A polymeric nanoenzyme material coupled with an antibody and its application in immunohistochemical colorimetric detection

Abstract

The present invention discloses a polymeric nanoenzyme material-coupled antibody and its application in immunohistochemical colorimetric detection, comprising a PAMAM dendrimer and an antibody covalently coupled thereto, wherein the PAMAM dendrimer is modified with a Ru-POD nanozyme on its external branches. The polymeric nanoenzyme material-coupled antibody of the present invention has excellent advantages such as high stability, simple preparation, long service life, and adjustable activity. Compared with the traditional horseradish peroxidase-based polymer enzyme secondary antibody detection system, it improves catalytic performance and storage stability. Its catalytic activity for DAB and H2O2 is comparable to that of natural horseradish peroxidase, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection technology, and specifically relates to a polymeric nanoenzyme material coupled with an antibody and its application 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 typically 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, choosing the right primary antibody 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 binding directly to the 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 largely been driven by advances in secondary antibody detection systems, with the main trends being the continuous improvement of detection sensitivity and the continuous reduction of detection interference factors. For example, the evolution from biotin detection systems (such as the ABC method and the SP method) to enhanced labeled polymer systems has eliminated the interference of endogenous biotin and improved detection sensitivity. However, early non-biotin secondary antibody detection systems primarily used secondary antibody-enzyme polymers linked to 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 simple to operate, avoid interference from endogenous biotin, and maintain high detection sensitivity, promoting the widespread application of immunohistochemistry in clinical pathology-assisted diagnosis. However, the disadvantages of these methods are the significant increase in molecular weight and the excessive space occupied. Due to steric hindrance and other factors, the efficiency of the polymers in entering tissue cells and binding to specific targets is affected, resulting in reduced specific staining intensity and the generation of nonspecific 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, the practical application of natural enzymes has been severely limited due to their high cost, poor stability, low yield, storage difficulties, and short half-life. Nanomaterials are materials with at least one dimension in the nanometer range (1–100 nm) in three dimensions, or materials composed of these materials as basic units. Nanozymes are a class of nanomaterials with enzyme-like properties and catalytic activity. Since the 2007 report that Fe₃O₄ nanoparticles mimic the activity of natural peroxidases, hundreds of nanomaterials have been discovered that mimic the activities of enzymes such as peroxidase (POD), oxidase (OXD), catalase (CAT), glucose oxidase (GOx), glutathione peroxidase (GPx), superoxide dismutase (SOD), and uricase. Furthermore, many nanomaterials have been reported to exhibit dual or multienzyme activities. As a new generation of artificial enzymes, nanozymes offer advantages over natural enzymes, such as low cost, high stability, simple preparation, long lifespan, and tunable activity.

[0005] To enable these nanomaterials to better replace natural horseradish peroxidase and achieve widespread application, researchers often manipulate the enzyme-like activity of the nanomaterials based on their properties to further enhance their catalase-like activity. Common strategies for enhancing the performance of nanomaterial-like enzymes include size manipulation, morphology manipulation, component manipulation, and surface and interface manipulation. Notably, surface and interface manipulation of nanomaterials can further enhance the synergistic effects between materials by combining different nanomaterials, which has important scientific significance in the construction of highly efficient enzyme-like catalytic materials. Summary of the Invention

[0006] The present invention aims 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 polymeric nanoenzyme material coupled with an antibody.

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

[0009] The technical solutions of the present invention are as follows:

[0010] A polymeric nanoenzyme material coupled with an antibody comprises a PAMAM dendrimer and an antibody covalently coupled thereto, wherein the external branches of the PAMAM dendrimer 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 by comprising the following steps:

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

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

[0016] The above-mentioned polymeric nanoenzyme material coupled with an 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 with the antibody of the present invention has excellent advantages such as high stability, simple preparation, long service life, and adjustable activity. 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 with antibodies of the present invention has low preparation and purification costs, providing a basis for its further development in secondary antibody detection systems;

[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 towards DAB and H2O2 is comparable to that of natural horseradish peroxidase, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This 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 immunohistochemical detection of 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 based on Comparative Example 1 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 goat anti-rabbit secondary antibody are shown.

[0028] Figure 7 The results of immunohistochemistry based on HRP goat anti-rabbit secondary antibody for detecting P53 protein in lung adenocarcinoma tissue in Example 3 of the present invention 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 methods in conjunction with the accompanying drawings.

[0031] The one-pot preparation in the following examples involved dissolving RuCl₃·nH₂O (0.53 g) and NaAc (1.5 g) in ethylene glycol (15 mL), followed by dissolving each in PSS (905 μL) to obtain Ru-POD nanozyme. (See 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 shake to mix thoroughly. Centrifuge at 13000 rpm for 5 min. Carefully discard the supernatant and retain the precipitate.

[0035] B. Add 200 μL of 50 mM MES buffer solution (containing 0.01% Tween 20) at pH 5.0-6.0 to the above precipitate, and wash it with 10% power ultrasonic cleaning for 1 min (ultrasonic cleaning for 2 seconds, stop for 2 seconds), mix it evenly, and then centrifuge and discard the supernatant. Repeat 2 times to obtain the washed Ru-POD nanozyme, the transmission electron microscopy photo of which 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 on a mixer at room temperature for 30 min to activate the carboxyl group.

[0037] D. Centrifuge the material obtained in step C at 13,000 rpm for 5 minutes, carefully discard the supernatant, then add 600 μL of 50 mM pH 5.0 MES buffer (containing 0.01% Tween 20) to the resulting precipitate, ultrasonically clean the precipitate for 1 minute using 10% power ultrasound (ultrasonication for 2 seconds, on / off for 2 seconds), then centrifuge at 13,000 rpm for 5 minutes, 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 PAMAM 5.0 solution (purchased from Macklin, product number: 3901821), vortexed and shaken overnight at 4°C on a mixer (>12h). Then, the mixture was incubated at room temperature for 30min, and then centrifuged at 1000rpm for 10min using a 10KD ultrafiltration tube to collect the PAMAM@Ru-POD nanozyme. The transmission electron microscope image is shown below. Figure 3 As shown, 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 freshly prepared 25 mM EDC solution and 100 μL of 25 mM NHS solution to 0.5 mg of antibody, vortex 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): The activated antibody obtained in step (1) was added to the PAMAM@Ru-POD nanozyme prepared in Example 1, vortexed and mixed, and shaken on a mixer at 4°C overnight (>12h), followed by incubation at room temperature for 30min, and then centrifuged at 1000rpm for 10min using a 10KD ultrafiltration tube 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): The activated antibody obtained in step (2) was added to the activated Ru-POD nanozyme obtained in step (1), vortexed and shaken overnight at 4°C on a mixer (>12h), followed by incubation at room temperature for 30min, and then centrifuged at 1000rpm for 10min using a 10KD ultrafiltration tube to collect the Ru-POD nanozyme material coupled with secondary antibody (IgG).

[0047] (4) Preparation of BSA-coupled secondary antibody (IgG): BSA was used to replace the activated Ru-POD nanozyme in step (3), and the rest was 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 shake to mix, centrifuge at 13000rpm for 5min, carefully discard the supernatant and retain the precipitate.

[0051] B. Add 200 μL of PBST buffer solution (0.01% Tween 20) to the above precipitate, sonicate at 10% power for 1 min (sonicate for 2 seconds, stop for 2 seconds), mix evenly, centrifuge and discard the supernatant, repeat twice to obtain the washed CeO2 nanozyme for cleaning.

[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 on a mixer at room temperature for 30 min to activate the carboxyl groups.

[0053] D. Centrifuge the material obtained in step C at 13,000 rpm for 5 minutes, carefully discard the supernatant, then add 600 μl of PBST buffer (0.01% Tween 20) to the resulting precipitate, ultrasonically clean the precipitate for 1 minute using 10% power ultrasound (ultrasonication for 2 seconds, on / off for 2 seconds), then centrifuge at 13,000 rpm for 5 minutes, carefully discard the supernatant and retain the precipitate to obtain the 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, product number: 3901821), vortexed and shaken on a mixer at 4°C overnight (>12h), followed by incubation at room temperature for 30min, and then centrifuged at 1000rpm for 10min using a 10KD ultrafiltration tube to collect the PAMAM@CeO2 nanozyme.

[0055] (3) Activation of the carboxyl groups of antibodies (IgG): Add 100 μl of 25 mM EDC solution and 100 μl of 25 mM NHS solution to 0.5 mg of antibody, vortex mix, and react on a mixer at room temperature for 30 min to activate the carboxyl groups on the antibody. Then, use a 30 KD 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): The activated antibody obtained in step (3) was added to the PAMAM@CeO2 nanozyme prepared in step (2), vortexed and shaken overnight at 4°C on a mixer (>12h), followed by incubation at room temperature for 30min, and then centrifuged at 1000rpm for 10min using a 10KD ultrafiltration tube to collect the PAMAM@CeO2 nanozyme material coupled with secondary antibody (IgG).

[0057] Example 3

[0058] 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, an HRP goat anti-rabbit secondary antibody (purchased from BioAolong), a commercially available goat anti-rabbit secondary antibody not coupled with HRP (purchased from BioAolong), and the BSA coupled secondary antibody prepared in Comparative Example 1, specifically comprising the following steps:

[0059] (1) Baking: Bake 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: Take out the cooled slides and wash them three times with PBST, each time for 30 minutes. Wipe 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 let it stand at room temperature for 20 minutes. Pour off the liquid and wash it 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 hour or at 4°C overnight, and wash with PBST three times, each wash for 3 minutes to ensure thorough cleaning.

[0064] (6) Secondary antibody incubation: Take out the slices, spin dry PBST, and add 150 μL of the corresponding secondary antibody reagent to 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 reagents in this step are 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 goat anti-rabbit secondary antibody, PBS coupled with goat anti-rabbit secondary antibody and BSA.

[0065] (7) Color development: Remove the sections, spin dry the PBS, add a drop of color developer to each section, and incubate at room temperature for 5 minutes. After color development is complete, soak the sections in PBS for 3 minutes each and wash three 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 minutes respectively, and dry them at room temperature.

[0068] (10) Mounting: 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 reading: Seal the slides with neutral gum. Once completed, the slides can be read under a microscope and photographed.

[0070] Result Analysis

[0071] The results are as follows Figures 4 to 8 As shown, compared with the two anti-antibodies coupled with Ru-POD nanozyme materials, the PAMAM@Ru-POD nanozyme material coupled with two anti-antibodies obtained in Example 2 when performing IHC staining, it is concluded that positive colored cells are more, and positive staining (brown) is stronger, and the staining result is similar to the staining effect of the currently commercially available HRP two-antibody product, and the result suggests that the PAMAM@Ru-POD nanozyme material coupled with two anti-antibodies for immunohistochemistry detection are more accurate and sensitive. The PAMAM@CeO2 nanozyme material coupled with two anti-antibodies obtained in Comparative Example 2 shows fewer positive colored cells, and positive staining (brown) is weak, and the result suggests 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 two anti-antibodies obtained by screening and specific preparation conditions optimization in the present embodiment 2 shows good specificity and catalytic effect in HIC detection.

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

Claims

1. Use of polymeric nanoenzyme material coupled with antibody as a secondary antibody in immunohistochemical colorimetric detection, characterized in that: The polymeric nanozyme material coupled antibody comprises a PAMAM dendrimer and an IgG covalently coupled thereto, wherein the external branches of the PAMAM dendrimer are modified and connected with a Ru-POD nanozyme, and the preparation method comprises the following steps: (1) preparing active Ru-POD nanozyme, washing the Ru-POD nanozyme, activating it with EDC and NHS, and then purifying it to obtain 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 the mixture to obtain PAMAM@Ru-POD nanozyme; (3) After the carboxyl group of the antibody is activated by EDC and NHS, it is mixed with the PAMAM@Ru-POD nanozyme obtained in step (2) for reaction, and then purified to obtain the polymer nanozyme material coupled with the antibody.

2. An immunohistochemical detection method, characterized in that: A polymeric nanozyme material coupled antibody is used as a secondary antibody. The polymeric nanozyme material coupled antibody includes a PAMAM dendrimer and an IgG covalently coupled thereto. The external branches of the PAMAM dendrimer are modified and connected with a Ru-POD nanozyme. The preparation method includes the following steps: (1) preparing active Ru-POD nanozyme, washing the Ru-POD nanozyme, activating it with EDC and NHS, and then purifying it to obtain 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 the mixture to obtain PAMAM@Ru-POD nanozyme; (3) After the carboxyl group of the antibody is activated by EDC and NHS, it is mixed with the PAMAM@Ru-POD nanozyme obtained in step (2) for reaction, and then purified to obtain the polymer nanozyme material coupled with the antibody.

Images