Polymerized nano-enzyme material coupled antibody and application thereof in immunohistochemical chromogenic detection

Abstract

The invention discloses a polymeric nano-enzyme material coupled antibody and application thereof in immunohistochemical chromogenic detection, the polymeric nano-enzyme material coupled antibody comprises a PAMAM dendrimer and an antibody covalently coupled with the PAMAM dendrimer, and an external branch of the PAMAM dendrimer is modified and connected with a Ru-POD nano-enzyme. The polymeric nano-enzyme material coupled antibody has the advantages of high stability, simple preparation, long service life, adjustable activity and the like, compared with a traditional horse radish peroxidase-based polymer enzyme secondary antibody detection system, the catalytic performance and storage stability are improved, the catalytic activity on DAB and H2O2 is equivalent to that of natural horse radish peroxidase, and the polymeric nano-enzyme material coupled antibody has the advantages of high stability, long service life, adjustable activity and the like. Wide application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and specifically relates to a polymeric nanoenzyme material coupled with an antibody and an application thereof in immunohistochemical color development detection. Background Art

[0002] Immunohistochemistry (IHC) is an antibody-based technique used to detect the presence of specific biomolecules and to analyze the expression of proteins in structures and tissues. This technique usually involves two different types of antibodies: primary antibodies and secondary antibodies. Primary antibodies bind to target molecules with their extremely high affinity and specificity, ensuring the accuracy and precision of detection. Therefore, the selection of appropriate primary antibodies is crucial for the successful detection and accurate quantification of target molecules in immunoassays. On the other hand, secondary antibodies recognize and bind to primary antibodies bound to target molecules rather than directly binding to target molecules. Secondary antibodies are often conjugated to enzymes (such as horseradish peroxidase, HRP), which can convert undetectable substrates into detectable products, thereby significantly amplifying the detection signal of low-abundance target molecules.

[0003] Since the invention of immunohistochemistry in the 1940s, its development has been largely reflected in the progress of secondary antibody detection systems, with the main trend being the continuous improvement of detection sensitivity and the continuous reduction of detection interference factors. For example, the development from biotin detection systems (such as ABC method and SP method) to enhanced labeled polymer systems (Enhanced Labelled Polymer System) has eliminated the interference of endogenous biotin and improved the detection sensitivity. However, early non-biotin secondary antibody detection systems mainly used secondary antibody-enzyme polymers connected by polydextran or polyamino acid backbones, or enzyme polymers coupled to larger dendritic backbones and then coupled to secondary antibodies to amplify specific signals. These methods are easy to operate, avoid the interference of endogenous biotin, and maintain a high detection sensitivity, which has promoted the widespread application of immunohistochemistry in clinical pathology auxiliary diagnosis. However, the disadvantages of these methods are the significant increase in molecular weight, the excessive space occupied, and the efficiency of polymers entering tissue cells to bind to specific targets due to steric hindrance and other reasons, resulting in a decrease in specific staining intensity and the easy generation of non-specific background. Therefore, it is necessary to develop a more efficient and simple new secondary antibody system so that the polymerized secondary antibodies can enter the interior of tissue cells and bind to specific targets, so as to improve the catalytic staining efficiency and amplify the signal while ensuring no nonspecific background staining.

[0004] In recent years, natural enzymes have been severely restricted in practical applications due to their high cost, poor stability, low yield, difficult preservation and short half-life. Nanomaterials refer to materials with at least one dimension in nanometer size (1–100nm) in three-dimensional space, or materials composed of them as basic units. Nanozymes are a class of nanomaterials with enzyme-like properties and catalytic activity. Since Fe3O4 nanoparticles were reported to have the activity of simulating natural peroxidase in 2007, hundreds of nanomaterials have been found to have enzyme activities such as peroxidase (POD), oxidase (OXD), catalase (CAT), glucose oxidase (GOx), glutathione peroxidase (GPx), superoxide dismutase (SOD), and uricase. In addition, many nanomaterials have been reported to exhibit dual or multi-enzyme activities at the same time. As a new generation of artificial enzymes, nanozymes have the advantages of low cost, high stability, simple preparation, long service life, and adjustable activity compared with natural enzymes.

[0005] In order to make the above-mentioned nanomaterials better replace natural horseradish peroxidase and gain wide application, researchers often regulate the enzyme-like activity of nanomaterials according to their characteristics to further enhance the catalase-like activity of nanomaterials. Common strategies for improving the performance of nanomaterial-like enzymes include size regulation, morphology regulation, component regulation, and surface interface regulation. It is worth noting that the surface interface regulation of nanomaterials can further exert the synergistic effect between materials by combining different nanomaterials, which has important scientific significance in the construction of efficient enzyme-like catalytic materials. Summary of the invention

[0006] The purpose of the present invention is to provide a polymeric nanoenzyme material coupled with an antibody.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned polymer nanoenzyme material coupled with an antibody.

[0008] Another object of the present invention is to provide an application of the above-mentioned polymer nanoenzyme material coupled with an antibody.

[0009] The technical solution of the present invention is as follows:

[0010] A polymer nanoenzyme material coupled with an antibody comprises a PAMAM dendritic polymer and an antibody covalently coupled thereto, wherein the external branches of the PAMAM dendritic polymer are modified and connected with a Ru-POD nanoenzyme.

[0011] In a preferred embodiment of the present invention, the antibody is IgG.

[0012] The method for preparing the polymeric nanoenzyme material coupled with an antibody is characterized in that it comprises the following steps:

[0013] (1) preparing an active Ru-POD nanozyme, washing the Ru-POD nanozyme, activating the Ru-POD nanozyme with EDC and NHS, and then purifying the Ru-POD nanozyme to obtain a purified activated Ru-POD nanozyme;

[0014] (2) mixing the purified activated Ru-POD nanozyme obtained in step (1) with a PAMAM 5.0 solution for reaction, and then purifying to obtain a PAMAM@Ru-POD nanozyme;

[0015] (3) Activating the carboxyl group of the antibody by EDC and NHS, and then reacting the mixture with the PAMAM@Ru-POD nanozyme obtained in step (2), and then purifying the mixture to obtain the polymer nanozyme material coupled with the antibody.

[0016] The above-mentioned polymeric nanoenzyme material coupled with antibody is used as a secondary antibody in immunohistochemical detection.

[0017] An immunohistochemical detection method uses the above-mentioned polymer nanoenzyme material coupled with an antibody as a secondary antibody.

[0018] The beneficial effects of the present invention are:

[0019] 1. The polymeric nanoenzyme material coupled antibody of the present invention has the advantages of high stability, simple preparation, long service life, adjustable activity, etc. Compared with the traditional secondary antibody detection system based on horseradish peroxidase or direct connection of nanomaterials, it improves the catalytic performance, detection sensitivity and storage stability;

[0020] 2. The polymeric nanoenzyme material coupled to the antibody of the present invention has a lower cost of preparation and purification, providing a basis for its further development in the secondary antibody detection system;

[0021] 3. The polymeric nanoenzyme material coupled with the antibody of the present invention has extremely high practical application value. As a nanomaterial with peroxidase-like activity, its catalytic activity for DAB and H2O2 is equivalent to that of natural horseradish peroxidase, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure is a schematic diagram of the preparation method and application of the polymer nanoenzyme material coupled with antibodies of the present invention.

[0023] Figure 2 This is a transmission electron microscopy photograph of the Ru-POD nanozyme obtained in Example 1 of the present invention.

[0024] Figure 3 This is a transmission electron microscope photograph of the PAMAM@Ru-POD nanozyme obtained in Example 1 of the present invention.

[0025] Figure 4The results of immunohistochemistry for detecting P53 protein in lung adenocarcinoma tissue based on PAMAM@Ru-POD nanozyme material coupled with secondary antibody in Example 3 of the present invention are shown.

[0026] Figure 5 The results of immunohistochemical detection of P53 protein in lung adenocarcinoma tissue using the Ru-POD nanozyme material coupled with a secondary antibody prepared in Example 3 of the present invention are shown.

[0027] Figure 6 The results of immunohistochemical detection of P53 protein in lung adenocarcinoma tissue using the PAMAM@CeO2 nanozyme material prepared in Example 3 of the present invention based on Comparative Example 2 and coupled with a sheep anti-rabbit secondary antibody are shown.

[0028] Figure 7 The results of immunohistochemistry based on HRP goat anti-rabbit secondary antibody in Example 3 of the present invention to detect P53 protein in lung adenocarcinoma tissue are shown.

[0029] Figure 8 The results of immunohistochemical detection of P53 protein in lung adenocarcinoma tissue using the BSA-coupled secondary antibody prepared in Comparative Example 1 in Example 3 of the present invention are shown. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0031] The one-pot preparation in the following example includes: dissolving RuCl3·nH2O (0.53 g) and NaAc (1.5 g) in ethylene glycol (15 mL), and then dissolving them in PSS (905 μL) to obtain Ru-POD nanozyme. (Refer to Surface ligand engineering ruthenium nanozyme superior to horseradish peroxidase for enhanced immunoassay, H Fan, J Zheng, J Xie, J Liu, X Gao, X Yan, K Fan, L Gao Advanced Materials, 2024)

[0032] Example 1

[0033] (1) Carboxyl activation of Ru-POD nanozyme:

[0034] A. Dilute the Ru-POD nanozyme prepared by the one-pot method with ddH2O to a concentration of 0.2 mg / mL, take 1 mL and oscillate to mix, centrifuge at 13000 rpm for 5 min, and carefully discard the supernatant and retain the precipitate;

[0035] B. Add 200 μL of MES buffer solution (containing 0.01% Tween20) with a pH of 5.0-6.0 and a concentration of 50 mM to the above precipitate, and wash it with 10% power ultrasound for 1 min (ultrasound for 2 seconds, stop for 2 seconds), mix it evenly, centrifuge it and discard the supernatant, repeat it twice, and obtain the washed Ru-POD nanozyme, whose transmission electron microscope photo is shown in the figure. Figure 2 As shown, it is in the form of uniformly dispersed spheres with a particle size of 10-15 nm;

[0036] C. Add 100 μL of 25 mM EDC solution and 100 μL of 25 mM NHS solution to the cleaned Ru-POD nanozyme, vortex mix, and react at room temperature for 30 min on a mixer to activate the carboxyl group;

[0037] D. Centrifuge the material obtained in step C at 13000 rpm for 5 min, carefully discard the supernatant, then add 600 μL 50 mM pH 5.0 MES buffer (containing 0.01% Tween 20) to the obtained precipitate, wash with 10% power ultrasound for 1 min (ultrasound for 2 s, stop for 2 s), then centrifuge at 13000 rpm for 5 min, carefully discard the supernatant and retain the precipitate to obtain the activated Ru-POD nanozyme;

[0038] (2) Preparation of PAMAM@Ru-POD nanozyme: The activated Ru-POD nanozyme obtained in step (1) was added to the PAMAM5.0 solution (purchased from Macklin, catalog number: 3901821), vortexed and mixed, and shaken overnight (>12 h) at 4°C on a mixer. Then, it was incubated at room temperature for 30 min, and then centrifuged at 1000 rpm for 10 min using a 10KD ultrafiltration tube to collect the PAMAM@Ru-POD nanozyme. The transmission electron microscope photo is shown in FIG. Figure 3 As shown in Figure 2, it can be seen that compared with the above-mentioned Ru-POD nanozyme, its overall size is significantly larger, indicating the successful preparation of PAMAM@Ru-POD nanozyme and the successful loading of Ru-POD nanozyme on the dendrimer PAMAM. The preparation principle is as follows: Figure 1 shown.

[0039] Example 2

[0040] (1) Activation of carboxyl groups of universal goat anti-rabbit secondary antibody (IgG): Add 100 μL of 25 mM EDC solution and 100 μL of 25 mM NHS solution to 0.5 mg of antibody, vortex to mix, and react on a mixer at room temperature for 30 min to activate the carboxyl groups on the antibody. Then use a 30KD ultrafiltration tube to centrifuge at 13,000 rpm for 5 min, discard the supernatant and collect the activated antibody.

[0041] (2) Preparation of PAMAM@Ru-POD nanozyme material coupled with secondary antibody (IgG): Add the activated antibody obtained in step (1) to the PAMAM@Ru-POD nanozyme prepared in Example 1, vortex mix, shake on a mixer at 4°C overnight (>12h), then incubate at room temperature for 30min, and then use a 10KD ultrafiltration tube to centrifuge at 1000rpm for 10min to collect the PAMAM@Ru-POD nanozyme material coupled with secondary antibody (IgG).

[0042] The PAMAM@Ru-POD nanozyme material prepared in this example coupled with a secondary antibody has good sensitivity for immunohistochemical staining of pathological specimens.

[0043] Comparative Example 1

[0044] (1) Activation of the carboxyl group of Ru-POD nanozyme: same as step (1) of Example 1;

[0045] (2) Activation of the carboxyl group of the antibody (IgG): same as step (1) of Example 2;

[0046] (3) Preparation of Ru-POD nanozyme material coupled with secondary antibody (IgG): Add the activated antibody obtained in step (2) to the activated Ru-POD nanozyme obtained in step (1), vortex to mix, shake on a mixer at 4°C overnight (>12h), then incubate at room temperature for 30min, and then use a 10KD ultrafiltration tube to centrifuge at 1000rpm for 10min to collect the Ru-POD nanozyme material coupled with secondary antibody (IgG).

[0047] (4) Preparation of BSA-coupled secondary antibody (IgG): Replace the activated Ru-POD nanozyme in step (3) with BSA, and the rest is the same as step (3).

[0048] Comparative Example 2

[0049] (1) Nano-cerium oxide (CeO2) carboxyl activation:

[0050] A. Dilute the CeO2 nanozyme prepared by the one-pot method with ddH2O, take 500μ0 and oscillate to mix, centrifuge at 13000rpm for 5min, carefully discard the supernatant and retain the precipitate. Then,

[0051] B. Add 200 μL of PBST buffer solution (0.01% Tween 20) to the above precipitate, and use 10% power ultrasound for 1 min (ultrasound for 2 seconds, stop for 2 seconds). After mixing evenly, centrifuge and discard the supernatant. Repeat twice to obtain the washed CeO2 nanozyme for washing.

[0052] C. Add 100 μl of 25 mM EDC solution and 100 μl of 25 mM NHS solution to the cleaned CeO2 nanozyme, vortex mix, and react at room temperature for 30 min on a mixer to activate the carboxyl group.

[0053] D. Centrifuge the material obtained in step C at 13000 rpm for 5 min, carefully discard the supernatant, then add 600 μl of PBST buffer (0.01% Tween 20) to the obtained precipitate, wash with 10% power ultrasound for 1 min (ultrasound for 2 s, stop for 2 s), then centrifuge at 13000 rpm for 5 min, carefully discard the supernatant and retain the precipitate to obtain activated CeO2 nanozyme;

[0054] (2) Preparation of PAMAM@CeO2 nanozyme: The activated CeO2 nanozyme obtained in step (1) was added to PAMAM 5.0 solution (purchased from Macklin, catalog number: 3901821), vortexed to mix, and shaken on a mixer at 4°C overnight (>12 h), followed by incubation at room temperature for 30 min, and then centrifuged at 1000 rpm for 10 min using a 10KD ultrafiltration tube to collect the PAMAM@CeO2 nanozyme.

[0055] (3) Activation of the carboxyl group of the antibody (IgG): Add freshly prepared 100 μl 25 mM EDC solution and 100 μl 25 mM NHS solution to 0.5 mg of the antibody, vortex to mix, and react on a mixer at room temperature for 30 min to activate the carboxyl group on the antibody. Then, use a 30KD ultrafiltration tube to centrifuge at 13,000 rpm for 5 min, and discard the supernatant to collect the activated antibody.

[0056] (4) Preparation of PAMAM@CeO2 nanozyme material coupled with secondary antibody (IgG): Add the activated antibody obtained in step (3) to the PAMAM@CeO2 nanozyme prepared in step (2), vortex to mix, shake on a mixer at 4°C overnight (>12h), then incubate at room temperature for 30min, and then use a 10KD ultrafiltration tube to centrifuge at 1000rpm for 10min to collect the PAMAM@CeO2 nanozyme material coupled with secondary antibody (IgG).

[0057] Example 3

[0058] The immunohistochemical detection of p53 protein in lung adenocarcinoma tissue was performed using the PAMAM@Ru-POD nanozyme material coupled with a secondary antibody prepared in Example 2, the Ru-POD nanozyme material coupled with a secondary antibody prepared in Comparative Example 1, the PAMAM@CeO2 nanozyme material coupled with a secondary antibody (IgG) prepared in Comparative Example 2, the HRP goat anti-rabbit secondary antibody (purchased from Biolong), the commercially available goat anti-rabbit secondary antibody not coupled with HRP (purchased from Biolong), and the BSA coupled secondary antibody prepared in Comparative Example 1, which specifically includes the following steps:

[0059] (1) Baking: baking in oven at 65℃ for 1h;

[0060] (2) Dewaxing: Soak the slide in a xylene dye vat for 10 min, then in another xylene dye vat for another 10 min; soak in anhydrous ethanol for 5 min; soak in 95% ethanol for 5 min; soak in 75% ethanol for 5 min; soak in water for 5 min;

[0061] (3) Antigen repair: Heat the water bath to about 95°C, prepare the antigen repair solution to the working concentration, pour it into the staining jar, put the dewaxed slide in, place it in a water bath, heat it in boiling water for 20 minutes, and then cool it naturally.

[0062] (4) Blocking: Remove the cooled slides and wash them three times with PBST, each time for 30 minutes. Dry the liquid around the tissue with a paper towel. Use an oil pen or paraffin circle, but do not touch the tissue. Add about 100 μL of peroxidase blocking solution (covering the tissue) and leave at room temperature for 20 minutes. Pour off the liquid and wash three times with PBST, each time for 3 minutes.

[0063] (5) Primary antibody incubation: Remove the slices, spin dry PBS, add about 100 μL of primary antibody to cover the tissue, incubate in a 37°C oven for 1 h or at 4°C overnight, and repeat the PBST wash three times, each wash for 3 min to ensure thorough washing.

[0064] (6) Secondary antibody incubation: Take out the slices, spin dry PBST, and drip 150 μL of the corresponding secondary antibody reagent on each slice to cover the tissue. Repeat the PBST washing three times, each washing for 3 minutes, to ensure that it is clean. The secondary antibody reagent in this step is the PAMAM@Ru-POD nanozyme material coupled with secondary antibody prepared in Example 2, the Ru-POD nanozyme material coupled with secondary antibody prepared in Comparative Example 1, the multimeric enzyme sheep anti-rabbit secondary antibody, PBS coupled with sheep anti-rabbit secondary antibody and BSA.

[0065] (7) Color development: Take out the slices, spin dry the PBS, add a drop of color developer to each slice, and incubate at room temperature for 5 min. After color development is completed, soak the slices in PBS for 3 min each and wash 3 times.

[0066] (8) Restaining: Remove the sections, spin dry the PBS, add 150 μL of hematoxylin to each section to cover the tissue on the section, incubate at room temperature for 1 min, spin dry the hematoxylin, and rinse with tap water for 3 min.

[0067] (9) Dehydration: Take out the slices, soak them in anhydrous ethanol and xylene for 3 min respectively, and dry them at room temperature.

[0068] (10) Sealing: Take out the slices, add one drop (about 0.02 mL) of mounting medium on each slice, cover with a coverslip, and place in a fume hood to dry.

[0069] (11) Film review: Seal the slides with neutral gum. Once completed, the slides can be reviewed under a microscope and photographed.

[0070] Results Analysis

[0071] The results are as follows Figures 4 to 8 As shown, compared with the second antibody coupled with the Ru-POD nanozyme material, the PAMAM@Ru-POD nanozyme material coupled with the second antibody obtained in Example 2, when performing IHC staining, it is concluded that there are more positively colored cells, and the positive staining (brown) is stronger, and the staining result is similar to the staining effect of the HRP second antibody product currently available on the market. The results indicate that the accuracy and sensitivity of the immunohistochemical detection by the PAMAM@Ru-POD nanozyme material coupled with the second antibody are both ideal. The PAMAM@CeO2 nanozyme material coupled with the second antibody obtained in Comparative Example 2 results in fewer positively colored cells, and the positive staining (brown) is weak, and the results indicate that even if there are nanozyme materials with POD activity, there may be differences in detection sensitivity after the same coupling method. The PAMAM@Ru-POD nanozyme material coupled with the second antibody obtained by screening and optimizing specific preparation conditions in this Example 2 shows good specificity and catalytic effect in HIC detection.

[0072] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A polymeric nanoenzyme material coupled with an antibody, characterized in that: The invention comprises a PAMAM dendritic polymer and an antibody covalently coupled thereto, wherein the external branches of the PAMAM dendritic polymer are modified and connected with a Ru-POD nanozyme.

2. The polymer nanoenzyme material coupled to an antibody according to claim 1, characterized in that: The antibody is IgG.

3. The method for preparing the polymeric nanoenzyme material coupled with an antibody according to claim 1 or 2, characterized in that: The steps include: (1) preparing an active Ru-POD nanozyme, washing the Ru-POD nanozyme, activating the Ru-POD nanozyme with EDC and NHS, and then purifying the Ru-POD nanozyme to obtain a purified activated Ru-POD nanozyme; (2) mixing the purified activated Ru-POD nanozyme obtained in step (1) with a PAMAM 5.0 solution for reaction, and then purifying to obtain a PAMAM@Ru-POD nanozyme; (3) Activating the carboxyl group of the antibody by EDC and NHS, and then reacting the mixture with the PAMAM@Ru-POD nanozyme obtained in step (2), and then purifying the mixture to obtain the polymer nanozyme material coupled with the antibody.

4. Use of the polymeric nanoenzyme material coupled with an antibody as claimed in claim 1 or 2 as a secondary antibody in immunohistochemical colorimetric detection.

5. An immunohistochemical detection method, characterized in that: The polymeric nanoenzyme material coupled with an antibody as described in claim 1 or 2 is used as a secondary antibody.

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