Immunoassay method based on CuCo PBA nano-enzyme instant synthesis

Abstract

The invention provides an immunoassay method based on instant synthesis of a nano-enzyme of a bimetallic CuCo Prussian blue analogue (CuCo PBA), and belongs to the field of chemical and biological sensing. According to the method, a sandwich type immune mode is formed by a glucose oxidase labeled antibody, a target object and an immobilized antibody, so that the concentration of the target object is in direct proportion to the amount of glucose oxidase. Hydrogen peroxide generated by catalyzing glucose by glucose oxidase catalyzes 3, 3 ', 5, 5'-tetramethyl benzidine (TMB) to oxidize and develop color under the assistance of CuCo PBA nano-enzyme (Cu < 2 + > and K3 [Co (CN) 6]) which is generated instantly (Cu < 2 + > and K3 [Co (CN) 6] are added instantly). And the color change of the detection liquid is recorded and is used for visual detection and quantitative detection by a microplate reader. The problems of advanced synthesis and storage of nano materials are avoided, the cost is low, the colorimetric detection method is simple in operation process, rapid in color development and high in sensitivity, and a novel method which is convenient and stable in performance is provided for clinical immunological detection.

Description

Technical Field

[0001] The present invention relates to an immunoassay method based on the instant synthesis of CuCo PBA nanozyme, belonging to the technical fields of nanomaterials and bioanalysis. Background Art

[0002] In recent years, colorimetric immunoassay technology has attracted much attention in the detection of biomarkers (such as carcinoembryonic antigen, CEA) due to its simple operation, high sensitivity and low cost. This technology relies on the catalytic activity of nanozymes to achieve color reaction, and its performance directly affects the sensitivity and accuracy of detection. However, in the prior art, the synthesis of nanozymes mostly relies on hydrothermal method, solvothermal method or co-precipitation method, which has problems such as complex process, long time consumption (several hours to several days), high temperature and high pressure or special equipment required, restricting its application in point-of-care testing. In addition, traditional synthesis methods are difficult to achieve the rapid on-demand preparation of nanozymes, resulting in insufficient response speed of the detection system and unable to meet the needs of clinical rapid diagnosis.

[0003] For CEA detection, existing methods (such as electrochemical immunoassay, fluorescence immunoassay) have certain sensitivity, but often rely on expensive instruments or complex operations, making it difficult to popularize to field testing. Although the colorimetric method has the advantages of intuitiveness and no need for complex equipment, its sensitivity is limited by the catalytic efficiency of nanozymes and the synthesis process. For example, reported nanozymes such as CuO, Fe 3 O 4 etc. have problems of insufficient catalytic activity or harsh synthesis conditions, and the preparation of traditional Prussian blue analogues still needs to optimize the reaction conditions, and the potential of their instant application in CEA detection has not been fully explored.

[0004] In view of the above problems, the present invention proposes an instant synthesis method of bimetallic CuCo PBA nanozyme, which can quickly generate nanozymes with high catalytic activity by simply mixing Cu 2+ with K 3 [Co(CN) 6 solution (<1 minute). This method does not require complex equipment or high temperature and high pressure conditions, significantly simplifying the synthesis process. Further, by combining the CuCo PNA nanozyme with a glucose oxidase-mediated hydrogen peroxide generation system, a highly sensitive colorimetric immunoassay platform is constructed to achieve rapid detection of CEA. Compared with the prior art, the present invention breaks through the limitations of traditional nanozyme synthesis and application, providing an efficient, low-cost and easy-to-operate solution for clinical point-of-care diagnosis. Summary of the Invention

[0005] The object of the present invention is to provide a colorimetric immunoassay method based on the in-situ synthesis of CuCo PBA nanozyme, which is mainly used for the detection of CEA in serum. Since CuCo PBA nanozyme has peroxidase-like activity and can catalyze hydrogen peroxide to oxidize substrates to produce a color reaction, a new colorimetric immunoassay method with high sensitivity and stable performance can be established by using this characteristic.

[0006] To achieve the above object, the present invention adopts the following technical solutions: An immunoassay method based on the in-situ synthesis of CuCo PBA nanozyme comprises the following steps: (1) Immobilize the coated antibody on an enzyme-linked immunosorbent assay (ELISA) plate with high affinity; (2) Combine glucose oxidase with the labeled antibody; (3) Add the test sample and the enzyme-labeled antibody to the ELISA plate immobilized with the coated antibody in sequence, incubate and wash to form an antibody-antigen-antibody sandwich-type immune complex; add glucose to the above micro-wells for reaction; (4) In the micro-wells of the ELISA plate where glucose has reacted, add the CuCoPBA solution formed by instantaneously mixing Cu 2+ and K 3 [Co(CN) 6 into it, then add the TMB chromogenic solution, and detect the absorbance value of each micro-well with an ELISA reader.

[0007] In step (1), the binding of the antibody-coated ELISA plate forms a stable solid-phase carrier through chemical bond interaction. The binding of the gold nanoparticles with the antibody and GOx mainly relies on electrostatic interaction, hydrophobic interaction and chemical bond binding; In step (2), the binding of the glucose oxidase with the antibody is a chemical bond binding; In step (3), the test sample is a macromolecular target; the completion of the sandwich-type immunoassay mode adopts a two-step method: that is, the test sample and the antibody immobilized on the ELISA plate are mixed to have an immune reaction to form an immune complex, and then mixed with the enzyme-labeled antibody nano-probe to have an immune reaction to form an antibody-antigen-antibody sandwich-type immune complex. The amount of glucose used is 50 μL and the concentration is 10 mM; In step (4), the dosage of the CuCo PBA nanozyme is 40 μL, the dosage of the TMB chromogenic solution is 100 μL, and the pH value is 4.0.

[0008] The detection principle of the colorimetric immunoassay method of the present invention is as follows: First, CEA in the sample to be detected binds to the immobilized antibody, and then incubates with the enzyme-labeled antibody nanoprobe to form an antibody-antigen-antibody sandwich complex. When the CEA concentration is low, fewer enzyme-labeled antibody nanoprobes bound to the carrier, the amount of hydrogen peroxide generated by glucose oxidase catalyzing glucose is low, and the oxidation degree of TMB catalyzed by CuCo PBA nanozyme is low, resulting in a weak color reaction and a low absorbance value; conversely, when the CEA concentration is high, the production of hydrogen peroxide increases, the catalytic reaction of CuCo PBA nanozyme is enhanced, the color reaction is enhanced, and the absorbance value increases. Quantitative analysis of CEA can be achieved through the change in absorbance.

[0009] The advantages of the present invention are as follows: (1) The present invention provides a method for instantaneously synthesizing CuCo PBA nanozyme without complex equipment and can be used for on-site rapid detection; (2) The present invention utilizes the high catalytic activity of CuCo PBA nanozyme to achieve ultrasensitive detection of CEA, with a detection limit as low as 22 pg / mL and a linear detection range of 0.05 - 60 ng / mL; (3) CuCo PBA nanozyme has high stability, is not affected by changes in pH value and temperature, avoids the problem of easy inactivation of traditional enzyme catalytic systems, can be stored for a long time and stably used under different conditions; (4) The method of the present invention is not only applicable to CEA detection, but also can be extended to the detection of other biomarkers, providing a new technical solution for early disease diagnosis and having significant economic and social value. Brief Description of the Drawings

[0010] Figure 1 is a schematic diagram of immunoassay based on instant synthesis of CuCo PBA nanozyme; Figure 2 is a diagram for exploring the mechanism of colorimetric immunoassay (a: glucose + glucose oxidase + CuCo PBA + TMB chromogenic solution; b: glucose + glucose oxidase + CuCo PBA; c: glucose oxidase + CuCo PBA + TMB chromogenic solution; d: glucose + CuCo PBA + TMB chromogenic solution; e: CuCo PBA + TMB chromogenic solution; f: glucose + glucose oxidase + TMB chromogenic solution.) (The inset is a photo of the corresponding solution); Figure 3 is a standard working curve of colorimetric immunoassay for detecting carcinoembryonic antigen based on instant synthesis of CuCo PBA nanozyme. Detailed Embodiments The technical solutions of the present invention will be further described below through specific implementation examples, but the scope of the present invention cannot be limited thereby. Example

[0012] (1) Preparation of antibody immobilized on the ELISA plate: 200 μL of CEA antibody (PBS at pH 7.4, 10 μg / mL) was added to the ELISA plate and incubated overnight. After washing three times with PBS buffer solution at pH 7.4, 300 μL of PBS buffer solution containing 1% BSA at pH 7.4 was added to each well as a blocking solution and incubated at 37 °C for 1 hour.

[0013] (2) Preparation of the enzyme-labeled antibody complex: First, 20 μg of CEA antibody and 20 U of glucose oxidase were dissolved together in 1.0 mL of PBS (pH 7.4). Second, 10 μL of 1% glutaraldehyde was added in the fume hood and shaken at 4 °C for 2 hours. Subsequently, sodium borohydride was added to remove the excess aldehydes and shaken at 4 °C for 1 hour. Finally, it was filtered through a 0.45 μm filter and purified by dialysis with PBS solution. The obtained enzyme-labeled antibody complex was dispersed in 4 mL of PBS buffer solution and stored at 4 °C.

[0014] (3) Investigation of the mechanism of colorimetric immunoassay: First, 10 µL of glucose oxidase (1 mg / mL) and 50 µL of glucose (4 mM) were added to the wells of the ELISA plate and incubated at 37 °C for 30 minutes. Subsequently, the instantaneously synthesized CuCo PBA nanozyme solution (20 µL Cu 2+ and 20 µL K 3 [Co(CN) 6 ) was added thereto, and 100 µL of TMB chromogenic solution (1 mM) was added. The reaction was carried out at room temperature for 30 minutes. Finally, the absorbance value was read at 652 nm with an ELISA reader. At the same time, detections were carried out in another five wells without adding TMB, CuCo PBA, glucose oxidase, and glucose respectively according to the above method process. As Figure 2 shown by the results, the solution can only develop color when glucose, glucose oxidase, CuCo PBA, and TMB are present simultaneously. Figure 2 The photo of the solution corresponding to the curve is shown in the illustration. The experimental results show that the source of the color development is the oxidation of TMB by hydrogen peroxide produced by the reaction of glucose and glucose oxidase with the assistance of CuCo PBA. Therefore, when the concentration of glucose oxidase changes, the absorbance changes due to the change in the amount of hydrogen peroxide produced. Therefore, this scheme can be applied to immunoassay.

[0015] (4) Colorimetric immunoassay for detecting CEA (carcinoembryonic antigen in this example is used as the model target analyte): Figure 1It is a schematic diagram of the immunoassay process based on the instant synthesis of CuCo PBA nanozyme in the present invention. First, different concentrations of CEA antigen were added to the wells of the enzyme-linked immunosorbent assay (ELISA) plate previously coated with CEA antibody, and incubated at 37 °C for 1 hour. Subsequently, it was washed three times with PBST washing solution (pH 7.4, 0.01 M, 0.05% Tween). Then, 100 μL of enzyme-labeled antibody complex (5 μg / mL) was added and incubated at 37 °C for 1 hour. After washing three times, 10 mM glucose was added and reacted for 30 minutes. Finally, the instantaneously synthesized mixed solution of CuCo PBA and TMB was added, reacted for 50 minutes, and the absorbance of the solution was measured with an enzyme-linked immunosorbent assay (ELISA) reader. As Figure 3 shown, the standard working curve of the change in CEA concentration and absorbance value was plotted. The experimental results showed that the detection limit of this method for carcinoembryonic antigen was 22 pg / mL, and the linear range was 0.05 to 60 ng / mL. Compared with the traditional enzyme-catalyzed colorimetric detection technology, this method innovatively adopted CuCo PBA nanozyme, realized instant synthesis and had excellent catalytic activity, effectively overcoming the limitations of traditional enzymes such as easy inactivation during storage and application and environmental influence on stability, providing more stable and efficient technical support for the fields of tumor marker detection and clinical diagnosis, etc.

[0016] The above are only the preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. An immunoassay method based on instant synthesis of CuCo PBA nanozymes, characterized in that: The method comprises the following steps: (1) Immobilize the coated antibody on a high-affinity ELISA plate; (2) combining glucose oxidase with labeled antibodies; (3) Add the sample to be tested and the enzyme-labeled antibody to the ELISA plate loaded with the coated antibody in sequence, incubate and wash to form an antibody-antigen-antibody sandwich immune complex; add glucose to the above microwells for reaction; (4) In the microwells of the ELISA plate containing glucose, place Cu 2+ The CuCo PBA solution formed by instant mixing with K3[Co(CN)6] is added thereto. Then, TMB colorimetric solution was added, and the absorbance value of each microwell was detected by an enzyme-labeled instrument.

2. The immunoassay method based on instant synthesis of CuCo PBA nanozymes according to claim 1, characterized in that: The instant synthesized CuCo PBA was used as a peroxidase mimic, avoiding the complex synthesis and storage problems of nanomaterials and achieving simple and rapid detection of the analyte.

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