A preparation method of a nano-enzyme-antibody complex based on site-specific enzyme catalytic coupling, products and applications thereof

Abstract

This invention discloses a method for preparing a nanozyme-antibody complex based on site-directed enzyme catalytic coupling, its products, and applications, belonging to the field of nanomedicine technology. The preparation method of this invention involves the site-directed introduction of an azide group at the Fc segment of the antibody's glycan chain through a cascade catalysis of N-glycosidase (PNGase F) and transglutaminase (TGase). Then, a click chemistry reaction is carried out between a platinum nanozyme modified with a DBCO group and the azide group, thereby constructing a nanozyme-antibody conjugate complex consisting of a single antibody coupled with two platinum nanozymes. During the coupling process, the structural integrity of the antibody and its antigen-targeting recognition function are maintained to the greatest extent, and the complex possesses highly efficient enzyme-like catalytic activity and good environmental stability, significantly improving the efficiency of converting the concentration information of the target substance into a color signal.

Description

Technical Field

[0001] This invention relates to a method for preparing a nanozyme-antibody complex based on site-directed enzyme catalytic coupling, its products and applications, belonging to the field of nanomedicine technology. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISA) is the most widely used enzyme immunoassay technique, offering advantages such as speed, sensitivity, simplicity, and ease of standardization. The basic principle of this method is the covalent binding of enzyme molecules to antibodies or anti-antibody molecules. These enzyme-labeled antibodies specifically bind to antigens or antibodies adsorbed on a solid-phase support. Upon addition of the substrate solution, the enzyme catalyzes a colorimetric reaction, with the color intensity directly proportional to the concentration of the target antigen or antibody in the sample, thus enabling qualitative or quantitative detection of the antigen or antibody. In other words, ELISA combines the high sensitivity of the enzymatic reaction with the high specificity of the antigen-antibody reaction to achieve highly efficient detection of target molecules.

[0003] Enzymes commonly used for labeling antibodies or anti-antibodies include horseradish peroxidase (HRP), alkaline phosphatase, and glucose oxidase, with HRP being the most widely used. However, while traditional natural enzymes, represented by HRP, possess high catalytic specificity, they still have some limitations in practical applications: poor stability, easily affected by environmental temperature, pH, and proteases; high preparation costs; demanding storage and handling conditions; and relatively limited functionality. These shortcomings restrict their further application in complex samples, extreme environments, or large-scale diagnostic scenarios.

[0004] In recent years, the emergence of nanozymes has provided a new direction for the development of immunoassay technology. Nanozymes are nanomaterials with at least one dimension ranging from 1 to 200 nm and possessing enzyme-like catalytic activity. Due to the low coordination number and dangling bonds on their surface atoms, they readily adsorb reactants and lower activation energies, giving them active centers and electron transfer structures similar to natural enzymes. This endows them with catalytic activity similar to natural enzymes under mild conditions. Compared to natural enzymes, nanozymes are mostly inorganic or metal-based nanomaterials, possessing excellent thermal stability, acid and alkali resistance, and resistance to protease degradation. They can be stored for long periods at room temperature and are resistant to interfering substances in complex biological samples. Furthermore, nanozymes can be prepared on a large scale and reproducibly through chemical synthesis pathways such as hydrothermal methods and co-precipitation methods, with significantly lower raw material costs than the biological extraction or recombinant expression processes of natural enzymes. Moreover, the nanostructure of nanozymes is easily functionalized, facilitating further attachment of target molecules.

[0005] In the development of antibody-nanozymes, traditional antibody-nanozyme conjugates are mainly multi-antibody conjugated single nanozymes, i.e., multiple antibody molecules are conjugated to the surface of a single nanozyme (typically with a particle size of 10-200 nm), based on a chemical conjugation strategy of carbodiimide (EDC) and N-hydroxysuccinimide (NHS). This strategy involves the EDC activating the carboxyl groups on the loading, which react with the ε-amino groups of lysine residues on the antibody surface to form amide bonds. However, this process is highly random and often has multiple negative impacts on the antigen recognition and binding ability of antibodies. First, the antibody Fab region has a high affinity for antigen binding, which depends on the integrity of its complementarity-determining region and its surrounding critical microenvironment. The antibody surface contains a large number of lysine residues, and the carboxyl groups activated by EDC will non-selectively attack these lysine residues. If conjugation occurs directly in the Fab region, especially on lysine residues within or near the complementarity-determining region, changes in side chain chemistry or local conformational perturbations may directly interfere with the specificity and binding force of antigen-antibody interactions, leading to the loss of targeting function in some antibodies. Even if coupling does not occur directly at the antigen-binding epitope, the carrier molecules attached to the antibody surface may physically obscure the antigen-binding interface due to steric hindrance, hindering the effective approach and correct orientation of the target antigen. Simultaneously, in the configuration of multiple antibody-conjugated single nanozymes, the proportion of inactivated antibodies is amplified. Furthermore, the dense distribution of multiple antibodies on the nanozyme surface can lead to spatial crowding between adjacent antibodies and between the antibody and the particle surface, resulting in the obscuring or obstruction of antigen-binding sites, ultimately causing a further decrease in overall targeting efficiency. Moreover, to reduce non-specific adsorption, this configuration often requires the use of macromolecules such as albumin to block the nanozyme, thereby weakening its catalytic activity.

[0006] Therefore, there is an urgent need to develop an enzyme-labeled antibody that combines excellent antigen recognition and binding capabilities, efficient enzyme-like catalytic activity, and good environmental stability, and can serve as a high-performance enzyme-labeled antibody. Summary of the Invention

[0007] Objective of this invention: The objective of this invention is to provide a method for preparing a nanozyme-antibody complex based on site-directed enzyme catalytic coupling, its product, and its applications. This method achieves site-directed and efficient coupling of platinum nanozymes to the Fc end of antibodies through a cascaded enzymatic reaction, constructing a universal and modular antibody-nanozyme conjugation platform. While retaining the complete targeting function of the antibody, it provides a novel antibody-nanozyme conjugated complex with high stability and high catalytic activity for immune detection and targeted therapy.

[0008] Technical solution: The present invention provides a method for preparing a nanozyme-antibody complex based on site-directed enzyme catalysis coupling, which involves the following steps: (1) The platinum precursor solution and the surface modifier containing dibenzocyclooctylene were mixed and the reducing agent was added dropwise while mechanically stirring in an acidic environment. After the reaction was completed, the dibenzocyclooctylene-functionalized platinum nanozyme was obtained. (2) Azide groups were introduced at specific sites on the Fc glycan chain of the antibody by a cascade catalysis method of N-glycosidase and transglutaminase to obtain azide-modified antibody; (3) The platinum nanozyme functionalized with dibenzocyclooctylene and the azide-modified antibody were incubated overnight to obtain the nanozyme-antibody complex.

[0009] Further, the platinum precursor solution mentioned in step (1) is a chloroplatinic acid solution with a concentration of 1.5~2.5mM.

[0010] Further, the surface modifier containing dibenzocyclooctylene mentioned in step (1) is DBCO-PEG-SH with a concentration of 0.1~0.3mM.

[0011] Furthermore, the acidic environment described in step (1) is provided by an acetic acid solution with a concentration of 2.5%.

[0012] Further, the reducing agent mentioned in step (1) is sodium borohydride with a concentration of 200~300mM.

[0013] Further, the molar ratio of N-glycosidase to antibody in step (2) is 1:2.5; the molar ratio of transglutaminase to antibody is 1:20.

[0014] Furthermore, the substrate of the azide used in step (2) is NH2-(CH2)5-N3, and its molar ratio with the antibody is 10:1.

[0015] Furthermore, the antibody mentioned in step (2) is an IgG antibody.

[0016] Furthermore, in step (3), the molar ratio of the dibenzocyclooctyne-functionalized platinum nanozyme and the azide-modified antibody is 2:1 to 3:1.

[0017] The nanozyme-antibody complex prepared by the preparation method described in this invention.

[0018] The nanozyme-antibody complex described in this invention is used as an enzyme-labeled antibody in enzyme-linked immunosorbent assay (ELISA).

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The preparation method of the present invention introduces azide groups at the Fc glycan chain of the antibody through the cascade catalysis of N-glycosidase (PNGase F) and transglutaminase (TGase), and then constructs a nanozyme-antibody conjugate complex consisting of a single antibody coupled with two platinum nanozymes by a click chemical reaction between the azide groups and the platinum nanozyme modified with DBCO groups. During the coupling process, the structural integrity of the antibody and the antigen-targeting recognition function can be maintained to the greatest extent, and it has high efficiency of enzyme-like catalytic activity and good environmental stability, which significantly improves the efficiency of converting the concentration information of the target substance into a color signal. Attached Figure Description

[0020] Figure 1 This is a graph showing the POD enzyme activity analysis of BSA@PtNPs.

[0021] Figure 2 The images show TEM images of BSA@PtNPs, where a and b represent the morphology of platinum nanoclusters obtained after a reaction of 40 min under TEM, and c-f represent the morphology of platinum nanoclusters obtained after a reaction of 90 min under TEM.

[0022] Figure 3 The images show TEM images of BSA@PtNPs, where a~c represent the morphology of platinum nanoclusters obtained after 120 min of reaction under TEM, and d~f represent the morphology of platinum nanoclusters obtained after 240 min of reaction under TEM.

[0023] Figure 4 Figure 1 shows the TEM image and hydrodynamic dimensions of BSA@PtNPs@SDS, where a~c are TEM images of BSA@PtNPs@SDS at different scales. Figure d is the DLS image of BSA@PtNPs@SDS.

[0024] Figure 5 The image shows the XRD pattern of HS-PEG@PtNPs.

[0025] Figure 6 XPS spectra of HS-PEG@PtNPs.

[0026] Figure 7 The images show the TEM images and elemental mapping diagrams of HS-PEG@PtNPs. A through c are TEM images at different scales, d is the distribution map of Pt, e is the distribution map of S, and f is the superimposed distribution map of C, O, S, and Pt.

[0027] Figure 8 This is an electrophoresis image of maleimide-modified antibody.

[0028] Figure 9Physical photograph (a) and structural diagram (b) of Pt@DBCO-PEG.

[0029] Figure 10 The images are transmission electron microscope (TEM) images of Pt@DBCO-PEG. a and b are TEM images of different regions at a scale of 20 nm, and c is a high-resolution TEM image at a scale of 5 nm.

[0030] Figure 11 XPS (a) and XRD (b) plots of Pt@DBCO-PEG.

[0031] Figure 12 The Fourier transform infrared spectrum of Pt@DBCO-PEG is shown.

[0032] Figure 13 The graph shows the peroxidase activity analysis of Pt@DBCO-PEG, where a represents the peroxidase activity under different pH conditions, b represents the peroxidase activity analysis of different concentrations of H2O2, and c represents the peroxidase activity analysis of different concentrations of TMB.

[0033] Figure 14 The graph shows the oxidase activity analysis of Pt@DBCO-PEG, where a represents the oxidase activity under different pH conditions and b represents the oxidase activity analysis of different concentrations of TMB.

[0034] Figure 15 Electrophoresis images of biotinylated and azidated antibodies.

[0035] Figure 16 This describes the antibody-platinum nanozyme synthesis pathway.

[0036] Figure 17 This is a transmission electron microscope image of the antibody-platinum nanozyme conjugate.

[0037] Figure 18 This is an electrophoresis image of the antibody-platinum nanozyme conjugate.

[0038] Figure 19 This is a graph showing the potency analysis of antibody-platinum nanozyme conjugates. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0040] Unless otherwise specified, all chemical reagents are commercially available and can be used directly without further purification.

[0041] H₂PtCl₆·6H₂O, 3,3',5,5'-Tetramethylbenzidine (TMB), DBCO-PEG-SH, and NH₂-(CH₂)₅-N₃ were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Glacial acetic acid, sodium borohydride, and hydrogen peroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. CD20 antibody was purchased from Roche Pharmaceuticals Ltd. (Shanghai). N-glycosidase (PNGase F) was purchased from Shanghai Yisheng Biotechnology Co., Ltd. Transglutaminase (TGase) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. All water with a resistivity higher than 18.2 MΩ·cm used in the experiments was purified using a laboratory water purification system (AW-2002-H, ICP).

[0042] Example 1: Investigation of methods for preparing platinum nanozymes (1) Since the substrate recognized by transglutaminase in the enzymatic catalysis method is a lysine analog, it is proposed to prepare a platinum nanozyme modified with BSA (bovine serum albumin) as the surface. This nanozyme would be used to catalyze a transamination reaction between lysine residues on BSA and glutamine residues on the antibody, thereby achieving coupling. The preparation method is as follows: Add 5.0 mL of 10.0 mM H₂PtCl₆ (0.05 mmol Pt) to 5.0 mL of 3–50 mg / mL BSA aqueous solution. Stir continuously at room temperature, and immediately after 10 minutes, add 5.0 mL of 100 mM NaOH to the mixture. Then stir at 70 °C for 30–360 minutes. Finally, cool the solution to room temperature and dialyze against ultrapure water for 24 h to remove all free Pt ions, obtaining pure BSA@PtNPs.

[0043] The POD enzyme activity of BSA@PtNPs prepared with different BSA feed amounts and reaction times is as follows: Figure 1 As shown, the TEM image is as follows Figure 2 and Figure 3 As shown. However, by continuously adjusting the BSA feed amount and reaction time, it was found that this method is difficult to synthesize platinum nanozymes with high activity.

[0044] (2) To improve the activity of platinum nanozymes, it was desired to synthesize platinum nanozymes in an acetic acid environment. However, BSA was difficult to dissolve under acidic conditions. Despite continuously adjusting the acetic acid concentration, BSA could not be dissolved. Finally, the only solution was to add SDS (sodium dodecyl sulfate) to disrupt the BSA structure, allowing BSA to dissolve in an acidic environment and participate in subsequent reactions. The specific preparation method is as follows: In 9 mL of 0.25% acetic acid solution, 14 mg SDS and 10 mg BSA were added and mixed thoroughly. Then, 1 mL of 10.0 mg / mL H₂PtCl₆ was added. The mixture was stirred continuously at room temperature for 10 minutes, after which 1 mL of 10 mg / mL NaBH₄ was immediately added. The mixture was then stirred at room temperature for 30 minutes. Finally, the solution was cooled to room temperature and dialyzed against ultrapure water for 24 h to remove all free Pt ions, yielding pure BSA@PtNPs@SDS.

[0045] TEM and DLS images of BSA@PtNPs are as follows: Figure 4 As shown, the prepared BSA@PtNPs@SDS nanozyme had a POD activity of only 20 U / mg, which does not meet our requirement of preparing a platinum nanozyme with strong POD activity.

[0046] (3) Through summary and analysis, it is believed that compared with BSA protein, using PEG macromolecules as surface modification of platinum nanozymes is more conducive to improving their POD activity. Since the Michael addition reaction between thiol and maleimide is a commonly used coupling method, it is planned to first synthesize HS-PEG-SH modified platinum nanozymes, then modify the Fc segment of the antibody with maleimide by enzyme catalysis, and finally achieve coupling through Michael addition reaction.

[0047] The preparation method of HS-PEG-SH modified platinum nanozymes is as follows: the platinum precursor solution and HS-PEG-SH are mixed, and NaBH4 is added dropwise while mechanically stirring. After the reaction is completed, the mixture is dialyzed to obtain HS-PEG@PtNPs.

[0048] By adjusting the feed amount of HS-PEG-SH, it was found that a feed amount of 2.5 mg yielded HS-PEG@PtNPs with the strongest POD activity, exhibiting an enzyme activity of 890 U / mg, which can be used for subsequent reactions. XRD analysis is shown below. Figure 5 As shown, the XPS plot is as follows Figure 6 As shown, the TEM image and element mapping diagram are as follows: Figure 7 As shown.

[0049] However, it was found that the maleimide-labeled antibody was difficult to maleimide using HS-FITC, therefore this approach remains difficult to implement. Figure 8 ).

[0050] Finally, the method of conjugating DBCO-functionalized platinum nanozymes with azidated antibodies was adopted.

[0051] Example 2 Preparation of Pt@DBCO-PEG nanozymes 10 mg of chloroplatinic acid hexahydrate (H₂PtCl₆·6H₂O) was weighed and dissolved in 4.5 mL of deionized water to prepare the platinum precursor solution. 7 mg of DBCO-PEG-SH was weighed and dissolved in 4.5 mL of deionized water, and 1 mL of a 2.5% acetic acid solution prepared with glacial acetic acid was added to provide an acidic environment, resulting in the DBCO-PEG-SH solution. The platinum precursor solution and the DBCO-PEG-SH solution were added to the reactor, and magnetic stirring was started at 600 rpm to ensure homogeneous mixing. 10 mg of sodium borohydride was weighed and dissolved in 1 mL of deionized water. While continuously stirring, the sodium borohydride solution was added dropwise to the above reaction system. After the addition was complete, the reaction conditions were maintained unchanged, and the reaction was continued at 25°C for 30 minutes to complete the in-situ reduction of platinum nanoparticles and the surface modification of DBCO-PEG-SH. After the reaction, the resulting product solution was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and purified by dialyzing in deionized water for 48 hours, with the dialysate changed every 8 hours. After dialysis, a stable Pt@DBCO-PEG nanozyme aqueous solution was obtained. The results are as follows... Figure 9 As shown in a.

[0052] Example 3 Morphology and particle size of Pt@DBCO-PEG nanozymes An aqueous solution of the Pt@DBCO-PEG nanozyme synthesized in Example 2 was dropped onto the surface of a copper mesh with a carbon film. After drying at room temperature, its morphology and particle size were observed using a transmission electron microscope (TEM). The results are as follows: Figure 10 As shown, the nanozyme exhibits a near-spherical morphology, with distinct lattice fringes observed under high-resolution field of view. Statistical calculations revealed an average particle size of approximately 2.67 nm. Then, another 1 mL of Pt@DBCO-PEG nanozyme aqueous solution was taken, and its hydrated particle size (hydrodynamic diameter) and surface Zeta potential were measured using dynamic light scattering. The results showed that the hydrated particle size of the nanozyme was 31 ± 4.3 nm, and the surface potential was -18.7 ± 0.7 mV. To assess its stability, the hydrated particle size of the freshly prepared nanozyme aqueous solution was measured after standing at 4 °C for one week. No significant change in hydrated particle size was observed within one week, demonstrating that the Pt@DBCO-PEG nanozyme possesses good stability.

[0053] Example 4: X-ray photoelectron spectroscopy and X-ray diffraction The Pt@DBCO-PEG nanozyme aqueous solution synthesized in Example 2 was freeze-dried to obtain a solid powder, which was then analyzed by X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). XPS analysis results showed that... Figure 11 a) The platinum element in nanozymes is mainly composed of metallic platinum (Pt). 0 ) and divalent platinum oxide (Pt)2+ Composed of ) where Pt 0 The binding energy peak corresponds to the metallic core of the platinum nanoparticle, Pt 2+ The signal mainly originates from the oxide layer or coordination environment formed on the surface of the nanoparticles. This result indicates that platinum ions in the precursor have been effectively reduced to platinum nanoparticles. XRD pattern ( Figure 11 b) shows four distinct diffraction peaks at approximately 39.9°, 46.3°, 68.0°, and 81.9° 2θ, corresponding to the (111), (200), (220), and (311) crystal planes of the face-centered cubic platinum crystal structure, respectively. This demonstrates that the prepared platinum nanozyme possesses a complete crystal structure and good crystallinity. The XPS and XRD results corroborate each other, jointly indicating that the method successfully synthesized a Pt@DBCO-PEG nanozyme with a platinum core and a regular crystal structure.

[0054] Example 5: Fourier Transform Infrared Spectroscopy The Pt@DBCO-PEG nanoenzyme aqueous solution synthesized in Example 2 was freeze-dried to obtain a solid powder. An appropriate amount of potassium bromide was placed in a mortar and ground thoroughly until powdered, then dried under an infrared lamp to remove interference from adsorbed moisture. A small amount of Pt@DBCO-PEG nanoenzyme powder was mixed with the pretreated potassium bromide powder in an appropriate ratio and ground thoroughly to ensure uniform dispersion of the sample in the potassium bromide. The uniformly mixed powder was transferred to a tableting mold and pressed into a transparent sheet under appropriate pressure. The resulting sheet was tested using a Fourier transform infrared spectrometer. The infrared spectrum results are shown below. Figure 12 As shown. At approximately 1100 cm -1 The strong absorption peaks nearby are attributed to the C–O–C stretching vibrations in the PEG chain; at approximately 2880 cm⁻¹ -1 Strong absorption peaks belonging to the symmetric and asymmetric stretching vibrations of –CH2– can be observed at approximately 1500 cm⁻¹; simultaneously, at approximately 1500 cm⁻¹... -1 and 1600cm -1 A moderate absorption peak belonging to the stretching vibration of the aromatic ring skeleton was observed. These characteristic peaks are consistent with the typical infrared absorption positions of the functional groups in the DBCO-PEG-SH molecule, indicating that the DBCO-PEG-SH molecule has been successfully modified onto the surface of the platinum nanozyme.

[0055] Example 6: Assay of Peroxidase Activity The Pt@DBCO-PEG nanozyme aqueous solution synthesized in Example 2 was used for peroxidase-like activity testing. First, the effect of pH on enzyme activity was tested. The reaction system contained 200 μL of acetate / sodium acetate buffer at different pH values ​​(3.6, 4.55, 5.35, 6.26, 7.13), 20 μL of Pt@DBCO-PEG nanozyme aqueous solution, 20 μL of H2O2 (2.4%) solution, and 10 μL of TMB (1.7 mM) solution. All components were added to a 96-well plate, mixed thoroughly, and the absorbance at 650 nm was immediately measured. The results are as follows: Figure 13 As shown in a, the absorbance is higher under acidic conditions and gradually decreases with increasing pH, indicating that the nanozyme has better peroxidase-like activity in an acidic environment.

[0056] Further, under optimal conditions of pH 3.6, its catalytic activity parameters were determined. The reaction system contained 200 μL of acetate / sodium acetate buffer, 20 μL of H2O2 (2.4%) solution, and 10 μL of TMB (1.7 mM) solution, as well as Pt@DBCO-PEG nanozyme solutions of different mass concentrations (corresponding to platinum masses of 0.084, 0.168, 0.337, 0.505, and 0.674 μg in the nanozyme). The absorbance at 650 nm was measured after the reaction, and the specific activity (a) of the nanozyme was calculated. nano The value was 1986.0 ± 203.4 U / mg. The initial reaction rate was determined under fixed nanozyme dosage and optimal pH conditions using different concentrations of H₂O₂ (80, 200, 400, 533, 800 mM) and TMB (2.08, 4.16, 10.40, 20.80, 41.61 mM) as substrates. The reaction rate was analyzed by Lineweaver-Burk double reciprocal plotting. Figure 13 (b, 13c) Fitting the kinetic parameters, we obtained the Michaelis constant Km = 356 mM and the maximum reaction rate Vmax = 0.44 mM / (L·min) for Pt@DBCO-PEG nanozyme with respect to H2O2; and Km = 1.27 mM and Vmax = 0.72 mM / (L·min) for TMB.

[0057] Example 7: Determination of Oxidase Activity Using TMB as a substrate, the oxidase-like activity of the Pt@DBCO-PEG nanozyme synthesized in Example 2 was determined. First, the effect of pH on the oxidase-like activity was tested. The reaction system contained 220 μL of acetate / sodium acetate buffer at different pH values ​​(3.6, 4.55, 5.35, 6.26, 7.13), 20 μL of Pt@DBCO-PEG nanozyme solution, and 10 μL of TMB (1.7 mM) solution. The absorbance was measured immediately after mixing at 650 nm. The results are as follows: Figure 14 As shown in a, the absorbance is high under acidic conditions and gradually decreases with increasing pH, indicating that the nanozyme exhibits good oxidase-like activity in an acidic environment.

[0058] Kinetic parameters were determined at pH 3.6. The reaction system contained 220 μL of acetate / sodium acetate buffer, 10 μL of TMB (1.7 mM) solution, and a series of Pt@DBCO-PEG nanozyme solutions at mass concentrations (corresponding to platinum masses of 0.084, 0.168, 0.337, 0.505, and 0.674 μg in the nanozyme). The absorbance change at 650 nm was measured, and the specific activity of the oxidase-like enzyme was calculated to be 6.4 ± 0.65 U / mg. Further, using different concentrations of TMB (2.08, 4.16, 10.40, 20.80, and 41.61 mM) as substrates, the initial reaction rate was determined, and the results were plotted using a double reciprocal plot. Figure 14 b) The Michaelis constant Km of the Pt@DBCO-PEG nanozyme for TMB was obtained by fitting, which was 2.27 mM and the maximum reaction rate Vmax was 0.68 mM / (L·min).

[0059] Example 8 Site-directed modification of CD20 antibody Add 320 μL of deionized water, 40 μL of 10× potassium-free PBS buffer (pH = 7.4), and 40 μL of 10 mg / mL CD20 antibody solution sequentially to a 2 mL centrifuge tube, and gently pipette to mix. Then add 40 μL of PNGase F enzyme solution (100,000 U / mL), mix well, and place in a metal bath. React at 37°C and 420 rpm for 24 hours to complete the deglycosylation of the antibody. After the reaction, add 200 μL of transglutaminase (TGase, 2 U) and 8 μL of 10 mg / mL amino-pentyl-azide (NH2-(CH2)5-N3) solution to the system, and continue incubation at 37°C and 420 rpm for 18 hours to achieve the specific introduction of azide groups into the Fc fragment of the antibody via TGase catalysis. Vortex the protein G magnetic beads for 30 seconds to fully resuspend them, then transfer 200 μl of the beads to a centrifuge tube. Add 200 μl of binding buffer (0.02 M, pH 7.4 PBST buffer) to the centrifuge tube, mix thoroughly, place the tube on a magnetic rack, and let it stand for about 1 minute to allow the magnetic beads to adhere completely to the tube wall. The supernatant should be clear and transparent; pipette and discard the supernatant. Repeat the above washing steps twice. Add the incubated antibody to the pre-washed magnetic beads and gently pipette to mix, avoiding foaming as much as possible. At room temperature, place the antibody mixture with the magnetic beads on a rotary mixer and incubate slowly by inverting for 10-15 minutes; then place the centrifuge tube on a magnetic rack and let it stand for about 1 minute to allow the magnetic beads to adhere completely to the tube wall; pipette and discard the supernatant. Add 200 μL of binding buffer to a centrifuge tube, mix thoroughly, place the tube on a magnetic rack for magnetic separation, and discard the supernatant. Add another 200 μL of binding buffer to the centrifuge tube, mix thoroughly, transfer to a clean centrifuge tube, magnetically separate, and discard the supernatant. Then add 80 μL of elution buffer (0.1 M, pH 2.5 citrate buffer) to the centrifuge tube, gently pipette to mix, avoiding foaming as much as possible. At room temperature, place the centrifuge tube on a rotary mixer and slowly invert for 5 min. Place the centrifuge tube on a magnetic rack for magnetic separation, carefully pipette the supernatant, and transfer it to a clean centrifuge tube. Immediately add 8 μL of neutralization buffer (1 M, pH 8 Tris-HCl buffer) to the eluted supernatant. Then transfer the resulting product solution to a dialysis bag with a molecular weight cutoff of 50 kDa and dialyze to PBS for purification for 48 hours, changing the dialysis buffer every 8 hours. After dialysis, azide-modified CD20 antibody is obtained.

[0060] To verify the presence of a modification in the Fc region of the antibody, fluorescent labeling was used. 10 μg of DBCO-FITC was added to the azide-modified CD20 antibody and incubated overnight at 4°C in the dark. After the reaction, the antibody was centrifuged at 2000 rpm for 10 min using an ultrafiltration tube with a molecular weight cutoff of 50 kDa, repeated three times to remove unreacted fluorescent dye, yielding the FITC-labeled antibody. 16 μl of the fluorescently labeled antibody was mixed with 4 μl of reducing loading buffer and heated at 100°C for 10 min to denature the protein. SDS-PAGE electrophoresis was then performed. After electrophoresis, the gel was gently rinsed three times with deionized water, and then imaged in the dark to obtain the desired results. Figure 15 The dark field image was obtained. Then, the same gel was stained with Coomassie Brilliant Blue and imaged under bright field to obtain... Figure 15 Bright-field images were obtained. Analysis of electrophoresis results under both dark-field and bright-field conditions revealed that a significant FITC fluorescence signal was observed only at molecular weights of 40–55 kDa (corresponding to the antibody heavy chain). Coomassie brilliant blue staining confirmed the integrity of the heavy and light chain bands. These results indicate that the azide group is specifically introduced into the antibody Fc segment (heavy chain), allowing only the heavy chain to undergo a click chemistry reaction with DBCO-FITC, further validating the site-specific modification of the antibody Fc segment using this method.

[0061] Example 9 Morphology and particle size of antibody-platinum nanozyme conjugates The Pt@DBCO-PEG nanozyme aqueous solution prepared in Example 2 was mixed with the azide-modified CD20 antibody prepared in Example 8 at antibody:nanozyme mass ratios of 1:2, 10:1, and 5:1. The mixture was allowed to react overnight at 4°C, allowing the DBCO groups on the nanozyme surface to covalently link with the azide groups introduced into the Fc segment of the antibody via click chemistry. After the reaction, the mixture was purified using the magnetic separation and dialysis methods described in Example 8 to remove uncoupled free nanozymes and other impurities, yielding an aqueous solution of the CD20 antibody-platinum nanozyme conjugate. The antibody-platinum nanozyme synthesis route is as follows: Figure 16 As shown in Table 1, the enzyme activity data for different antibody nanozyme mass ratios are presented.

[0062] Table 1

[0063] Based on enzyme activity data, a CD20 antibody-platinum nanozyme conjugate prepared at an antibody-nanozyme mass ratio of 1:2 was selected for subsequent experiments. An appropriate amount of the purified antibody-platinum nanozyme conjugate solution was dropped onto the surface of a copper mesh covered with a carbon support film. After drying at room temperature, the morphology, particle size distribution, and elemental distribution of the sample were observed using transmission electron microscopy (TEM). The results are as follows: Figure 17As shown, compared with the uncoupled Pt@DBCO-PEG nanozyme in Comparative Example 2, the distribution of platinum nanoparticles in the coupled sample was significantly looser, with increased interparticle spacing. This is presumably due to the steric hindrance effect of the antibody molecules hindering nanoparticle aggregation. Furthermore, observation of the mapping diagram revealed a high degree of overlap between the platinum enrichment region and the nitrogen distribution region. Given that the antibody molecules are rich in peptide bonds (-CO-NH-), their nitrogen abundance is significantly higher than that of the nanozyme. This spatial co-localization result provides direct evidence for the successful coupling of the antibody and the platinum nanozyme. Another 1 mL of the antibody-platinum nanozyme conjugate aqueous solution was used to measure its hydrated particle size and surface Zeta potential using a dynamic light scattering instrument. The results showed that the hydrated particle size of the conjugate increased to 101±42.6 nm, and the surface potential also changed significantly, rising to -1.9±0.2 mV. The significant increase in hydrated particle size and the positive migration of surface potential mentioned above are consistent with the increase in hydrodynamic size and changes in surface chemical environment caused by the successful modification of antibody molecules onto the surface of nanozymes, further confirming the effective coupling of CD20 antibody and Pt@DBCO-PEG nanozymes from a physicochemical perspective.

[0064] Example 10: SDS-PAGE electrophoresis of antibody-platinum nanozyme conjugate The antibody-platinum nanozyme conjugate prepared in Example 9 was subjected to SDS-PAGE electrophoresis according to the method of Example 8, with unmodified CD20 antibody as a control. Coomassie brilliant blue staining and gel imaging were performed. The electrophoresis results are as follows: Figure 18 As shown, in the lanes of the CD20 antibody (Ab), two distinct bands are clearly visible at molecular weights of approximately 50 kDa and 25 kDa, corresponding to the heavy and light chains of the antibody, respectively, indicating that the antibody structure is intact. In the lanes of the antibody-platinum nanozyme conjugate (Ab-PEG@Pt), only the light chain band is observed, while no obvious band is visible at the molecular weight corresponding to the heavy chain region. These phenomena indicate that the platinum nanozyme is successfully coupled to the Fc segment of the antibody (located in the heavy chain). Due to the high molecular weight and dense metal core structure of the platinum nanozyme, coupling leads to a significant increase in the molecular size and mass of the heavy chain, resulting in a sharp decrease in its migration rate in polyacrylamide gel electrophoresis. In contrast, the light chain, not participating in coupling, is unaffected in its electrophoretic behavior, thus exhibiting clear and intact bands. This result further confirms that the method described in this invention achieves site-specific coupling of platinum nanozymes to the Fc segment of the antibody.

[0065] Example 11: Potency Analysis of Antibody-Platinum Nanozyme Conjugates The titer of the antibody-platinum nanozyme conjugate prepared in Example 9 was measured.

[0066] Preparation and Coating: Dilute CD20 antigen to 0.5 μg / mL with carbonate buffer (pH 9.6). Add 100 μL to each well of a high-binding ELISA plate and incubate overnight at 4°C. After incubation, discard the liquid in the wells and wash three times with washing buffer (PBST) for 3-5 minutes each time to remove unbound antigen. Then add 200 μL of blocking buffer (PBST containing 1% BSA) to each well and block at 37°C for 90 minutes to occupy non-specific binding sites. Wash three more times after blocking for 3-5 minutes each time. Then serially dilute the antibody-platinum nanozyme conjugate with PBST (1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024). Add 100 μL of each dilution of antibody-platinum nanozyme conjugate to each well. Incubate at 37°C for 60 minutes. After incubation, discard the liquid in the wells and wash five times with PBST to completely remove unbound enzyme-labeled antibody. Add 100 μL of freshly prepared substrate solution (10 mg / ml TMB: 30% H2O2: pH 3.6 HAc-NaAc buffer = 1:2:20) to each well. Incubate at 37°C in the dark for 10-30 minutes. Add 50 μL of stop solution (2M H2SO4) to each well to terminate the enzymatic reaction. Read the absorbance of each well at 450 nm using a microplate reader. The results are as follows: Figure 19 As shown, its potency was measured to be 1:128.

Claims

1. A method for preparing a nanozyme-antibody complex based on site-directed enzyme catalytic coupling, characterized in that, Prepared by the following steps: (1) The platinum precursor solution and the surface modifier containing dibenzocyclooctylene were mixed and the reducing agent was added dropwise while mechanically stirring in an acidic environment. After the reaction was completed, the dibenzocyclooctylene-functionalized platinum nanozyme was obtained. (2) Azide groups were introduced at specific sites on the Fc glycan chain of the antibody by a cascade catalysis method of N-glycosidase and transglutaminase to obtain azide-modified antibody; (3) The platinum nanozyme functionalized with dibenzocyclooctylene and the azide-modified antibody were incubated overnight to obtain the nanozyme-antibody complex.

2. The method for preparing nanozyme-antibody complexes based on site-directed enzyme catalytic coupling according to claim 1, characterized in that, The platinum precursor solution mentioned in step (1) is a chloroplatinic acid solution with a concentration of 1.5~2.5mM.

3. The method for preparing nanozyme-antibody complexes based on site-directed enzyme catalytic coupling according to claim 1, characterized in that, The surface modifier containing dibenzocyclooctylene mentioned in step (1) is DBCO-PEG-SH with a concentration of 0.1~0.3mM.

4. The method for preparing nanozyme-antibody complexes based on site-directed enzyme catalytic coupling according to claim 1, characterized in that, The reducing agent mentioned in step (1) is sodium borohydride with a concentration of 200~300mM.

5. The method for preparing nanozyme-antibody complexes based on site-directed enzyme catalytic coupling according to claim 1, characterized in that, The molar ratio of N-glycosidase to antibody in step (2) is 1:2.5; the molar ratio of transglutaminase to antibody is 1:

20.

6. The method for preparing nanozyme-antibody complexes based on site-directed enzyme catalytic coupling according to claim 1, characterized in that, The substrate for the azide used in step (2) is NH2-(CH2)5-N3, and its molar ratio with the antibody is 10:

1.

7. The method for preparing nanozyme-antibody complexes based on site-directed enzyme catalytic coupling according to claim 1, characterized in that, The antibody mentioned in step (2) is an IgG antibody.

8. The method for preparing nanozyme-antibody complexes based on site-directed enzyme catalytic coupling according to claim 1, characterized in that, The molar ratio of the dibenzocyclooctyne-functionalized platinum nanozyme and the azide-modified antibody in step (3) is 2:1 to 3:

1.

9. The nanozyme-antibody complex prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the nanozyme-antibody complex according to claim 9 as an enzyme-labeled antibody in enzyme-linked immunosorbent assay (ELISA).

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