Preparation method of osmium citrate nano-enzyme and application of osmium citrate nano-enzyme in immunoadsorption analysis

Abstract

The invention discloses a preparation method of osmium citrate nano-enzyme and application of the osmium citrate nano-enzyme in immunoadsorption analysis, relates to the technical field of immunodetection, and aims to solve the problems of long operation time and complex flow of the existing enzyme-linked immunosorbent assay. The method comprises the following steps: 1, mixing K2OsCl6 and trisodium citrate with deionized water to obtain a mixed solution A; 2, mixing sodium borohydride with deionized water to obtain a sodium borohydride solution, and dropwise adding the sodium borohydride solution into the mixed solution A to obtain a mixed solution B; stirring and reacting the mixed solution B under a dark condition to obtain the osmium citrate nano-enzyme. The nano-enzyme with the particle size range prepared by the method has high catalytic efficiency and better stability and dispersity. The osmium citrate nano enzyme is used for immunoassay, so that the time required by immunoassay is greatly shortened. The osmium citrate nano-enzyme provided by the invention can be used for preparing an osmium citrate nano-enzyme-antigen / antibody compound for immunoadsorption analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and particularly relates to a wash-free and highly efficient enzyme-linked immunosorbent assay method based on nanozyme. Background Art

[0002] Immunoassay is a method for identifying target analytes based on specific antigens and antibodies. This method has the advantage of being able to sensitively analyze a large number of samples. Among them, enzyme-linked immunosorbent assay (ELISA for short) is an immunoassay technique widely used in the fields of biology and medicine.

[0003] The operation steps of enzyme-linked immunosorbent assay include: (1) Coating: Adsorbing antigens or antibodies on the surface of a solid-phase carrier, usually using a polystyrene microplate as the solid-phase carrier. (2) Blocking: Blocking the unoccupied sites on the solid-phase carrier with irrelevant proteins (such as bovine serum albumin) to reduce non-specific adsorption. (3) Sampling: Adding the sample to be detected and the enzyme-labeled substance to make a specific binding reaction occur on the surface of the solid-phase carrier. (4) Washing: Removing the unbound substances by washing to reduce background interference. (5) Color development: Adding the substrate of the enzyme, and under the catalytic action of the enzyme, the substrate undergoes a color development reaction. (6) Terminating the reaction: Adding a termination solution to stop the enzyme reaction, and then measuring the absorbance value with an enzyme-labeled instrument, and judging the content or positive / negative result of the target substance in the sample according to the standard curve or the critical value. Therefore, enzyme-linked immunosorbent assay has problems of long time and complex process in the actual operation process. Summary of the Invention

[0004] The present invention aims to solve the problems of long operation time and complex process of the existing enzyme-linked immunosorbent assay, and provides a wash-free and highly efficient immunoassay method based on nanozyme.

[0005] The preparation method of the osmium citrate nanozyme of the present invention includes the following steps:

[0006] Step 1: Mix K2OsCl6 and trisodium citrate with deionized water, and stir evenly at 10°C to 30°C to obtain a mixed solution A;

[0007] Step 2: Mix sodium borohydride with deionized water to obtain a sodium borohydride solution, and drip the sodium borohydride solution into the mixed solution A prepared in Step 1 at a dripping rate of 100 - 300 μL / min to obtain a mixed solution B; make the mixed solution B stir and react under dark conditions at 10°C to 30°C to obtain osmium citrate nanozyme; wherein the particle size of the osmium citrate nanozyme is 60 - 200 nm.

[0008] The present invention also provides the application of the above-mentioned osmium citrate nanozyme in immunoabsorbent assay

[0009] Furthermore, an immunosorbent assay is performed using the osmic citrate nanozyme-prepared osmic citrate nanozyme-antigen / antibody complex.

[0010] The preparation method of the osmic citrate nanozyme-antigen / antibody complex is as follows:

[0011] Step 1: Vacuum freeze-dry the osmic citrate nanozyme to constant weight, and then add deionized water to disperse it evenly to obtain an osmic citrate nanozyme solution;

[0012] Step 2: Dilute the osmic citrate nanozyme solution with PBS, and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), and mix well by ultrasonic treatment to obtain a mixed solution; the concentration of EDC is 20 mg / mL, and the concentration of NHS is 48 mg / mL;

[0013] Step 3: Add the antigen or antibody to the mixed solution prepared in Step 2, incubate overnight at 4°C, and then centrifuge. After removing the supernatant, resuspend it in a blocking solution for blocking;

[0014] Step 4: Then continue to centrifuge, remove the supernatant, retain the precipitate, and then add phosphate buffer to obtain the osmic citrate nanozyme-antigen / antibody complex.

[0015] The method for immunosorbent assay using the osmic citrate nanozyme-antigen / antibody complex in the present invention includes the following steps:

[0016] Add the antibody or antigen to be tested diluted with PBS into a 96-well plate, add the osmic citrate nanozyme-antigen / antibody complex into the 96-well plate, and oscillate at room temperature;

[0017] Add a mixed solution of TMB and hydrogen peroxide to the 96-well plate, then incubate in an environment at 37°C, add a termination solution, and measure the immunoassay result.

[0018] The beneficial effects of the present invention:

[0019] To overcome the problems of long operation time and complex process in enzyme-linked immunosorbent assay and obtain better immunoassay results, the present invention uses K2OsCl6, trisodium citrate, sodium borohydride, and antigen / antibody as raw materials to prepare a functionalized osmic citrate nanozyme, which shows good peroxidase-like activity. Moreover, the osmic citrate nanozyme and the antigen are specifically combined by covalent bonds to achieve the purpose of immunoassay.

[0020] The particle size of the osmium citrate nanozyme of the present invention is 60 - 200 nm. The nanozymes with the particle size range prepared by the present invention have high catalytic efficiency, good stability and dispersibility. The principle lies in that the smaller particle size ensures a larger specific surface area, increases the contact area between the nanozyme and the substrate, and is conducive to the progress of catalysis. At the same time, the nanoparticles in this particle size range take into account both stability and dispersibility, avoiding the problems of easy aggregation of too small particles or sedimentation of too large particles, and ensuring the uniformity and repeatability of the detection system.

[0021] The active site of the osmium citrate nanozyme of the present invention is blocked after being conjugated with the antigen. Only in the presence of the specific antibody, the antigen will be removed from the surface of the nanozyme through the high-affinity binding of antigen-antibody, and the active site will be exposed again. In this way, through the specific binding of antigen and antibody, the selective opening of the immunoassay signal can be achieved without a washing step to remove unbound antibody or antigen, thus simplifying the operation process. Specifically, when there is no specific antibody, the peroxidase activity of the nanozyme is at a low level and will not cause significant color change in the hydrogen peroxide / TMB system. In the presence of the specific antibody, the antibody removes the antigen from the surface of the nanozyme, and the active site is exposed again, enabling the nanozyme to catalyze the reaction in the hydrogen peroxide / TMB system to generate a blue product. Therefore, this method relies on the presence or absence of the specific target analyte to regulate the enzyme activity, rather than removing excess substances by washing the plate.

[0022] The washing-free and highly efficient immunoassay method of the present invention greatly shortens the time required for immunoassay. The operation process of traditional ELISA detection is 147 minutes, while the method of the present invention takes about 24 minutes in total, greatly improving the detection efficiency and simplifying the cumbersome operation process.

[0023] The functionalized nanozyme for immunoassay prepared by the present invention can show a proportional absorbance in the TMB and hydrogen peroxide system with the increase of the concentration of the analyte, and does not interfere with other proteins. It is proved that the functionalized nanozyme for immunoassay can be used for specific detection.

[0024] The functionalized nanozyme for immunoassay prepared by the present invention has a wide range of temperature stability, pH stability and specificity, can be used as an enzyme in some immunoassays, and has good application prospects. Brief Description of the Drawings

[0025] Figure 1 It is a comparison diagram of the catalytic mechanisms of oxidase and peroxidase;

[0026] Figure 2 It is a scanning electron microscope image of the osmium citrate nanozyme prepared by the present invention at 40,000 times magnification;

[0027] Figure 3FT-IR comparison chart of osmic citrate nanozyme prepared according to the present invention and potassium hexachloroosmate;

[0028] Figure 4 EDS energy spectrum diagram of osmic citrate nanozyme prepared according to the present invention;

[0029] Figure 5 Results of temperature stability determination of osmic citrate nanozyme prepared according to the present invention and horseradish peroxidase;

[0030] Figure 6 Results of pH stability determination of osmic citrate nanozyme prepared according to the present invention and horseradish peroxidase;

[0031] Figure 7 Results of linear range determination of osmic citrate nanozyme prepared according to the present invention for murine IgG immunoassay;

[0032] Figure 8 Results of specificity determination of osmic citrate nanozyme prepared according to the present invention for murine IgG immunoassay;

[0033] Figure 9 Results of stability determination of osmic citrate nanozyme prepared according to the present invention for murine IgG immunoassay;

[0034] Figure 10 Results of linear range determination of osmic citrate nanozyme prepared according to the present invention for ALV antigen immunoassay;

[0035] Figure 11 Results of specificity determination of osmic citrate nanozyme prepared according to the present invention for ALV antigen immunoassay;

[0036] Figure 12 Results of stability determination of osmic citrate nanozyme prepared according to the present invention for ALV antigen immunoassay. Detailed implementation manners

[0037] The technical solution of the present invention is not limited to the specific implementation manners listed below, and also includes any combination between the specific implementation manners.

[0038] Detailed implementation manner one: The preparation method of osmic citrate nanozyme in this implementation manner includes the following steps:

[0039] Step one: Mix K2OsCl6 and trisodium citrate with deionized water, and stir evenly at 10°C to 30°C to obtain a mixed solution A;

[0040] Step 2: Mix sodium borohydride with deionized water to obtain a sodium borohydride solution, and drop the sodium borohydride solution into the mixed solution A prepared in Step 1 at a dropping rate of 100 - 300 μL / min to obtain a mixed solution B; stir and react the mixed solution B under dark conditions at 10°C - 30°C to obtain osmium citrate nanozyme with controllable particle size; wherein the particle size of the osmium citrate nanozyme is 60 - 200 nm.

[0041] The existing applications of osmium nanozyme mainly focus on its oxidase activity (such as Figure 1 a), and heavy metal ions such as mercury are introduced as ligands to enhance its activity. Its catalytic process relies on oxygen as an electron acceptor. Since osmium directly participates in the redox reaction in this case, a relatively high concentration of osmium nanoparticles is required to compensate for the insufficient reaction rate, which leads to a significant increase in the amount of nanoparticles used in the reaction system. This not only increases the cost but also poses challenges to the biosafety of the detection system and the stability of proteins in the system due to the excessive heavy metal ions such as mercury.

[0042] In addition to oxidase activity, osmium nanozyme also has peroxidase-like activity ( Figure 1 b), which is superior to most nanozymes. Research shows that in the hydrogen peroxide, TMB, osmium nanozyme system, this catalytic color development mechanism may follow the Fenton reaction, that is, tetravalent osmium ions combine with hydrogen peroxide to generate hydroxyl radicals (·OH), thereby making the reaction system have strong oxidizing properties. And the ESR results confirm the existence of ·OH in the reaction system under acidic conditions. Therefore, the catalytic mechanism of osmium nanozyme is to first interact with H2O2 to generate ·OH, and then ·OH reacts with TMB to develop color. In this reaction, osmium nanozyme only acts as a catalyst, that is, a trace amount of osmium nanozyme can efficiently drive the oxidation and color development of TMB, and the nanozyme acts as a catalyst and is recycled in the reaction. There is no need for additional sensitizers or high-concentration catalysts in the system, which also avoids the dosage redundancy caused by heavy metal ligand dependence in the oxidase system.

[0043] The particle size of the osmium citrate nanozyme of the present invention is 60 - 200 nm. The nanozyme prepared in the particle size range of the present invention has high catalytic efficiency, good stability and dispersibility. The principle is that a smaller particle size ensures a larger specific surface area, increases the contact area between the nanozyme and the substrate, and is conducive to catalysis. At the same time, the nanoparticles in this particle size range take into account both stability and dispersibility, avoiding the problems of easy aggregation of too small particles or sedimentation of too large particles, and ensuring the uniformity and repeatability of the detection system.

[0044] The particle size of the present invention can be controlled within such a range, which is mainly related to the following factors: (1) The ratio of potassium osmate hexachloride to trisodium citrate controls the complexation rate of osmium ions, affects the nucleation process, and thus regulates the final particle size. (2) The dropping rate and addition amount of sodium borohydride. As a strong reducing agent, the addition amount of sodium borohydride directly affects the intensity of the reduction reaction. Slowly dropping can avoid local over-reduction and ensure uniform growth of particles. (3) Control of the reaction environment. Reacting in a dark environment can prevent photosensitive side reactions, while at a relatively mild temperature, the reaction rate can be controlled to promote particle size uniformity.

[0045] Specific Embodiment 2: The mass ratio of K2OsCl6 to the volume of deionized water described in Step 1 of this embodiment is (4 - 6) mg∶(30 - 50) mL. Other steps and parameters are the same as those in Specific Embodiment 1.

[0046] Specific Embodiment 3: The mass ratio of trisodium citrate to the volume of deionized water described in Step 1 of this embodiment is (5 - 15) mg∶(30 - 50) mL. Other steps and parameters are the same as those in Specific Embodiment 1 or 2.

[0047] Specific Embodiment 4: The mass ratio of sodium borohydride to the volume of the deionized water mixture described in Step 2 of this embodiment is (10 - 20) mg∶(10 - 15) mL. Other steps and parameters are the same as those in any one of Specific Embodiments 1 to 3.

[0048] Specific Embodiment 5: The volume ratio of the sodium borohydride solution to mixture A described in Step 2 of this embodiment is (100 - 300) μL∶(30 - 50) mL. Other steps and parameters are the same as those in any one of Specific Embodiments 1 to 4.

[0049] Specific Embodiment 6: The stirring reaction time in Step 2 of this embodiment is 60 - 70 min. Other steps and parameters are the same as those in any one of Specific Embodiments 1 to 5.

[0050] Specific Embodiment 7: Application of osmic citrate nanozyme in immunosorbent assay.

[0051] An immunosorbent assay is carried out using the osmic citrate nanozyme - antigen / antibody complex prepared from osmic citrate nanozyme.

[0052] The preparation method of the osmic citrate nanozyme - antigen / antibody complex is as follows:

[0053] Step 1: Vacuum freeze-dry the osmic citrate nanozyme to constant weight, and then add deionized water to make it uniformly dispersed to obtain an osmic citrate nanozyme solution;

[0054] Step 2: Dilute the osmium citrate nanozyme solution with PBS, and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), then mix them evenly by ultrasonic treatment to obtain a mixed solution. The concentration of EDC is 20 mg / mL, and the concentration of NHS is 48 mg / mL.

[0055] Step 3: Add the antigen or antibody into the mixed solution prepared in Step 2, incubate overnight at 4°C, then centrifuge, remove the supernatant, and resuspend it in the blocking solution for blocking.

[0056] Step 4: Then continue to centrifuge, remove the supernatant, retain the precipitate, and then add phosphate buffer solution to obtain the osmium citrate nanozyme-antigen / antibody complex.

[0057] Taking the osmium citrate nanozyme-antigen complex as an example for illustration:

[0058] Due to the coupling of the antigen, the surface active sites of the nanozyme in the osmium citrate nanozyme-antigen complex are blocked, and its peroxidase activity is significantly suppressed. The peroxidase activity of the osmium nanozyme can be selectively restored in the presence of the specific antibody modified on the surface of the antigen. This is due to the high affinity between the antigen and the antibody, which causes them to displace from the surface of the osmium nanozyme and re-expose the active sites on the surface of the osmium nanozyme. In the hydrogen peroxide / TMB system, TMB is oxidized into a blue product. In the absence of the specific antibody, its peroxidase activity remains at a relatively low level, and it will not cause a significant change in absorbance in the hydrogen peroxide / TMB system, thus achieving the purpose of specific detection.

[0059] Based on this property, in the present invention, the osmium citrate nanozyme-antigen complex is directly added to the appropriately diluted specific antibody to be detected. After shaking and mixing evenly, the mixed solution of TMB and hydrogen peroxide is added. After a short incubation, a terminator is added, and then the absorbance is measured. The absorbance measured by this method increases with the increase of the concentration of the analyte, thereby achieving the purpose of quantitative detection. Compared with the traditional ELISA detection method, this method does not require plate washing and shortens the long incubation time, shortening the ELISA detection time from about 150 minutes to within 30 minutes, greatly improving the detection efficiency.

[0060] Specific Embodiment 8: In Step 1 of this embodiment, the mass ratio of the osmium citrate nanozyme to the volume of deionized water is (0.8 - 1.2) mg∶(0.8 - 1.2) mL. Other steps and parameters are the same as those in Specific Embodiment 7.

[0061] Specific Embodiment 9: In Step 2 of this embodiment, the volume ratio of the osmium citrate nanozyme solution to PBS is (10 - 20) μL∶(0.8 - 1) mL. Other steps and parameters are the same as those in Specific Embodiment 7 or 8.

[0062] Embodiment 10: The volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide to the osmium citrate nanozyme solution described in step 2 of this embodiment is (8-10) μL∶(8-10) μL∶(0.8-1) mL. Other steps and parameters are the same as those in any one of Embodiments 7 to 9.

[0063] Embodiment 11: The volume ratio of the antigen or antibody to the mixture described in step 3 of this embodiment is (15-50) μL∶(0.8-1) mL. Other steps and parameters are the same as those in any one of Embodiments 7 to 10.

[0064] Embodiment 12: The incubation time described in step 3 of this embodiment is 8-12 h. Other steps and parameters are the same as those in any one of Embodiments 7 to 11.

[0065] Embodiment 13: The blocking time described in step 3 of this embodiment is 8-12 h. Other steps and parameters are the same as those in any one of Embodiments 7 to 12.

[0066] Embodiment 14: The volume ratio of the precipitate retained after centrifugation in step 4 of this embodiment to the phosphate buffer solution is (15-20) μL∶(0.8-1) mL. Other steps and parameters are the same as those in any one of Embodiments 7 to 13.

[0067] Embodiment 15: The method for immunoassay using the osmium citrate nanozyme-antigen / antibody complex includes the following steps:

[0068] Add the antibody or antigen to be tested diluted with PBS into a 96-well plate, add the osmium citrate nanozyme-antigen / antibody complex into the 96-well plate, and shake at room temperature;

[0069] Add the TMB and hydrogen peroxide mixture into the 96-well plate, then incubate in an environment at 37 °C, add the termination solution, and measure the immunoassay result.

[0070] Compared with the traditional ELISA detection method, this method does not require plate washing and shortens the long incubation time, reducing the detection time from about 150 min (ELISA detection) to within 30 min, greatly improving the detection efficiency.

[0071] Embodiment 16: The mass ratio of the antibody or antigen to be tested to the volume of PBS is (1-100) pg∶(0.8-1.2) mL. Other steps and parameters are the same as those in Embodiment 15.

[0072] Specific Embodiment XVII: The added volume of the antibody or antigen to be measured after dilution in this embodiment is 50 - 120 μL / well. Other steps and parameters are the same as those in Specific Embodiment XV or XVI.

[0073] Specific Embodiment XVIII: The added volume of the citric acid osmium nanozyme - antigen / antibody complex in this embodiment is 20 - 30 μL / well. Other steps and parameters are the same as those in any one of Specific Embodiments XV to XVII.

[0074] Specific Embodiment XIX: The oscillation time at room temperature in this embodiment is 2 - 5 min. Other steps and parameters are the same as those in any one of Specific Embodiments XV to XVIII.

[0075] Specific Embodiment XX: The incubation time in this embodiment is 10 - 15 min. Other steps and parameters are the same as those in any one of Specific Embodiments XV to XIX.

[0076] The following is a detailed description of the embodiments of the present invention. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0077] Example 1:

[0078] The preparation method of the citric acid osmium nanozyme in this example includes the following steps:

[0079] Step 1: Mix 5 mg of K2OsCl6, 10 mg of trisodium citrate, and 40 mL of deionized water, and stir at a speed of 600 r / min at 25 °C for 30 min to obtain a mixed solution A;

[0080] Step 2: Mix 20 mg of sodium borohydride with 15 mL of deionized water to obtain a sodium borohydride solution; drop 300 μL of the sodium borohydride solution into the mixed solution A prepared in Step 1 at a dropping speed of 300 μL / min to obtain a mixed solution B; stir and react the mixed solution B in a dark environment at 20 °C for 60 min to obtain a citric acid osmium nanozyme with controllable particle size; the average particle size of the citric acid osmium nanozyme is 150 ± 20 nm.

[0081] The following detections are carried out on the citric acid osmium nanozyme prepared in this example:

[0082] (I) Morphological observation of the citric acid osmium nanozyme

[0083] The morphology of the citric acid osmium nanozyme prepared in this example is observed under a scanning electron microscope at a magnification of 40000 times, as Figure 2 shown, the particle size is 150 ± 20 nm, and the particle size distribution is relatively uniform.

[0084] (II) Characterization of the citric acid osmium nanozyme

[0085] The osmium citrate nanozyme prepared in this example was characterized by Fourier transform infrared spectroscopy and energy-dispersive X-ray spectroscopy.

[0086] The Fourier transform infrared spectroscopy analysis results of osmium citrate nanozyme and potassium hexachloroiridate are as Figure 3 shown. From Figure 3 it can be analyzed that the peak at 604.86 cm -1 is the Os-O stretching vibration peak, the peak at 1068.87 cm -1 belongs to the C-O stretching vibration peak of carboxylate, and the peaks at 1263.22 cm -1 , 1399.8 cm -1 and 1590.66 cm -1 are the COO- stretching vibration peaks of carboxylate, and the broad peak at 3257.88 cm -1 is the stretching vibration peak of hydroxyl group. According to these characteristic peaks, FT-IR analysis confirmed that the synthesized product was osmium citrate, indicating that citric acid had successfully coordinated with osmium to form an osmium-citric acid complex.

[0087] The energy-dispersive X-ray spectroscopy analysis results are as Figure 4 shown. From Figure 4 analysis, it can be obtained that a large number of Os elements are distributed in the material, while the K and Cl elements may come from the incompletely reacted potassium hexachloroiridate, and the Na and C elements may come from sodium citrate.

[0088] (III) Temperature stability experiment of osmium citrate nanozyme and horseradish peroxidase

[0089] The osmium citrate nanozyme of this example was vacuum freeze-dried to constant weight, and then deionized water was added to prepare an osmium citrate nanozyme solution with a concentration of 5 μg / mL. The osmium citrate nanozyme solution was respectively filled into 7 tubes, 2 mL per tube; the horseradish peroxidase solution (5 μg / mL) was respectively filled into 7 tubes, 2 mL per tube. Seven environmental temperatures of 0 °C, 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, and 60 °C were set respectively. One tube of osmium citrate nanozyme solution and one tube of horseradish peroxidase solution were placed at each environmental temperature and incubated for 1 h. Subsequently, a mixed solution composed of 100 μL of TMB (1.4 mg / mL), 200 μL of H2O2 (34 μg / mL), and 1.7 mL of HAc-NaAc buffer (0.2 mol / L, pH 6) was quickly added in sequence and mixed evenly. The ultraviolet spectrophotometer was used to record the absorbance change at 652 nm. Among them, TMB was dissolved in DMSO. Among them, TMB was dissolved in DMSO.

[0090] The temperature stability determination results of osmium citrate nanozyme and horseradish peroxidase are asFigure 5 as shown. Among them represents osmium citrate nanozyme, represents horseradish peroxidase. It can be seen from Figure 5 that osmium citrate nanozyme has the best enzyme activity at 20 °C, about 950 U / mg, and has a wide pH adaptation range, and its enzyme activity far exceeds that of horseradish peroxidase.

[0091] (4) pH stability experiment of osmium citrate nanozyme and horseradish peroxidase

[0092] Take 2 mL of osmium citrate nanozyme solution (5 μg / mL) or 2 mL of horseradish peroxidase solution (5 μg / mL), and then quickly add a mixture composed of 100 μL of TMB (1.4 mg / mL), 200 μL of H2O2 (34 μg / mL) and 1.7 mL of buffer solution (pH 2, pH 4, pH 6, pH 8, pH 10) in sequence. Keep it in a constant temperature water bath at 37 °C for 1 min, and record the change in absorbance at 652 nm. Set the temperature at 37 °C.

[0093] The pH stability determination results of osmium citrate nanozyme and horseradish peroxidase are as Figure 6 shown. Among them represents osmium citrate nanozyme, represents horseradish peroxidase. It can be seen from Figure 6 that osmium citrate nanozyme has the best enzyme activity at pH = 2, about 950 U / mg, and has a wide temperature adaptation range, and its enzyme activity far exceeds that of horseradish peroxidase.

[0094] Example 2:

[0095] The preparation method of the osmium citrate nanozyme-antigen complex in this example includes the following steps:

[0096] Step 1: Vacuum freeze-dry the osmium citrate nanozyme prepared in Example 1 to constant weight, and then add deionized water to disperse it evenly to obtain an osmium citrate nanozyme solution. The mass ratio of osmium citrate nanozyme to the volume of deionized water is 1 mg∶1 mL;

[0097] Step 2: Take 20 μL of the osmium citrate nanozyme solution prepared in Step 1 and dilute it with 1 mL of PBS, and then add 10 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 10 μL of N-hydroxysuccinimide (NHS), and ultrasonically mix evenly for 30 min for carboxyl activation to obtain a mixture; the concentration of EDC is 20 mg / mL, and the concentration of NHS is 48 mg / mL;

[0098] Step 3: Add the OVA antigen to the mixture prepared in Step 2. The volume ratio of the OVA antigen to the mixed solution is 20 μL∶1 mL. Incubate it in a refrigerator at 4 °C for 12 h, then centrifuge at a speed of 8000 r / min for 15 min. After removing the supernatant, resuspend it in a 5% BSA solution and block for 12 h. The concentration of the OVA antigen is 14 mg / mL, and the OVA antigen is purchased.

[0099] Step 4: Then continue to centrifuge, remove the supernatant, retain the precipitate and then add phosphate buffer to obtain a citrate osmium nanozyme-antigen complex. The volume ratio of the precipitate retained after centrifugation to the phosphate buffer is 20 μL∶1 mL.

[0100] Example 3:

[0101] Perform immunosorbent assay using the citrate osmium nanozyme-antigen complex prepared in Example 2. The specific method is as follows:

[0102] Dilute 50 pg of the antibody to be detected with 1 mL of PBS. Add the PBS-diluted antibody to be detected into a 96-well plate, 100 μL per well. Add the citrate osmium nanozyme-antigen complex to the 96-well plate, 20 μL per well, and oscillate at room temperature for 3 minutes. The antibody to be detected is a mouse IgG antibody induced by OVA.

[0103] Add the TMB and hydrogen peroxide mixture to the 96-well plate, then incubate in an environment at 37 °C for 10 minutes, add the stop solution, and measure the OD with an enzyme-linked immunosorbent assay reader. 450 The TMB and hydrogen peroxide mixture is composed of 20 μL of TMB (1.4 mg / mL) and 80 μL of hydrogen peroxide (34 μg / mL).

[0104] The immunoassay detection steps of this example are divided into four procedures, and the time used is statistically as follows:

[0105] 1. Add the sample to be detected (3 minutes); 2. Add the nanozyme-labeled antigen (3 minutes); 3. Add the chromogenic solution and wait (15 minutes); 4. Add the stop solution (3 minutes); a total of 24 minutes.

[0106] The operation process of the existing ELISA detection method takes the following time:

[0107] 1. Add the sample to be detected (3 minutes); 2. Incubate (40 minutes); 3. Wash the plate (10 minutes); 4. Add the primary antibody (3 minutes); 5. Incubate (30 minutes); 6. Wash the plate (10 minutes); 7. Add the enzyme-labeled secondary antibody (3 minutes); 8. Incubate (20 minutes); 9. Wash the plate (10 minutes); 10. Add the chromogenic solution and wait (15 minutes); 11. Add the stop solution (3 minutes); a total of 147 minutes.

[0108] By comparison, it can be seen that the method of the present invention greatly shortens the time required for immunoassay.

[0109] In order to verify the sensitivity, specificity, and stability of the osmium citrate nanozyme of the present invention in immunosorbent assay, the following experiments were carried out:

[0110] (I) Experiment on the sensitivity of osmium citrate nanozyme for murine IgG immunoassay

[0111] Prepare murine IgG antibodies induced by OVA with concentrations of 40, 20, 10, 5, 2.5, and 0.625 pg / mL, and add 50 μL of the above-mentioned different concentrations of antibodies into each well of a 96-well plate. Subsequently, directly add the osmium citrate nanozyme-antigen complex prepared in Example 2 into the wells. After shaking and mixing evenly, add a pre-prepared mixture composed of 20 μL of TMB (1.4 mg / mL) and 80 μL of hydrogen peroxide (34 μg / mL), mix evenly, and react in the dark at 37 °C for 10 min. Subsequently, add 100 μL of the termination solution to each well, mix evenly, and measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader within 30 min.

[0112] Figure 7 are the results of the sensitivity of murine IgG immunoassay. From Figure 7 it can be seen that the functionalized osmium citrate nanozyme has excellent sensitivity when applied to immunoassay and shows a good linear relationship in the range of 0.625 pg / mL to 40 pg / mL.

[0113] (II) Experiment on the specificity of osmium citrate nanozyme for murine IgG immunoassay

[0114] Sequentially add 100 pg / mL of murine IgG, goat anti-rabbit IgG, goat anti-bovine IgG, goat anti-pig IgG antibodies induced by OVA, and BSA protein, and add 50 μL of the diluted antibody sample to each detection well. Subsequently, add the osmium citrate nanozyme-antigen complex prepared in Example 2, shake and mix evenly, add a pre-prepared mixture composed of 20 μL of TMB (1.4 mg / mL) and 80 μL of hydrogen peroxide (34 μg / mL), mix evenly, and react in the dark at 37 °C for 10 min. Subsequently, add 100 μL of the termination solution to each well, mix evenly, and measure the absorbance at 450 nm with an ELISA reader within 30 min.

[0115] Figure 8 are the results of the specificity experiment of murine IgG immunoassay. From Figure 8 it can be seen that the functionalized osmium citrate nanozyme has good specificity when applied to immunoassay and does not show non-specific binding with other antibodies or proteins.

[0116] (III) Experiment on the stability of osmium citrate nanozyme for murine IgG immunoassay

[0117] Mouse IgG antibodies induced by OVA with a configured concentration of 100 pg / mL were added to each well of a 96-well plate at 50 μL per well. Subsequently, the osmium citrate nanozyme-antigen complexes prepared in Example 2 with a synthesis time of 7 days, 15 days, and 30 days were directly added to the wells. After shaking and mixing evenly, 100 μL of a pre-prepared mixture of TMB and hydrogen peroxide was added, and after mixing evenly, the reaction was carried out in the dark at 37 °C for 10 minutes. Subsequently, 100 μL of the termination solution was added to each well, mixed evenly, and the absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay reader within 30 minutes.

[0118] Figure 9 For the stability experiment results of mouse IgG immunoassay. From Figure 9 It can be seen that the functionalized osmium citrate nanozyme can still exhibit good detection effects after being synthesized for a period of time, and the functionalized osmium citrate nanozymes with synthesis times of 7 days, 15 days, and 30 days have an OD 450 of about 3 when applied in detection, indicating that it has good stability when applied in immunoassay.

[0119] Example 4:

[0120] The preparation method of the osmium citrate nanozyme-antibody complex in this example includes the following steps:

[0121] Step 1: Vacuum freeze-dry the osmium citrate nanozyme prepared in Example 1 until it reaches a constant weight, and then add deionized water to disperse it evenly to obtain an osmium citrate nanozyme solution. The mass ratio of the osmium citrate nanozyme to the volume of deionized water is 1 mg∶1 mL;

[0122] Step 2: Take 20 μL of the osmium citrate nanozyme solution prepared in Step 1 and dilute it with 1 mL of PBS, then add 10 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 10 μL of N-hydroxysuccinimide (NHS), and ultrasonically mix evenly for 30 min for carboxyl activation to obtain a mixed solution; the concentration of EDC is 20 mg / mL, and the concentration of NHS is 48 mg / mL;

[0123] Step 3: Add avian leukosis (ALV) monoclonal antibody to the mixed solution prepared in Step 2. The volume ratio of the avian leukosis monoclonal antibody to the mixed solution is 50 μL∶1 mL, incubate it in a refrigerator at 4 °C for 12 h, then centrifuge it at a speed of 8000 r / min for 15 min, remove the supernatant, and resuspend it in a 5% BSA solution for blocking for 12 h; the concentration of the avian leukosis monoclonal antibody is 0.1 mg / mL, and the avian leukosis monoclonal antibody is purchased;

[0124] Step 4: Then continue centrifugation, remove the supernatant, retain the precipitate, and then add phosphate buffer to obtain the citrate osmium nanozyme-antibody complex; the volume ratio of the precipitate retained after centrifugation to the phosphate buffer is 20 μL∶1 mL.

[0125] Example 5:

[0126] Use the above-prepared citrate osmium nanozyme-antibody complex for immunosorbent assay, and the specific method is as follows:

[0127] Dilute 50 pg of the antigen to be tested with 1 mL of PBS, add the antigen to be tested diluted with PBS into a 96-well plate, 100 μL per well, add the citrate osmium nanozyme-antibody complex to the 96-well plate, 20 μL per well, and oscillate for 3 minutes at room temperature; the antigen to be tested is the ALV antigen;

[0128] Add the TMB and hydrogen peroxide mixture to the 96-well plate, then incubate in an environment at 37 °C for 10 minutes, add the termination solution, and measure the OD with an enzyme-linked immunosorbent assay (ELISA) reader 450 . The TMB and hydrogen peroxide mixture is composed of 20 μL of TMB (1.4 mg / mL) and 80 μL of hydrogen peroxide (34 μg / mL).

[0129] In order to verify the sensitivity, specificity, and stability of the citrate osmium nanozyme of the present invention in immunosorbent assay, the following experiments were carried out:

[0130] (I) Citrate osmium nanozyme for ALV antigen immunodetection sensitivity experiment

[0131] Prepare ALV antigens with concentrations of 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95, and 0.98 pg / mL, and add 50 μL of the above different concentrations of antigens into a 96-well plate respectively. Subsequently, directly add the citrate osmium nanozyme-antibody complex prepared in Example 4 into the wells. After shaking and mixing evenly, add the pre-prepared 20 μL of TMB (1.4 mg / mL) and 80 μL of hydrogen peroxide (34 μg / mL) mixture, mix evenly, and react in the dark at 37 °C for 10 min. Subsequently, add 100 μL of the termination solution to each well, mix evenly, and measure the OD with an enzyme-linked immunosorbent assay (ELISA) reader within 30 min 450 .

[0132] Figure 10 This is the result of the immunodetection sensitivity of the functionalized citrate osmium nanozyme prepared in the present invention for ALV antigen. As Figure 10 can be seen, the functionalized citrate osmium nanozyme has excellent sensitivity when applied to immunoassay, and shows a good linear relationship in the range of 0.98 pg / mL to 500 pg / mL.

[0133] (2) Specific experiment on the immunodetection of ALV antigen using osmium citrate nanozyme

[0134] Add 50 μL of diluted samples of 400 pg / mL ALV, H5 AIV, H7 AIV, H9 AIV, and AILTV antigens to each detection well in sequence. Then add 100 μL of the osmium citrate nanozyme-antibody complex prepared in Example 4, mix well, react in the dark at 37 °C for 10 min, add the chromogenic solution / hydrogen peroxide, and incubate for 15 min. Subsequently, add 100 μL of the termination solution to each well, mix well, and measure the OD with an enzyme-linked immunosorbent assay (ELISA) reader within 30 min 450 。

[0135] Figure 11 This is the result of the specific experiment on the immunodetection of ALV antigen using the functionalized osmium citrate nanozyme prepared in the present invention. As can be seen from Figure 11 it, the functionalized osmium citrate nanozyme has good specificity when applied to immunoassay and does not non-specifically bind to other antigens

[0136] (3) Stability experiment on the immunodetection of ALV antigen using osmium citrate nanozyme

[0137] Prepare an ALV antigen with a concentration of 400 pg / mL and add 50 μL to each well in a 96-well plate. Then directly add the osmium citrate nanozyme-antibody complex prepared in Example 4 with synthesis times of 7 days, 15 days, and 30 days to the wells. After shaking and mixing evenly, add 100 μL of the pre-prepared mixture of TMB and hydrogen peroxide, mix well, and react in the dark at 37 °C for 10 min. Subsequently, add 100 μL of the termination solution to each well, mix well, and measure the OD with an enzyme-linked immunosorbent assay (ELISA) reader within 30 min 450 。

[0138] Figure 12 This is the result of the stability experiment on the immunodetection of mouse IgG using the functionalized osmium citrate nanozyme prepared in the present invention. As can be seen from Figure 12 it, the functionalized osmium citrate nanozyme can still show good detection effects after being synthesized for a period of time, and the OD values of the functionalized osmium citrate nanozymes with synthesis times of 7 days, 15 days, and 30 days 450 all remain at a relatively high level, indicating that it has good stability when applied to immunoassay

Claims

1. Preparation method of osmium citrate nanozyme, characterized in that The method comprises the following steps: Step 1: Mix K2OsCl6 and trisodium citrate with deionized water, and stir evenly at 10°C to 30°C to obtain a mixed solution A; Step 2: Mix sodium borohydride with deionized water to obtain a sodium borohydride solution, and drip the sodium borohydride solution into the mixed solution A prepared in Step 1 at a dripping rate of 100 - 300 μL / min to obtain a mixed solution B; Stir and react the mixed solution B under dark conditions at 10°C to 30°C to obtain osmium citrate nanozyme; wherein the particle size of the osmium citrate nanozyme is 60 - 200 nm.

2. The preparation method of the osmium citrate nanozyme according to claim 1, characterized in that, The mass ratio of K2OsCl6 described in Step 1 to the volume of deionized water is (4 - 6) mg : (30 - 50) mL.

3. The preparation method of the osmium citrate nanozyme according to claim 1 or 2, characterized in that, The mass ratio of trisodium citrate described in Step 1 to the volume of deionized water is (5 - 15) mg : (30 - 50) mL.

4. The preparation method of the osmium citrate nanozyme according to claim 3, characterized in that, The mass ratio of sodium borohydride described in Step 2 to the volume of the deionized water mixed solution is (10 - 20) mg : (10 - 15) mL.

5. The preparation method of the osmium citrate nanozyme according to claim 4, wherein, The volume ratio of the sodium borohydride solution to the mixed solution A described in Step 2 is (100 - 300) μL : (30 - 50) mL.

6. The preparation method of the osmium citrate nanozyme according to claim 5, characterized in that, The stirring reaction time in Step 2 is 60 - 70 min.

7. Application of the osmium citrate nanozyme prepared by the method according to claim 1 in immunoassay.

8. The application according to claim 7, characterized in that, Perform immunoassay using the osmium citrate nanozyme-antigen / antibody complex prepared with the osmium citrate nanozyme.

9. The application according to claim 8, characterized in that, The preparation method of the osmium citrate nanozyme-antigen / antibody complex is as follows: Step 1: Vacuum freeze-dry the osmium citrate nanozyme to constant weight, and then add deionized water to disperse it evenly to obtain an osmium citrate nanozyme solution; Step 2: Dilute the osmium citrate nanozyme solution with PBS, and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and mix evenly by ultrasonic to obtain a mixed solution; wherein the concentration of EDC is 20 mg / mL and the concentration of NHS is 48 mg / mL; Step 3: Add an antigen or an antibody to the mixed solution prepared in Step 2, incubate overnight at 4°C, then centrifuge, remove the supernatant and resuspend it in a blocking solution for blocking; Step 4: Then continue to centrifuge, remove the supernatant, retain the precipitate and then add phosphate buffer to obtain an osmium citrate nanozyme-antigen / antibody complex.

10. The application according to claim 8 or 9, characterized in that, The method for performing immunoassay using the osmium citrate nanozyme-antigen / antibody complex comprises the following steps: Add the antibody or antigen to be tested diluted with PBS into a 96-well plate, add the osmium citrate nanozyme-antigen / antibody complex into the 96-well plate, and oscillate at room temperature; Add a mixed solution of TMB and hydrogen peroxide into the 96-well plate, then incubate in an environment at 37°C, add a termination solution, and measure the immunoassay result.

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