A sandwich colorimetric assay method based on epitope-based molecularly imprinted polymers and nanoscale enzyme

Abstract

The application relates to a sandwich colorimetric analysis method based on an epitope-based molecular imprinting polymer and a nanoenzyme. The sandwich method is used to ensure high specificity and high sensitivity of target analyte detection through the synergistic effect of a capture antibody and a labeled antibody, and is particularly suitable for the analysis of glycoproteins in serum. Boric acid can form a reversible covalent bond with a compound containing cis-diol under a higher pH condition. The combination is more stable than electrostatic interaction, hydrogen bond and other non-covalent bonds, and is very suitable for recognizing glycoproteins containing cis-diol structures.

Description

Technical Field

[0001] This application relates to the fields of biomimetic molecular recognition materials and molecular imprinting technology, and in particular to a sandwich colorimetric analysis method based on epitope-imprinted polymers and nanozymes. Background Technology

[0002] Glycoproteins are an indispensable part of the protein family, accounting for more than 50% of the total protein in the human body. They are formed by the covalent linkage of oligosaccharides and polypeptides, playing a crucial role in physiological systems, encompassing intercellular interactions, immune responses, and tumor development. In life sciences, medical research, and clinical diagnostics, glycoproteins are of great significance as biomarkers and therapeutic targets. Abnormal glycosylation patterns of proteins are closely related to the occurrence and development of many diseases, such as cancer, autoimmune diseases, cardiovascular diseases, and neurodegenerative diseases. Currently, the vast majority of tumor markers monitored clinically are glycoproteins. For example, alpha-fetoprotein (AFP) is the preferred marker for primary liver cancer and is often used in the early screening of hepatocellular carcinoma; carcinoembryonic antigen (CEA) is a broad-spectrum tumor marker; CA15-3 is an important marker for breast cancer; and prostate-specific antigen (PSA) is a specific marker for prostate cancer. These glycoprotein markers are the foundation for achieving early tumor diagnosis. However, glycoproteins are usually present in low concentrations in the human body and coexist with a large number of high concentrations of cellular material, non-glycoproteins, peptides, small molecules and other interfering substances, which greatly hinders the sensitive detection of glycoproteins.

[0003] Mass spectrometry and high-performance liquid chromatography (HPLC) are powerful analytical tools for detecting glycoproteins. However, these devices require specialized instruments and skilled operators, resulting in high detection costs and unsuitability for the point-of-care testing of glycoprotein biomarkers. Furthermore, while antibodies and lectins offer unique advantages in recognizing glycoproteins, antibody preparation is complex and costly, and antibodies suffer from poor stability and reproducibility. Lectins, as affinity tools for glycans, can recognize specific glycan structures, but their specificity and affinity remain relatively limited. Therefore, developing alternative methods with selective recognition, rapid and sensitive response, and low cost for detecting trace amounts of glycoproteins in biological samples is essential.

[0004] Enzyme-linked immunosorbent assay (ELISA) is a highly sensitive immunological technique that combines the specific reaction of antigens and antibodies with the highly efficient catalytic action of enzymes on substrates. The double-antibody sandwich method utilizes two antibodies targeting different epitopes to detect antigens. However, antibodies and enzymes face challenges in practical applications, such as difficulty in obtaining them and demanding operating conditions. To address the issue of insufficient antibody availability and reduce detection costs, researchers are dedicated to developing alternatives to capture and labeled antibodies.

[0005] Molecularly imprinted polymers (MIPs), often used as "artificial antibodies," can selectively recognize and bind to target substances. MIPs prepared by imprinting protein epitopes can achieve specific capture of target proteins, showing broad application prospects in the field of biosensing.

[0006] Nanozymes, as nanomaterials, can mimic the activity of natural enzymes. As a promising alternative to natural enzymes, nanozymes are well-suited for colorimetric detection due to their high catalytic efficiency and good stability. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a sandwich colorimetric analysis method based on epitope-imprinted polymers and nanozymes.

[0008] This invention combines the specific recognition ability of MIPs with the enzyme-like catalytic ability of nanozymes to establish a sandwich colorimetric analysis method for AFP. Figure 1The N-terminal nonapeptide of AFP was screened as an epitope. Using boron affinity immobilization, the glycosylated epitope was directionally immobilized on the surface of a capillary glass tube, preparing epitope-based microinjections (MIPs) to specifically extract AFP from complex matrices. Then, a Mn3O4-Au nanozyme with oxidase activity was prepared as a signal tag, and its glycosyl recognition ability was endowed by modification with 4-mercaptophenylboronic acid (4-MPBA). During glycoprotein assays, the epitope MIPs specifically captured target glycoproteins in complex matrices; simultaneously, the captured glycoproteins were labeled with the 4-MPBA-modified Mn3O4 nanozyme, forming a sandwich structure of MIPs / AFP / nanozyme. This nanozyme can directly catalyze the oxidation of the substrate 3,3',5,5'-tetramethylbenzidine (TMB) to a blue product, enabling colorimetric detection. Finally, the method was compared with the traditional ELISA method, demonstrating the reliability of the established sandwich colorimetric method in the analysis of human serum samples.

[0009] The first object of the present invention is to provide a composition.

[0010] A second object of the present invention is to provide the use of any of the compositions in the preparation of a detection kit for the target protein or in the establishment of a method for detecting the target protein for non-diagnostic purposes.

[0011] A third objective of this invention is to provide a detection kit for a target protein.

[0012] The fourth objective of this invention is to provide a method for detecting target proteins for non-diagnostic purposes.

[0013] To achieve the above objectives, the present invention is implemented through the following technical solution: The present invention first claims a composition comprising an epitope-based molecularly imprinted polymer and a nanozyme; The epitope-based molecularly imprinted polymer is obtained by using the terminal polypeptide of the target protein as a characteristic epitope, the glycosylated polypeptide obtained after glycosylation as an imprint template, and selecting different types and proportions of monomeric silanizing reagents according to the amino acid type of the epitope sequence for non-covalent imprinting. The thickness of the imprint layer is precisely imprinted by adjusting the imprinting time, resulting in a molecularly imprinted polymer. The nanozyme is a boric acid-functionalized Mn3O4-Au nanozyme, which is a Mn3O4 nanozyme with Au nanoparticles deposited on its surface.

[0014] Preferably, the epitope-based molecularly imprinted polymer is coated onto a boric acid-functionalized substrate material via boron affinity.

[0015] In one or more embodiments, the target protein is AFP, the characteristic epitope is the N-terminal nonapeptide of AFP, and the monomeric silanizing agent is 3-aminopropyltriethoxysilane, 3-ureopropyltriethoxysilane, benzyltriethoxysilane, isobutyltriethoxysilane, and tetraethyl orthosilicate, in a molar ratio of 18–22:18–22:9–11:18–22:27–33.

[0016] More preferably, the molar ratio of 3-aminopropyltriethoxysilane, 3-ureopropyltriethoxysilane, benzyltriethoxysilane, isobutyltriethoxysilane and tetraethyl orthosilicate is 20:20:9-11:18-22:27-33.

[0017] Preferably, the imprinting time is 45 to 55 minutes.

[0018] More preferably, the imprinting time is 50 minutes.

[0019] Preferably, the substrate material is gold-plated glass, magnetic nanomaterials, a single-layer gold nanoparticle self-assembled glass sheet, or silver nanomaterials.

[0020] In one specific embodiment, the substrate material is a gold-plated capillary glass tube.

[0021] Preferably, the monosaccharide combined in the saccharification process is fructose.

[0022] Preferably, the substituted boric acids used in the boric acid functionalization process include 4-formylphenylboronic acid, 2,4-difluoro-3-formylphenylboronic acid, aldehyde phenylboronic acid, aminophenylboronic acid, carboxyphenylboronic acid, mercaptophenylboronic acid, and alkenylphenylboronic acid.

[0023] In one specific embodiment, the substituted boric acid used in the boric acid functionalization process is 4-formylphenylboronic acid.

[0024] As a specific embodiment, the method for preparing the epitope-based molecularly imprinted polymer coated onto a boric acid-functionalized substrate material via boron affinity is as follows: S1. The substrate material is immersed in a methanol solution containing substituted boric acid and sodium cyanoborohydride for full reaction, thoroughly cleaned and dried to obtain a borate-treated substrate material; S2. The borate-treated substrate material is immersed in an ammonium bicarbonate buffer solution of glycosylated peptides and reacted fully. After thorough washing, it is dried to obtain a borate-treated substrate material with glycosylated peptides immobilized. S3. Immerse the boronized substrate material immobilized with glycosylated peptides in a mixture containing anhydrous ethanol, ammonia and water, then mix it with anhydrous ethanol solution containing monomer silanizing reagent, allow it to react fully, wash it thoroughly and dry it to obtain the final product.

[0025] As a specific embodiment, the preparation method of nanozymes is as follows: A hydrothermal reaction is carried out in an ethanol solution of manganese acetate, the product is collected, thoroughly washed, and dried to obtain Mn3O4 nanoparticles; Mn3O4 nanoparticles are thoroughly dispersed in an organic solvent, thoroughly mixed with HAuCl4, then mixed with an aqueous solution of sodium borohydride, reacted thoroughly, thoroughly washed, and dried to obtain Mn3O4-Au nanoparticles; Mn3O4-Au nanoparticles are dispersed in ethanol, mixed with an ethanol solution containing substituted boric acid, reacted thoroughly, thoroughly washed, and dried to obtain the final product.

[0026] Preferably, Mn3O4-Au nanoparticles are dispersed in ethanol and mixed with an ethanol solution containing substituted boric acid, wherein the concentration of substituted boric acid in the system is (1 / 5.2) to (1 / 5.0) mmol / L.

[0027] More preferably, Mn3O4-Au nanoparticles are dispersed in ethanol and mixed with an ethanol solution containing substituted boric acid, wherein the concentration of substituted boric acid in the system is (1 / 5.1) mmol / L.

[0028] Preferably, the Mn3O4 nanoparticles are fully dispersed in an organic solvent and thoroughly mixed with HAuCl4, with the concentration of HAuCl4 in the system being (1 / 6.5) to (1 / 7.5) g / L.

[0029] As a specific implementation method, Mn3O4 nanoparticles are fully dispersed in an organic solvent and thoroughly mixed with HAuCl4. The concentration of HAuCl4 in the system is (1 / 7) g / L.

[0030] Preferably, the composition further contains 3,3',5,5'-tetramethylbenzidine.

[0031] The present invention also claims the use of any of the described compositions in the preparation of a detection kit for the target protein or in the establishment of a method for detecting the target protein for non-diagnostic purposes.

[0032] The present invention also claims a detection kit for a target protein, comprising any of the compositions described herein.

[0033] The present invention also claims a method for detecting a target protein for non-diagnostic purposes, using any of the compositions described herein, wherein an epitope-based molecularly imprinted polymer is mixed with and fully reacted with the sample, washed, then mixed with a nanozyme and fully reacted, washed again, reacted with TMB, and the absorbance value is measured at a wavelength of 652 nm.

[0034] Compared with the prior art, the present invention has the following beneficial effects: (1) Glycosylated alpha-fetoprotein N-terminal nonapeptide was immobilized on the surface of a capillary glass tube, and MIPs based on AFP epitopes were prepared. This enabled the capture and enrichment of AFP from complex samples.

[0035] (2) Mn3O4-Au nanozymes with oxidase activity were prepared as signal tags. The nanozymes were further modified with 4-MPBA by borylation. This functionalization not only preserved the enzyme-like catalytic function of the nanozymes, but also endowed the nanozymes with the ability to recognize protein glycosyl groups.

[0036] (3) The developed sandwich colorimetric method showed good linearity for AFP in the range of 1 to 100 ng / mL, with a detection limit of 0.78 ng / mL, and demonstrated good stability and anti-interference ability. The method was used to detect AFP in human blood samples and showed good accuracy.

[0037] (4) Based on the specific capture ability of epitope MIPs and the enzyme-like catalytic effect of nanozymes, the formed epitope MIPs / AFP / nanozyme structure has good specificity. This detection strategy based on epitope MIPs and nanozymes not only enhances the specificity of the analytical method, but also effectively reduces the detection cost.

[0038] This invention utilizes a sandwich method, leveraging the synergistic effect of capture and labeled antibodies to ensure high specificity and sensitivity for the detection of target analytes, making it particularly suitable for the analysis of glycoproteins in serum. Boric acid, under high pH conditions, can form reversible covalent bonds with compounds containing cis-diols. This binding is more stable than non-covalent bonds such as electrostatic interactions and hydrogen bonds, making it highly suitable for recognizing glycoproteins containing cis-diol structures.

[0039] Compared to existing conventional ELISA methods, this invention does not require the use of natural antibodies as the recognition medium for the target protein. The recognition medium used in this invention is epitope-based MIPs and borate-functionalized Mn3O4Au nanozymes, which exhibit unique advantages in cost and stability compared to natural antibodies and enzymes, reducing reagent storage requirements. Conventional ELISA methods require long detection times and cumbersome procedures. In contrast, the method of this invention effectively saves analysis time and improves detection efficiency. Attached Figure Description

[0040] Figure 1 A schematic diagram illustrating the principle of the colorimetric method for establishing AFP-based epitope MIPs and boric acid-functionalized Mn3O4-Au nanozymes.

[0041] Figure 2This is a schematic diagram of the synthesis of MIPs based on AFP epitopes.

[0042] Figure 3 This is a schematic diagram illustrating the synthesis of boric acid-functionalized Mn3O4-Au nanozymes.

[0043] Figure 4 TEM images of Mn3O4 nanozymes; (a) TEM image; (b) high-resolution TEM image.

[0044] Figure 5 TEM images of Mn3O4-Au nanozymes; (a) TEM image; (b) high-resolution TEM image; (c) elemental distribution.

[0045] Figure 6 XPS characterization of Mn3O4-Au nanozymes: (a) XPS spectrum of Mn3O4-Au nanozymes; (b) XPS spectrum of Mn 2p in Mn3O4-Au nanozymes; (c) XPS spectrum of O 1s in Mn3O4-Au nanozymes; (d) XPS spectrum of Au4f in Mn3O4-Au nanozymes.

[0046] Figure 7 The images show the full-wavelength UV-Vis absorption spectra of different reaction systems.

[0047] Figure 8 Effects of different reaction conditions on the activity of Mn3O4-Au nanoparticles (n=3): (a) pH value; (b) reaction time; (c) TMB concentration; (d) nanozyme concentration.

[0048] Figure 9 The effects of different detection conditions (n ​​= 3); (a) extraction time; (b) labeling time.

[0049] Figure 10 The standard linear curve for AFP (n = 3).

[0050] Figure 11 For the specificity test of the sandwich colorimetric analysis method (n = 3).

[0051] Figure 12 To evaluate the performance of the sandwich colorimetric analysis method (n = 3).

[0052] Figure 13 The results of AFP detection in serum samples (n = 3). Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0054] Example 1: Preparation of molecularly imprinted polymers based on alpha-fetoprotein epitopes (1) Amination treatment of glass capillary (introduction of amino functional groups) A glass capillary tube (0.9–1.1 mm in diameter) sealed at one end was repeatedly rinsed with deionized water, then vertically immersed in a 1 mol / L sodium hydroxide solution for 8 h, followed by washing with large amounts of deionized water and ethanol. The treated glass capillary tube was then placed at 150 °C for 1 h to activate the silanol groups on its surface. Subsequently, the activated glass capillary tube was vertically immersed in a 4% (v / v) ethanol solution of 3-aminopropyltriethoxysilane and incubated at room temperature for 12 h. Afterward, the glass capillary tube was washed repeatedly with ethanol and dried at 100 °C for 2 h.

[0055] (2) Gold plating treatment of glass capillary An aqueous solution containing 12 mmol / L HAuCl4, 0.5 mol / L potassium bicarbonate, and 25 mmol / L glucose was prepared as the gold plating solution. The amination-treated glass capillary was immersed in the gold plating solution and placed at 35 °C for 5 h, forming a thin gold film on the surface of the glass capillary. Subsequently, the treated glass capillary was washed three times with deionized water and anhydrous ethanol, respectively, and stored at room temperature.

[0056] (3) Boricating treatment of glass capillary The gold-plated glass capillary was vertically inserted into a 4% (v / v) ethanol solution of 3-aminopropyltriethoxysilane and incubated at room temperature for 2 h. The capillary was then repeatedly rinsed with anhydrous ethanol and deionized water to remove unreacted 3-aminopropyltriethoxysilane. Subsequently, the capillary was immersed in a methanol solution containing 5.0 mg / mL 4-formylphenylboronic acid and 1 mg / mL sodium cyanoborohydride and incubated at room temperature for 24 h. The solution was changed three times during this period to prevent solvent evaporation. After incubation, the capillary was rinsed with deionized water and ethanol, and then dried under vacuum at 40 °C for 12 h. The treated capillary was stored at 4 °C to obtain a boron-treated gold-plated glass capillary for subsequent use. The boric acid on the surface of the boron-treated capillary covalently interacts with the glycosyl groups of the glycoprotein, resulting in a boron affinity reaction.

[0057] (4) Immobilization of glycosylated epitopes on gold-plated glass capillaries Fructose-modified alpha-fetoprotein (with the N-terminal sequence RTLHRNEYG, denoted as Fru-RTLHRNEYG, synthesized by Shanghai Taopu Biotechnology Co., Ltd.) was dissolved in ammonium bicarbonate buffer (50 mmol / L, pH 8.5) to prepare a 1.0 mg / mL solution. The gold-plated glass was immersed in the fructose-modified epitope solution and incubated at room temperature for 2 h. After incubation, the glass capillary was rinsed three times with ammonium bicarbonate buffer (50 mmol / L, pH 8.5) to obtain a gold-plated glass capillary with immobilized glycosylated epitopes.

[0058] (5) Preparation of epitope-imprinted polymers First, 3 mL of anhydrous ethanol, 90 μL of ammonia, and 2 mL of water were mixed to obtain mixture 1. Next, 40 μL of 3-aminopropyltriethoxysilane (CAS No. 919-30-2), 80 μL of 3-ureopropyltriethoxysilane (CAS No. 116912-64-2), 20 μL of benzyltriethoxysilane (CAS No. 2549-99-7), 40 μL of isobutyltriethoxysilane (CAS No. 17980-47-1), and 60 μL of tetraethyl orthosilicate (CAS No. 78-10-4) (molar ratio 20:20:10:20:30) were dissolved in 40 mL of anhydrous ethanol to obtain mixture 2. Subsequently, the gold-plated glass capillary with fixed glycosylated epitopes was immersed in 120 μL of mixture 1, and then 32 μL of mixture 2 was added. The mixture was shaken and reacted at 25 °C for 50 min.

[0059] After the reaction was complete, the resulting glass capillary was washed three times with ethanol to remove unreacted monomers. Then, the capillary was immersed in a mixed solution of acetonitrile, water, and acetic acid (50:49:1, volume ratio) for 3 h at room temperature to remove template molecules. Afterward, it was washed three times with ethanol and deionized water to obtain glass capillary tubes coated with molecularly imprinted polymers (MIPs) based on alpha-fetoprotein epitopes, which were stored at 4 °C.

[0060] In addition, control NIPs were prepared. The preparation process was the same as that of MIPs, but no template molecules were fixed on the glass capillary, that is, step "(4) Fixation of glycosylated epitopes on gold-plated glass capillary" was not performed.

[0061] Example 2: Preparation of boric acid-functionalized Mn3O4-Au nanozymes (1) Preparation of Mn3O4 nanoparticles 0.75 g of manganese acetate (Mn(CH3COO)2·4H2O) was dissolved in 30 mL of ethanol by stirring for 30 min until the manganese acetate was completely dissolved. Then, the manganese acetate solution was added to a polytetrafluoroethylene liner. The mixture was heated to 120 °C in a high-pressure reactor and maintained at this temperature for 24 h. After the reaction was complete, the product was allowed to cool naturally to room temperature. The product was collected by centrifugation, and the resulting Mn3O4 nanoparticles were washed multiple times with deionized water and ethanol. Finally, the Mn3O4 nanoparticles were vacuum dried overnight at 60 °C to obtain Mn3O4 nanoparticles.

[0062] (2) Preparation of Mn3O4-Au nanozymes To deposit Au nanoparticles on the surface of Mn3O4, 20 mg of Mn3O4 nanoparticles were first ultrasonically dispersed in 6 mL of tetrahydrofuran, followed by the addition of 1 mL of 0.1% HAuCl4 solution. After stirring at room temperature for 2 h, 2 mL of 0.53 mol / L sodium borohydride aqueous solution was rapidly added, and stirring was continued for 12 h. After the reaction was complete, the product Mn3O4-Au nanozyme was collected by centrifugation. The obtained Mn3O4-Au nanozyme was washed several times with deionized water and ethanol. Finally, the obtained Mn3O4-Au nanozyme was vacuum dried overnight at 60 °C to obtain Mn3O4-Au nanoparticles.

[0063] (3) Preparation of boric acid-functionalized Mn3O4-Au nanozymes Two mg of Mn3O4-Au nanozyme was sonicated for a period of time to disperse it in 5 mL of ethanol. Then, 0.1 mL of a 10 mmol / L ethanol solution of 4-mercaptophenylboronic acid was added, and the mixture was slowly stirred at room temperature for 2 h. After the reaction was complete, the product was collected by centrifugation, washed sequentially with ethanol, and redispersed in 2 mL of PBS (0.01 mol / L, pH 7.4) to obtain borate-functionalized Mn3O4-Au nanozyme.

[0064] Example 3 Morphological characterization of Mn3O4-Au nanozymes I. Experimental Methods The morphology of the Mn3O4 nanoparticles and Mn3O4-Au nanozymes prepared in Example 2 was analyzed using TEM technology.

[0065] II. Experimental Results See results Figure 4 , Figure 4 a and Figure 4 Figure b shows the morphology of the Mn3O4 nanoparticles, with an average particle size of approximately 12 nm.

[0066] Au was further deposited on the surface of Mn3O4 nanoparticles to obtain Mn3O4-Au nanozymes, the TEM results of which are as follows. Figure 5 As shown in a, the overall size of the Mn3O4-Au nanozyme did not change significantly.

[0067] Further analysis of the Mn3O4-Au nanoparticles was performed using high-resolution TEM, and the obtained images are as follows. Figure 5 As shown in b, the average particle size of Au nanoparticles in Mn3O4-Au nanoparticles is 5 nm, and the lattice spacing of Au nanoparticles is 0.376 nm.

[0068] Example 4: Elemental composition and distribution of Mn3O4-Au nanozymes I. Experimental Methods The elemental composition and distribution of the prepared Mn3O4-Au nanoparticles were analyzed using TEM combined with EDS; the elemental composition of the Mn3O4-Au nanoparticles was analyzed using X-ray photoelectron spectroscopy (XPS). The specific methods are as follows: II. Experimental Results The results are as follows Figure 5 As shown in c in the figure, the Mn3O4-Au nanozyme is composed of Mn, O, and Au elements, all of which are distributed on the surface of the nanomaterial. EDS analysis results show that the contents of Mn, O, and Au are approximately 42.2%, 57.5%, and 0.3%, respectively.

[0069] XPS results of Mn3O4-Au nanoparticles are as follows: Figure 6 As shown in Figure 'a', the spectral peaks exhibiting Mn 2p, O 1s, C 1s, and Au 4f are displayed. This indicates that the nanoparticles are mainly composed of Mn, O, C, and Au elements. Further peak fitting was performed on the spectral peaks of Mn 2p, O 1s, C 1s, and Au 4f.

[0070] The peak separation results of Mn element are as follows Figure 6 As shown in b, Mn 2p 3 / 2 and Mn 2p 1 / 2 The binding energies of the peaks are located at 641.2 eV and 653.1 eV, with a doublet spacing of 11.9 eV, indicating that Mn exists in its oxidized state. Furthermore, Mn 2p 3 / 2 The peak deconvolution resulted in three sets of peaks with binding energies of 643.4 eV, 641.8 eV, and 640.5 eV, corresponding to Mn, respectively. 4+ Mn 3+ and Mn 2 + Price state.

[0071] The peak separation results of element O are as follows: Figure 6 As shown in c, the XPS spectrum of O element can be fitted to three main oxygen components. The peak with a binding energy of 529.8 eV is attributed to lattice oxygen, the intermediate peak with a binding energy of 531.3 eV corresponds to low-coordinated defect oxygen sites, and the peak with a binding energy of 532.5 eV originates from water molecules adsorbed on the material surface.

[0072] The peak separation results of Au element are as follows: Figure 6 As shown in d, the peaks with binding energies at 87.7 eV and 84.1 eV are attributed to Au 4f in the Mn3O4-Au nanozyme. 7 / 2 and Au 4f 5 / 2 Characteristic peaks.

[0073] Based on the above component analysis and morphological characterization, these results demonstrate the successful preparation of Mn3O4-Au nanozymes.

[0074] Example 5: Mn3O4-Au nanoparticles possess oxidase-mimicking activity. I. Experimental Methods 5 μL of the dispersion of Mn3O4-Au nanozyme obtained in Example 2 (1 mg / L) and 40 μL of TMB solution (10 mmol / L) were added to 955 μL of acetate-sodium acetate buffer (0.2 mmol / L, pH 5.0) and mixed. After the mixture was kept in the reaction at room temperature for 150 s, the UV-Vis absorption spectrum of the mixture was recorded using a UV-Vis spectrophotometer. The absorbance of the reaction solution at a wavelength of 652 nm was read to detect the oxidase activity of the Mn3O4-Au nanozyme prepared in Example 2.

[0075] II. Experimental Results The results are as follows Figure 7 As shown, under acidic conditions, when only Mn3O4-Au nanoparticles and TMB are present in the system, TMB can be directly and rapidly oxidized by the Mn3O4-Au nanoparticles to generate the oxidation product oxTMB. At this time, the solution changes from colorless to blue, and the solution is scanned across the entire wavelength range of the UV-Vis spectrum.

[0076] Ultraviolet (UV) detection revealed a distinct UV absorption peak at 652 nm, corresponding to the characteristic absorption of the TMB oxidation product. Therefore, the detection wavelength for oxTMB was set at 652 nm. In contrast, the aforementioned colorimetric reaction did not occur when only Mn3O4-Au nanoparticles or TMB were present in the system. Furthermore, a full-wavelength UV-Vis spectral scan of the solution showed no significant UV absorption peak at 652 nm. This result demonstrates that Mn3O4-Au nanoparticles possess oxidase-like activity, enabling them to catalyze the oxidation of the substrate and produce a colorimetric product without the need for hydrogen peroxide.

[0077] Example 6 Effect of different reaction conditions on the catalytic activity of Mn3O4-Au nanoparticles I. Effect of buffer pH on the catalytic activity of Mn3O4-Au nanoparticles 1. Experimental Methods 5 μL of Mn3O4-Au dispersion (1 mg / L) and 40 μL of TMB solution (10 mmol / L) were added to 955 μL of acetate-sodium acetate buffer (0.2 mmol / L, pH 5.0) and mixed. After the mixture was allowed to react at room temperature for 300 s, the UV-Vis absorption spectrum of the mixture was recorded using a UV-Vis spectrophotometer. The absorbance of the reaction solution at 652 nm was read to detect the effect of buffer pH on the oxidase activity of the Mn3O4-Au nanozyme prepared in Example 2.

[0078] 2. Experimental Results The results are as follows Figure 8As shown in Figure a, the catalytic performance of Mn3O4-Au nanozymes exhibits a phenomenon of first increasing and then decreasing within the pH range of 3.0 to 8.0. Mn3O4-Au nanozymes show higher catalytic activity under acidic conditions. The absorbance of the solution is highest and the color is deepest when the buffer solution pH is 5.0. However, the catalytic activity of Mn3O4-Au nanozymes decreases significantly under alkaline conditions. This indicates that the catalytic performance of the nanozyme is closely related to the pH of the buffer solution. Therefore, the optimal reaction pH for this nanozyme is 5.0.

[0079] II. Effect of reaction time on the catalytic effect of Mn3O4-Au nanozymes 1. Experimental Methods 5 μL of Mn3O4-Au dispersion (1 mg / L) and 40 μL of TMB solution (10 mmol / L) were added to 955 μL of acetate-sodium acetate buffer (0.2 mmol / L, pH 5.0) and mixed. The mixture was kept at room temperature, and the absorbance of the reaction solution at 652 nm was read every 10 s using a microplate reader to measure the effect of reaction time on the reaction.

[0080] 2. Experimental Results The results are as follows Figure 8 As shown in b, the absorbance value of the system increases with the gradual increase of reaction time, reaching reaction equilibrium at 150 s. Therefore, the optimal catalytic reaction time is selected as 150 s.

[0081] III. Effect of TMB concentration on the absorbance value of the system 1. Experimental Methods 5 μL of Mn3O4-Au dispersion (1 mg / L) and different volumes (1 μL, 5 μL, 10, 15, 20, 30, 40, 50, 60) of TMB solution (10 mmol / L) were mixed and then brought to a final volume of 1000 μL with acetate-sodium acetate buffer (0.2 mmol / L, pH 5.0). After incubating the mixture at room temperature for 300 s, the UV-Vis absorption spectrum of the mixture was recorded using a UV-Vis spectrophotometer. The absorbance at 652 nm was measured to determine the effect of reaction time on the TMB concentration on the absorbance value of the system.

[0082] 2. Experimental Results The results are as follows Figure 8 As shown in c, the absorbance value of the system gradually increases with the increase of TMB concentration. When the TMB concentration is 0.4 mmol / L, the catalytic reaction reaches dynamic equilibrium, and the absorbance value is basically stable. Therefore, the optimal TMB concentration is 0.4 mmol / L.

[0083] IV. Effect of Mn3O4-Au nanozyme concentration on the absorbance value of the system 1. Experimental Methods Different volumes (1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, and 10 μL) of Mn3O4-Au dispersion (1 mg / L) and 40 μL of TMB solution were mixed and then brought to a final volume of 1000 μL with acetate-sodium acetate buffer (0.2 mmol / L, pH 5.0). After the mixture was allowed to react at room temperature for 150 s, the UV-Vis absorption spectrum of the mixture was recorded using a UV-Vis spectrophotometer. The absorbance of the reaction solution at 652 nm was read to determine the effect of the concentration of the Mn3O4-Au nanozyme prepared in Example 2 on the absorbance value of the system.

[0084] 2. Experimental Results The results are as follows Figure 8 As shown in d, the absorbance value of the system gradually increases with the continuous increase of the concentration of Mn3O4-Au nanozyme. The concentration of Mn3O4-Au nanozyme and the absorbance value of the system show a good linear relationship, which indicates that the reaction rate increases with the increase of nanozyme concentration.

[0085] Example 7: The Influence of Detection Conditions on Detection Sensitivity I. The effect of protein extraction time on detection sensitivity 1. Experimental Methods First, the glass capillaries prepared in Example 1, coated with molecularly imprinted polymers (MIPs) based on alpha-fetoprotein epitopes, were immersed in AFP solutions of different concentrations (PBS, 0.01 mol / L, pH 7.4) and incubated with shaking for 5, 10, 15, 20, 25, and 30 min. After incubation, the glass capillaries were washed with PBS (0.01 mol / L, pH 7.4) to remove unbound AFP from the surface.

[0086] Subsequently, the glass capillary was placed in the boric acid-functionalized Mn3O4-Au nanozyme dispersion (PBS, 0.01 mol / L, pH 7.4) prepared in Example 2, and incubated with shaking for another 20 min. After incubation, the glass capillary was washed again with PBS (0.01 mol / L, pH 7.4) to remove unbound boric acid-functionalized Mn3O4-Au nanozymes.

[0087] Next, the glass capillary was immersed in 1 mM TMB solution (0.2 mol / L acetate-sodium acetate buffer, pH 5.0) and incubated with shaking. After the reaction was complete, the glass capillary was removed from the reaction solution. The absorbance of the reaction solution at 652 nm was measured using a microplate reader.

[0088] 2. Experimental Results The results are as follows Figure 9 As shown in Figure a, within the range of 5 to 20 min, the absorbance value of the system gradually increased with increasing extraction time, and then stabilized after 20 min. This indicates that with increasing protein extraction time, more AFP is specifically bound to the MIPs, resulting in a gradual increase in the number of labeled Mn3O4-Au nanozymes and further enhancement of catalytic activity. Therefore, the optimal extraction time was selected as 20 min.

[0089] II. The effect of protein labeling time on detection sensitivity 1. Experimental Methods First, the glass capillaries prepared in Example 1, coated with molecularly imprinted polymers (MIPs) based on alpha-fetoprotein epitopes, were immersed in AFP solutions of different concentrations (PBS, 0.01 mol / L, pH 7.4) and incubated with shaking for 20 min. After incubation, the glass capillaries were washed with PBS (0.01 mol / L, pH 7.4) to remove unbound AFP from the surface.

[0090] Subsequently, the glass capillary was placed in the boric acid-functionalized Mn3O4-Au nanozyme dispersion (PBS, 0.01 mol / L, pH 7.4) prepared in Example 2, and incubated with shaking for 5, 10, 15, 20, 25, and 30 min. After incubation, the glass capillary was washed again with PBS (0.01 mol / L, pH 7.4) to remove unbound boric acid-functionalized Mn3O4-Au nanozymes.

[0091] Next, the glass capillary was immersed in 1 mM TMB solution (0.2 mol / L acetate-sodium acetate buffer, pH 5.0) and incubated with shaking. After the reaction was complete, the glass capillary was removed from the reaction solution. The absorbance of the reaction solution at 652 nm was measured using a microplate reader.

[0092] 2. Experimental Results The results are as follows Figure 9As shown in b, within the range of 5–15 min, the absorbance value of the system gradually increases with the increase of nanozyme labeling time. This indicates that with the increase of labeling time, the number of labeled Mn3O4-Au nanozymes gradually increases, forming more and more MIPs / AFP / nanozyme sandwich structures, reaching a dynamic equilibrium after 15 min. At this point, the catalytic activity is enhanced, catalyzing the oxidation of more TMB and generating more oxTMB. Therefore, 15 min is selected as the optimal labeling time.

[0093] Example 8: A sandwich colorimetric analysis method for detecting AFP A sandwich colorimetric method for AFP detection was constructed using epitope-based microplatelets (MIPs) and boric acid-functionalized Mn3O4-Au nanozymes. The method first uses a capillary glass tube modified with epitope MIPs to specifically capture free AFP in the sample. Then, the captured AFP is labeled with boric acid-functionalized Mn3O4-Au nanozymes, forming a sandwich structure of AFP-eptope-based MIPs-AFP-boric acid-functionalized Mn3O4-Au nanozymes. Next, the capillary glass tube is immersed in a detection solution containing TMB; the labeled nanozymes directly catalyze the oxidation of the TMB substrate, generating a blue oxidation product. Finally, the absorbance at 652 nm is read using a microplate reader for colorimetric detection.

[0094] 1. Experimental Methods The performance of the sandwich colorimetric method established for the epitopes of alpha-fetoprotein (MIPs) prepared in Example 1 and the boric acid-functionalized Mn3O4-Au nanozyme prepared in Example 2 was evaluated. The specific method is as follows: Glass capillaries coated with MIPs based on alpha-fetoprotein epitopes were immersed in AFP solutions (PBS, 0.01 mol / L, pH 7.4) at different concentrations (1, 10, 20, 40, 60, 80, and 100 ng / mL) and incubated with shaking for 20 min. After incubation, the glass capillaries were washed with PBS (0.01 mol / L, pH 7.4) to remove unbound AFP from the surface. The glass capillary was placed in a 1 mg / L boric acid-functionalized Mn3O4-Au nanozyme dispersion (PBS, 0.01 mol / L, pH 7.4) and incubated with shaking for 15 min. After incubation, the glass capillary was washed again with PBS (0.01 mol / L, pH 7.4) to remove unbound borate-functionalized Mn3O4-Au nanozymes. Immerse the glass capillary in 1 mM TMB solution (0.2 mol / L acetate-sodium acetate buffer, pH 5.0) and incubate with shaking. After the reaction was complete, the glass capillary tube in the reaction solution was removed. The absorbance of the reaction solution at a wavelength of 652 nm was measured using a microplate reader.

[0095] II. Experimental Results Experimental results are as follows Figure 10 As shown, with the gradual increase of AFP protein concentration in the sample, the absorbance value gradually increases, and the color of the system gradually turns blue. This is because the more free AFP in the sample is specifically captured by the epitope MIPs on the capillary glass tube, the more Mn3O4-Au nanozymes labeled with AFP glycosyl groups are produced, ultimately enhancing the catalytic activity of the system. Therefore, the absorbance value of the system gradually increases with the AFP concentration.

[0096] The results showed that within the range of 1–100 ng / mL, the absorbance value of the system had a good linear relationship with the concentration of AFP protein, exhibiting a positive correlation. After linear fitting, the linear equation for AFP was: y = 0.00087 x + 0.074 ( R 2 =0.9685), and the detection limit was 0.78 ng / mL (S / N = 3). This indicates that the prepared AFP-based epitope-based MIPs and boric acid-functionalized Mn3O4-Au nanozymes can effectively recognize the N-terminal epitope and glycosyl group of AFP, thus enabling the specific detection of AFP through the sandwich method.

[0097] Example 9: Specificity of a sandwich colorimetric analysis method for detecting AFP I. Experimental Methods According to the sandwich colorimetric analysis method in Example 8, 200 ng / mL of interfering proteins were detected, with 100 ng / mL of AFP protein as a positive control and NIPs as a negative control.

[0098] Interfering proteins include human angiotensin-converting enzyme 2 (ACE2), human serum albumin (HSA), C-reactive protein (CRP, glycoprotein), interleukin-6 (IL-6, glycoprotein), and procalcitonin (PCT, glycoprotein).

[0099] II. Experimental Results The results are as follows Figure 11 As shown, within the 99% confidence interval, the sandwich colorimetric analysis method of Example 7 exhibited a significant difference in the response to AFP and interfering proteins. p <0.01). It exhibits a significant response to AFP, while the response to other interfering proteins is negligible. p >0.05).

[0100] Meanwhile, control experiments were conducted using NIPs. However, the sandwich assay constructed based on NIPs and boric acid-functionalized Mn3O4-Au nanozymes showed essentially the same response to all proteins, failing to effectively distinguish the target protein. This is because the N-terminal epitope of AFP was used as a template to prepare MIPs, forming an imprinted cavity complementary to the template peptide structure during polymerization. This endows the MIPs with selective recognition of the target protein AFP, while preventing the recognition of interfering proteins.

[0101] Furthermore, the imprinting factor (IF) was determined by calculating the absorbance ratio of AFP-based epitopes MIPs to NIPs, thus reflecting the selective recognition ability of the MIPs. The results showed that the analytical method achieved the highest IF value (3.7) for the target protein AFP. This excellent selectivity stems from the highest specificity of AFP-to-AFP recognition, enabling efficient enrichment of free AFP in the sample. Simultaneously, the boric acid-functionalized Mn3O4-Au nanozyme rapidly recognizes and labels the glycosyl groups of glycoproteins. The sandwich structure constructed using these two materials exhibits good specificity and selectivity for the target protein.

[0102] Example 10: Reproducibility of a sandwich colorimetric analysis method for detecting AFP I. Experimental Methods Following the methods of Examples 1 and 2, five batches of molecularly imprinted polymers based on alpha-fetoprotein epitopes and borate-functionalized Mn3O4-Au nanozymes were prepared, respectively. Subsequently, 100 ng / mL of AFP was detected using the sandwich colorimetric analysis method described in Example 8.

[0103] II. Experimental Results See results Figure 12 The results showed that the RSD of the absorbance values ​​at 625 nm in the five measurements was 2.01%, which demonstrated satisfactory reproducibility.

[0104] Example 11: Long-term stability of a sandwich colorimetric analysis method for detecting AFP I. Experimental Methods Following the methods of Examples 1 and 2, molecularly imprinted polymers based on alpha-fetoprotein epitopes and borate-functionalized Mn3O4-Au nanozymes were prepared, respectively, and stored in a 4 °C refrigerator. The absorbance of 100 ng / mL AFP was measured every 7 days according to the sandwich colorimetric analysis method of Example 8.

[0105] II. Experimental Results The results are as follows Figure 12 As shown in b, after 21 days of storage, the sandwich colorimetric method established using molecularly imprinted polymers based on alpha-fetoprotein epitopes and boric acid-functionalized Mn3O4-Au nanozymes showed little attenuation in the absorbance value of AFP, and no significant change was observed.

[0106] In summary, the results demonstrate that the sandwich colorimetric analysis method constructed in Example 7 exhibits good anti-interference ability, long-term stability, and repeatability in the detection of AFP.

[0107] Example 12: Sandwich colorimetric analysis method for detecting AFP – detection of actual samples I. Experimental Methods 50 μL of human serum samples from different volunteers were diluted to 100 μL with PBS (0.01 mol / L, pH 7.4), and AFP was measured according to the sandwich colorimetric method in Example 8.

[0108] II. Experimental Results The results showed that the AFP concentration range determined by the sandwich colorimetric analysis method in Example 8 was 25.35 ~ 68.43 ng / mL, indicating certain individual differences.

[0109] Example 13: Comparison of sandwich colorimetric analysis method for AFP detection with ELISA kit I. Experimental Methods For the same three serum samples, AFP was detected using the sandwich colorimetric analysis method described in Example 8 and the double antibody sandwich ELISA kit, respectively.

[0110] In addition, AFP solutions of different concentrations (1, 10, 20, 40, 60, 80 and 100 ng / mL) were detected using a double-antibody sandwich ELISA kit, and a standard curve was constructed to obtain its detection limit.

[0111] II. Experimental Results The results are as follows Figure 13As shown in Figure 8 and Table 1, there was no significant difference (p > 0.05) between the sandwich colorimetric analysis method of Example 8 and the ELISA method, indicating that the developed method exhibits good accuracy and reliability in actual sample testing. Compared with the ELISA method, the sandwich colorimetric analysis method of Example 7 also has the advantages of lower cost and shorter reaction time. Therefore, the sandwich colorimetric analysis method of Example 7 has good application value in practical applications.

[0112] Table 1 Comparison of the method of the present invention with the double-antibody sandwich ELISA method

Claims

1. A composition, characterized in that, It contains epitope-based molecularly imprinted polymers and nanozymes; The epitope-based molecularly imprinted polymer is obtained by using the terminal polypeptide of the target protein as a characteristic epitope, the glycosylated polypeptide obtained after glycosylation as an imprint template, and selecting different types and proportions of monomeric silanizing reagents according to the amino acid type of the epitope sequence for non-covalent imprinting. The thickness of the imprint layer is precisely imprinted by adjusting the imprinting time, resulting in a molecularly imprinted polymer. The nanozyme is a boric acid-functionalized Mn3O4-Au nanozyme, which is a Mn3O4 nanozyme with Au nanoparticles deposited on its surface.

2. The composition according to claim 1, characterized in that, The epitope-based molecularly imprinted polymer is coated onto a boric acid-functionalized substrate material via boron affinity.

3. The composition according to claim 1 or 2, characterized in that, The target protein is AFP, the characteristic epitope is the N-terminal nonapeptide of AFP, and the monomeric silanizing agent is 3-aminopropyltriethoxysilane, 3-ureopropyltriethoxysilane, benzyltriethoxysilane, isobutyltriethoxysilane, and tetraethyl orthosilicate, with a molar ratio of 18-22:18-22:9-11:18-22:27-33.

4. The composition according to claim 1, characterized in that, The monosaccharide combined in the saccharification process is selected as fructose.

5. The composition according to claim 2, characterized in that, The substituted boric acids used in the boric acid functionalization process include 4-formylphenylboronic acid, 2,4-difluoro-3-formylphenylboronic acid, aldehyde phenylboronic acid, aminophenylboronic acid, carboxyphenylboronic acid, mercaptophenylboronic acid, and alkenylphenylboronic acid.

6. The composition according to claim 2, characterized in that, The method for preparing the epitope-based molecularly imprinted polymer by coating a boric acid-functionalized substrate material with boron affinity is as follows: S1. The substrate material is immersed in a methanol solution containing substituted boric acid and sodium cyanoborohydride for full reaction, thoroughly cleaned and dried to obtain a borate-treated substrate material; S2. The borate-treated substrate material is immersed in an ammonium bicarbonate buffer solution of glycosylated peptides and reacted fully. After thorough washing, it is dried to obtain a borate-treated substrate material with glycosylated peptides immobilized. S3. Immerse the boronized substrate material immobilized with glycosylated peptides in a mixture containing anhydrous ethanol, ammonia and water, then mix it with anhydrous ethanol solution containing monomer silanizing reagent, allow it to react fully, wash it thoroughly and dry it to obtain the final product.

7. The composition according to claim 1, characterized in that, The preparation method of nanozymes is as follows: A hydrothermal reaction is carried out in an ethanol solution of manganese acetate. The product is collected, thoroughly washed, and dried to obtain Mn3O4 nanoparticles. Mn3O4 nanoparticles are then fully dispersed in an organic solvent, thoroughly mixed with HAuCl4, and then mixed with an aqueous solution of sodium borohydride. After thorough reaction, the mixture is thoroughly washed and dried to obtain Mn3O4-Au nanoparticles. Finally, Mn3O4-Au nanoparticles are dispersed in ethanol, mixed with an ethanol solution containing substituted boric acid, thoroughly reacted, thoroughly washed, and dried to obtain the final product.

8. The use of the composition according to any one of claims 1 to 7 in the preparation of a detection kit for the target protein or in the establishment of a method for detecting the target protein for non-diagnostic purposes.

9. A detection kit for a target protein, characterized in that, Contains the composition according to any one of claims 1 to 7.

10. A method for detecting a target protein for non-diagnostic purposes, characterized in that, Using the composition of any one of claims 1 to 7, the epitope-based molecularly imprinted polymer is mixed with the sample to be tested and reacted fully, washed, mixed with nanozyme and reacted fully, washed, reacted with TMB, and the absorbance value at a wavelength of 652 nm is measured.

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