A method for preparing copper-doped NiFe PBA nanozyme probes and its application

Abstract

This invention discloses a method for preparing a copper-doped NiFe PBA nanozyme probe and its application, comprising (1) preparing a copper-doped NiFe PBA nanozyme and connecting it with a secondary antibody Ab2 to obtain a Cu-NiFe PBA-Ab2 nanozyme probe; (2) dropping the antigen molecule and the Cu-NiFe PBA-Ab2 nanozyme probe onto the surface of an epoxy-activated carrier sheet modified with a primary antibody Ab1 and streptavidin, and incubating to form a chemiluminescent imaging immunosensor with a double antibody sandwich core, thereby achieving highly sensitive detection of antigen molecules with a detection limit down to the femtogram level.

Description

Technical Field

[0001] This invention relates to immunological analysis and detection technology, specifically to a method for preparing a copper-doped NiFe PBA nanozyme probe for detecting protein molecules in bodily fluids such as blood, urine, saliva, pleural effusion, and peritoneal fluid, and its application. Background Technology

[0002] Chemiluminescence imaging immunoassay (CLIIA) combines chemiluminescence immunoassay with imaging technology, offering the advantages of both. It boasts high throughput, a wide linear range, and fast analysis speed, making it suitable for detecting trace medical substances such as proteins, hormones, and bacteria. It also plays a crucial role in disease diagnosis, monitoring treatment effectiveness, and disease progression. In chemiluminescence systems, catalysts play a vital role in enhancing chemiluminescence intensity. While natural enzymes are commonly used catalysts to improve the detection sensitivity of chemiluminescence analysis, their susceptibility to inactivation, high preparation costs, and difficulty in storage severely hinder the development and practical application of chemiluminescence immunoassay methods. Compared to natural enzymes, nanozymes offer advantages such as low cost, high stability, and good biocompatibility, and have been widely applied in the food, chemical, and medical fields.

[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 energy and environmental fields, including energy conversion and storage, adsorption, electrochemical sensors and biosensors.

[0004] Compared to monometallic nanozymes, bimetallic nanozymes exhibit superior catalytic performance and stability. On one hand, the efficient charge transfer between the different elements in a bimetallic nanozyme contributes to enhanced catalytic activity; on the other hand, the construction of a bimetallic center can increase the number of catalytic centers. High chemical surface area, more exposed active sites, and appropriate lattice strain have been identified as influencing factors that enhance intrinsic activity and promote kinetic reaction rates. Therefore, there is an urgent need to develop a nanozyme probe with high catalytic activity for highly sensitive detection of analyte antigens. Summary of the Invention

[0005] The purpose of this invention is to prepare a chemiluminescent imaging immunosensor by providing a method for preparing a copper-doped NiFe PBA nanozyme probe, and then combining it with a double antibody sandwich method to achieve high-throughput, high-specificity, and high-sensitivity detection of target antigen molecules, with a detection limit down to the femtogram level.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

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

[0008] S1: Preparation of copper-doped NiFe PBA nanozymes;

[0009] S2: The surface of copper-doped NiFe PBA nanozymes was modified with carboxyl groups and linked with secondary antibody Ab2 to prepare Cu@NiFePBA-Ab2 nanozyme probes.

[0010] Preferably, S1 includes the following steps:

[0011] Ni(NO3)2·6H2O, C6H5Na3O7, and Cu(NO3)2·3H2O were weighed and dispersed in 50 mL of deionized water. The solution was dissolved completely by sonication to form solution A, which was blue-green. K3[Fe(CN)6] was then dissolved in 50 mL of deionized water to form solution B, which was yellow. Solution B was added dropwise to solution A, stirred at room temperature for 10 min, and aged for 24 h. The solution was then collected by centrifugation at 8000 r / min for 5 min, washed three times alternately with ethanol and water, and finally vacuum dried 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] Copper-doped NiFe PBA nanozymes were dissolved in phosphate buffer solution, and 11-mercaptoundecanoic acid (MUA) solution was added to modify the carboxyl groups on the surface of copper-doped NiFe PBA. Then, the carboxyl groups were activated with a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The mixture was stirred at room temperature for 1 h, and then a secondary antibody (Ab2) was added. The mixture was stirred at 4 °C for 3 h, and then centrifuged and washed at 4 °C to obtain the Cu@NiFe PBA-Ab2 nanozyme probe, which was dispersed in 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 objective of this invention, this invention also provides a method for detecting antigens using a chemiluminescence imaging immunosensor based on a copper-doped NiFe PBA nanozyme probe, comprising the following steps:

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

[0018] (b) Mix equal volumes of chitosan solution and streptavidin solution. Add 5 μL of the mixture into the microwells of the reaction array each time and let it air dry naturally. Then add 5 μL of primary antibody solution to each well, let it air dry for 30 min, and incubate at 4°C for 12 hours.

[0019] (c) Bovine serum albumin solution was dropped onto the surface of the micropores, and the unbound sites were sealed and incubated at 4°C for 6 hours. Then, the micropores were rinsed with phosphate buffer solution and air-dried at room temperature.

[0020] (d) Add antigen solution to the surface of the micropores, incubate at room temperature for 25 min, then rinse with phosphate buffer solution and air dry at room temperature;

[0021] (e) Cu@NiFe PBA-Ab2 nanozyme probe was dropped onto the surface of the micropores and reacted with the antigen. The mixture was incubated at room temperature for 30 min, then rinsed with phosphate buffer solution and air-dried at room temperature.

[0022] (f) Prepare the luminescent substrate, drop it onto the surface of the micropores, and quickly place it into a fully automated luminescence imaging system to detect the luminescence intensity.

[0023] Furthermore, the sensor array prepared in step (a) has a 4×12 array of micropores with a diameter of 4 mm and a spacing of 1.5 mm between the pores and their edges. The micropores are covered with a green insulating paint that has a hydrophobic effect.

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

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

[0026] Furthermore, in step (f), the luminescent substrate 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.

[0027] In the copper-doped NiFe PBA nanozyme of this invention, Ni and Fe act as bimetallic centers, synergistically enhancing the catalytic activity of the nanozyme. Simultaneously, the incorporation of Cu alters the electronic structure, further enhancing the catalytic activity. Furthermore, the core-shell and hollow structure provides a higher cavity volume and surface area, exposing more active sites.

[0028] Using a chemiluminescent system based on copper-doped NiFe PBA nanozymes as a probe to label antibodies can effectively overcome the drawbacks of natural enzymes, such as easy inactivation, high preparation cost, and difficulty in storage. Simultaneously, the copper-doped NiFe PBA nanozyme exhibits peroxidase-like activity, catalyzing hydrogen peroxide to generate a strong oxidizing hydroxyl radical, which triggers luminol to produce a chemiluminescent signal. The luminescent signal is collected by a charge-coupled device (CCD), and the linear relationship between the chemiluminescent signal intensity and antigen concentration enables the detection of the analyte antigen. The addition of copper ions to the copper-doped NiFe PBA nanozyme enhances the peroxidase-like catalytic activity and stability of NiFe PBA. The addition of streptavidin to the reaction system amplifies the chemiluminescent signal. Combined with the double-antibody sandwich method, this improves the sensitivity and specificity of protein molecule detection. Attached Figure Description

[0029] Figure 1 A schematic diagram of the process for fabricating a chemiluminescent imaging immunosensor based on copper-doped NiFe PBA nanozymes.

[0030] Figure 2 Scanning electron microscope (SEM) and transmission electron microscope (TEM) images of NiFePBA and copper-doped NiFePBA nanozymes.

[0031] Figure 3 The graph shows the peroxidase-like performance of NiFe PBA doped with different amounts of copper, evaluated using a UV spectrophotometer.

[0032] Figure 4 Chemiluminescence imaging image and its linear regression curve for detecting BTN3A2 standard using the chemiluminescence imaging immunosensor based on copper-doped NiFe PBA nanozyme of this invention.

[0033] Figure 5 Correlation analysis of serum BTN3A2 concentration in gastric cancer patients at Yizheng People's Hospital based on the chemiluminescent imaging immunosensor and ELISA kit of the present invention;

[0034] Figure 6 Correlation analysis of serum BTN3A2 concentration in gastric cancer patients at Subei People's Hospital based on the chemiluminescent imaging immunosensor and ELISA kit of the present invention;

[0035] Figure 7 This study investigates the correlation between the chemiluminescence imaging immunosensor and ELISA kit based on copper-doped NiFe PBA nanozyme used in this invention for determining serum BTN3A2 concentration in gastric cancer patients at Anhui Provincial Hospital. Detailed Implementation

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

[0037] Example 1

[0038] like Figure 1 The process shown is used to prepare a chemiluminescent imaging immunosensor based on a copper-doped NiFe PBA nanozyme probe.

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

[0040] (1) Weigh Ni(NO3)2·6H2O, C6H5Na3O7 and Cu(NO3)2·3H2O, disperse them in 50 mL of deionized water, and sonicate them to dissolve them completely 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. Add solution B dropwise to solution A, stir at room temperature for 10 min, age for 24 h, centrifuge at 8000 r / min for 5 min to collect, wash three times with ethanol and water alternately, and then vacuum dry at 60 °C for 12 h to obtain copper-doped NiFe PBA nanozyme;

[0041] (2) Copper-doped NiFe PBA nanozymes were dissolved in phosphate buffer solution, and 11-mercaptoundecanoic acid (MUA) solution was added to modify carboxyl groups on the surface of copper-doped NiFe PBA. Then, the carboxyl groups were activated with a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The mixture was stirred at room temperature for 1 h, and then a secondary antibody (Ab2) was added. The mixture was stirred at 4 °C for 3 h, and then centrifuged and washed at 4 °C to obtain Cu@NiFe PBA-Ab2 nanozyme probe, which was dispersed in phosphate buffer solution and stored at low temperature.

[0042] Then, a chemiluminescent imaging immunosensor based on a copper-doped NiFe PBA nanozyme probe was prepared for antigen detection, specifically including the following steps:

[0043] (a) A reaction array is fabricated on the surface of an epoxy-silanized glass slide using screen printing technology and with the aid of a template;

[0044] (b) Mix equal volumes of chitosan solution and streptavidin solution. Add 5 μL of the mixture into the microwells of the reaction array each time and let it air dry naturally. Then add 5 μL of primary antibody solution to each well, let it air dry for 30 min, and incubate at 4°C for 12 hours.

[0045] (c) Bovine serum albumin solution was dropped onto the surface of the micropores, and the unbound sites were sealed and incubated at 4°C for 6 hours. Then, the micropores were rinsed with phosphate buffer solution and air-dried at room temperature.

[0046] (d) Add antigen solution to the surface of the micropores, incubate at room temperature for 25 min, then rinse with phosphate buffer solution and air dry at room temperature;

[0047] (e) The probe was dropped onto the surface of the micropore and reacted with the antigen. The mixture was incubated at room temperature for 30 min, then rinsed with phosphate buffer solution and air-dried at room temperature.

[0048] (f) Prepare the luminescent substrate, drop it onto the surface of the micropores, and quickly place it into a fully automated luminescence imaging system to detect the luminescence intensity.

[0049] The prepared NiFe PBA and copper-doped NiFe PBA were analyzed by scanning electron microscopy as follows: Figure 2 As shown, Figure 2 A, Figure 2 C shows the scanning and transmission electron microscopy images of NiFe PBA, revealing its cubic shape and particle size of approximately 150 nm. Figure 2 B Figure 2 D shows the scanning and transmission electron microscopy images of copper-doped NiFe PBA. It can be observed that its shape remains unchanged, still cubic, but its size has decreased, with a particle size of about 100 nm.

[0050] The peroxidase-like properties of NiFe PBA and NiFe PBA with different amounts of copper doping were analyzed using a UV-Vis spectrophotometer. Figure 3 As shown, the solution systems are (a) 3.0 mM Cu-NiFe PBA+, (b) 1.5 mM Cu-NiFe PBA+, (c) NiFe PBA+, (d) 6.0 mM Cu-NiFe PBA+, and (e) 4.5 mM Cu-NiFe PBA. The inset shows the color changes of the solution. NiFe PBA and NiFe PBA with different amounts of copper doping have strong absorption at 652 nm, and the signal of 3.0 mM Cu-NiFe PBA is stronger, which confirms that 3.0 mM Cu-NiFe PBA has strong peroxidase-like catalytic performance.

[0051] Figure 4This image shows the chemiluminescence imaging pattern and linear regression curve of the BTN3A2 standard sample detected by the chemiluminescence imaging immunosensor based on the copper-doped NiFe PBA nanozyme probe of this invention. Figure 4 It can be clearly seen that the chemiluminescence signal increases with increasing BTN3A2 concentration, with a linear range of 0.5-5000 pg / mL. The linear regression equation is I = 33103.73 + 4500.13 logC. BTN3A2 (pg / mL), with a linear correlation coefficient of 0.9942. Based on the above data, the chemiluminescent imaging immunosensor based on the copper-doped NiFe PBA nanozyme probe exhibits high sensitivity and a low detection limit for BTN3A2.

[0052] Example 2

[0053] like Figure 5-7 As shown, this embodiment uses the chemiluminescence imaging immunosensor prepared in Example 1 to detect BTN3A2 in serum samples from gastric cancer patients in three hospitals: Yizheng People's Hospital (n=20), Subei People's Hospital (n=20), and Anhui Provincial Hospital (n=20). Before detection, the serum was diluted 10 times with PBS (10mM, pH 7.4) without further pretreatment. The 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 embodiments listed in this application are merely examples to more clearly illustrate the present invention, and are not intended to limit the implementation of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A preparation method of a copper-doped NiFe PBA nanoszyme probe, characterized in that, Comprising the following steps: S1: Preparation of copper-doped NiFe PBA nanoscale enzyme: Take Ni(NO3)2·6H2O, C6H5Na3O7 and Cu(NO3)2·3H2O, disperse them in 50 mL of deionized water, and ultrasonic them to completely dissolve to form A liquid, the solution is blue-green, then dissolve K3[Fe(CN)6] in 50 mL of deionized water to form B liquid, the solution is yellow; add B liquid drop by drop into A liquid, stir at room temperature for 10 min, age for 24 h, collect by centrifugation at 8000 r / min for 5 min, wash with ethanol and water alternately for three times, and then vacuum dry at 60℃ for 12 h to obtain copper-doped NiFe PBA nanoscale enzyme; the concentrations of Ni(NO3)2·6H2O, C6H5Na3O7 and Cu(NO3)2·3H2O in A liquid are 30 mmol / L, 45 mmol / L and 6 mmol / L respectively, and the concentration of K3[Fe(CN)6] in B liquid is 20 mmol / L; S2: Carboxyl modification on the surface of copper-doped NiFe PBA nanoscale enzyme and connection of secondary antibody Ab2 to prepare Cu@NiFe PBA-Ab2 nanoscale enzyme probe: Dissolve copper-doped NiFe PBA nanoscale enzyme in phosphate buffer solution, add 11-mercaptoundecanoic acid solution to modify carboxyl on the surface of copper-doped NiFe PBA, then activate the carboxyl with a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stir at room temperature for 1 h, then add secondary antibody Ab2, stir at 4℃ for 3 h, and then continue to centrifuge and wash at 4℃ to obtain Cu@NiFe PBA-Ab2 nanoscale enzyme probe, which is dispersed in phosphate buffer solution and stored at low temperature; The concentration of 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 secondary antibody Ab2 is 500 µg / mL.

2. A method for detecting antigen by a copper-doped NiFe PBA nanoscale enzyme probe-based chemiluminescence imaging immunosensor, comprising the following steps: (a) Using screen printing technology and with the help of a template, a reaction array is prepared on the surface of an epoxy silanized glass slide; (b) Mix the same volume of chitosan solution and streptavidin solution, take 5 μL of the mixed solution each time, drop it into the microwells of the reaction array, and wait for natural air drying, then add 5 μL of primary antibody solution to each well, air dry for 30 min, and incubate in a 4℃ refrigerator for 12 hours; (c) Drop bovine serum albumin solution on the surface of the microwells, seal and incubate in a 4℃ environment for 6 hours to block unbound sites, then rinse with phosphate buffer solution and air dry at room temperature; (d) Drop antigen solution on the surface of the microwells, incubate at room temperature for 25 min, then rinse with phosphate buffer solution and air dry at room temperature; (e) The Cu@NiFe PBA-Ab2 nanoscale enzyme probe prepared in step (a) was added dropwise to the micro-well surface and reacted with the antigen, and incubated at room temperature for 30 min, then washed with phosphate buffer solution and air-dried at room temperature; (f) The luminescent substrate was prepared and added dropwise to the micro-well surface, and then quickly placed into the full-automatic luminescence imaging system to detect the luminescence intensity.

3. The method of claim 2, wherein, The sensing array prepared in step (a) has a 4×12 array of micro-wells, with a micro-well diameter of 4 mm and a well-to-well edge distance of 1.5 mm, and the micro-wells are surrounded by green insulating paint with hydrophobic properties.

4. The method of claim 2, wherein, In step (b), the chitosan mass concentration is 0.5%, the streptavidin solution concentration is 50 μg / mL, and the concentration of the primary antibody is 5 μg / mL.

5. The method of claim 2, wherein, In step (c), the bovine serum albumin solution has a mass concentration of 2%, and in step (e), the probe concentration is 2 mg / mL.

6. The method of claim 2, wherein, In step (f), the luminescent substrate 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 in steps (a)-(f), the phosphate buffer solution is 0.01 mol / L.

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