare Au@CuZn-ZIF nanozyme and modify its surface with carboxyl groups, then connect it to the secondary antibody Ab2 to prepare the Au@CuZn-ZIF-Ab2 nanozyme probe, which includes the following steps: 1.1 First, Zn(NO3)2·6H2O and Cu(NO3)2·3H2O were dissolved in methanol to form solution A. Then, 2-methylimidazole was dissolved in methanol to form solution B. Solution A was added dropwise to solution B. The mixture was stirred at room temperature for 30-60 minutes. The solid phase product in the mixture was then collected and washed by centrifugation to obtain CuZn-ZIF nanoparticles. 1.2 Dissolve CuZn-ZIF nanoparticles in water to form a solution, add it to the block polyether F-127 aqueous solution, stir at room temperature for 30-60 minutes, add HAuCl4 solution, stir at room temperature for another 30-60 minutes, then add NaBH4 solution, continue stirring for two hours, and then centrifuge to collect the solid phase product in the washing mixture to obtain Au@CuZn-ZIF nanozyme; 1.3 The Au@CuZn-ZIF nanozyme was dissolved in phosphate buffer solution, and 11-mercaptoundecanoic acid (MUA) solution was added to modify the carboxyl groups on the Au@CuZn-ZIF surface. The carboxyl groups were then activated with a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. The secondary antibody was then added and stirred at 4°C for 3 h. The mixture was then centrifuged and washed at 4°C to obtain the Au@CuZn-ZIF-Ab2 nanozyme probe, which was dispersed in phosphate buffer solution and stored at low temperature. In the second step, the antigen molecules and the Au@CuZn-ZIF-Ab2 nanozyme probe are added dropwise to the surface of the epoxy-activated carrier modified with the primary antibody Ab1, and incubated to form a sandwich chemiluminescent imaging immunosensor to detect the antigen molecules. This includes the following steps: 2.1 Using screen printing technology and templates to fabricate reaction microwell arrays on the surface of epoxy-silanized glass slides; 2.2 Mix the primary antibody Ab1 solution with the chitosan solution, shake well, and then drop into the microwells of the reaction array and store at 4°C for 12 hours; 2.3 Add bovine serum albumin solution to the micropore surface, seal and incubate at 4°C for 12 hours to block unbound sites, then rinse with phosphate buffer solution and dry at room temperature; 2.4 Add a series of antigen solutions of known concentrations and antigen solutions of unknown concentrations to different microwell surfaces to react with Ab1, incubate at room temperature for half an hour, then rinse with phosphate buffer solution and dry at room temperature; 2.5 Add Au@CuZn-ZIF-Ab2 nanozyme probe to the micropore surface to react with the antigen, incubate at room temperature for half an hour, then rinse with phosphate buffer solution and dry at room temperature; 2.6 Prepare the luminescent substrate, drop it onto the microwell surface, and immediately place it in the fully automatic luminescence imaging system to detect the luminescence intensity; 2.7 Based on the luminescence intensity measured in step 2.6 for the antigen solution of known concentration, a standard curve is constructed to show the linear relationship between the luminescence signal and the antigen concentration. The luminescence intensity corresponding to the unknown antigen concentration is then plotted against the coordinates corresponding to the standard curve to determine the antigen concentration.

2. The chemiluminescence imaging immunoassay method based on the Au@CuZn-ZIF nanozyme probe according to claim 1, characterized in that In step 1.1, the concentrations of Zn(NO3)2·6H2O and Cu(NO3)2·3H2O in solution A are 0.013 mol / L and 0.05 mol / L, respectively, the concentration of 2-methylimidazole in solution B is 0.55 mol / L, and the volume ratio of solution A to solution B is 1:

2.

3. The chemiluminescence imaging immunoassay method based on the Au@CuZn-ZIF nanozyme probe according to claim 1, characterized in that: In step 1.2, the concentration of the CuZn-ZIF solution was 4 mg / mL, the concentration of the F-127 aqueous solution was 3 mg / mL, the concentration of the HAuCl4 solution was 10 mmol / L, and the concentration of the NaBH4 solution was 0.1 mol / L. The volume ratio of the CuZn-ZIF, F-127, HAuCl4, and NaBH4 solutions was 50:100:20:

3.

4. The chemiluminescence imaging immunoassay method based on the Au@CuZn-ZIF nanozyme probe according to claim 1, wherein In step 1.3, the concentration of the Au@CuZn-ZIF solution was 2 mg / mL, the concentration of the MUA solution was 0.01 mol / L, the concentrations of the EDC and NHS solutions were both 10 mg / mL, and the concentration of the secondary antibody Ab2 solution was 1 mg / mL; the volume ratio of the Au@CuZn-ZIF, MUA, EDC, NHS, and Ab2 solutions was 100:20:10:7:

4.

5. The chemiluminescence imaging immunoassay method based on the Au@CuZn-ZIF nanozyme probe according to claim 4, characterized in that: In step 2.2, the concentration of the primary antibody Ab1 is 200 µg / mL, the mass concentration of the chitosan is 0.8%, and they are mixed in equal volumes.

6. The chemiluminescence imaging immunoassay method based on the Au@CuZn-ZIF nanozyme probe according to claim 4, characterized in that: In step 2.3, the mass concentration of the bovine serum albumin solution is 1%.

7. The chemiluminescence imaging immunoassay method based on the Au@CuZn-ZIF nanozyme probe according to claim 4, characterized in that: In step 2.5, the Au@CuZn-ZIF nanozyme probe solution was a PBS solution with a concentration of 2 mg / mL.

8. The chemiluminescence imaging immunoassay method based on the Au@CuZn-ZIF nanozyme probe according to claim 4, characterized in that: In step 2.6, the luminescent substrate is prepared from 5 mM luminol, 0.6 mM p-iodophenol, 4 mM hydrogen peroxide, and 0.1 mol / L Tris-HCl buffer solution, with the volume ratio of each solution being 50:60:1:

889. In steps 2.1 to 2.6, the phosphate buffer solution is 0.01 mol / L.

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