Prussian blue nano-enzyme based on molecular imprinting technology, preparation method of Prussian blue nano-enzyme and application of Prussian blue nano-enzyme in detection of small extracellular vesicles

Abstract

The invention discloses a Prussian blue nano-enzyme based on a molecular imprinting technology, a preparation method of the Prussian blue nano-enzyme and application of the Prussian blue nano-enzyme to detection of small extracellular vesicles. The nano enzyme disclosed by the invention can be used for effectively enriching small extracellular vesicles (sEVs) and simultaneously realizing the detection of three proteins, namely CD20, CD19 and PD-L1. The Fe3O4-coated TiO2 magnetic beads are combined with phosphate groups in the sEVs phospholipid bilayer, and effective separation of the Fe3O4-coated TiO2 magnetic beads and a complex sample is achieved under the action of an external magnetic field. The molecularly imprinted nano enzyme PB-MIP-PEG has excellent peroxidase activity, and shows specific binding capacity to 3, 3 ', 5, 5'-tetramethyl benzidine (TMB), so that the catalytic efficiency is enhanced, and non-specific adsorption is reduced.

Description

Technical Field

[0001] This invention relates to a Prussian blue nanozyme based on molecular imprinting technology, its preparation method, and its application in the detection of small extracellular vesicles, belonging to the field of nanomaterials and biomedical nanotechnology. Background Technology

[0002] In hematologic malignancies, lymphoma, due to its significant heterogeneity, makes accurate subtyping and dynamic monitoring of treatment efficacy crucial aspects of clinical diagnosis and treatment. In recent years, small extracellular vesicles (sEVs) have attracted widespread attention as novel biomarkers for disease diagnosis. These nanoscale vesicles, secreted by cells, not only exist stably in bodily fluids such as blood and urine, but also carry various functional molecules, including specific proteins and nucleic acids, enabling them to reflect tumor burden, microenvironment characteristics, and treatment response status in real time. Notably, the expression of related proteins on sEVs has significant clinical guiding value. In B-cell lymphoma, high expression of CD20 and CD19 provides important evidence for disease subtyping and can also serve as monitoring indicators for the efficacy of targeted therapies such as rituximab; while the expression level of the immune checkpoint protein PD-L1 is directly related to tumor immune escape ability, providing guidance for the formulation of immunotherapy regimens. Therefore, quantitative detection of the levels of proteins such as CD20, CD19, and PD-L1 has important clinical guiding significance for disease subtyping, treatment efficacy evaluation, and postoperative follow-up.

[0003] Accurate quantitative analysis of proteins on sEVs relies primarily on establishing efficient separation and enrichment methods. Currently used separation techniques include ultracentrifugation, size exclusion chromatography, and polymer precipitation. While widely applied, these methods generally suffer from high equipment requirements, cumbersome and time-consuming procedures, limited separation efficiency, and insufficient specificity. Immunorecognition-based separation methods capture sEVs by targeting specific surface markers such as CD63, CD81, and CD9; however, their widespread application is limited by the high cost of antibodies and their limited capture efficiency. Considering that the membrane structure of sEVs is mainly composed of a phospholipid bilayer, titanium dioxide (TiO2), a metal oxide with strong Lewis properties, can effectively capture sEVs by forming Ti-OP coordination bonds with phosphate groups in phospholipids. Furthermore, Fe3O4@TiO2 magnetic beads, prepared by modifying TiO2 with highly paramagnetic Fe3O4, can rapidly separate captured sEVs under an external magnetic field. For quantitative analysis of proteins on sEVs, current techniques mainly employ chemiluminescence (CL), fluorescence analysis, electrochemistry (EC), and surface plasmon resonance (SPR). While each of these methods has its advantages, they generally require complex operating procedures and sophisticated instrumentation. In contrast, colorimetric sensing methods based on color changes have gained widespread attention due to their ease of operation, low cost, and intuitive results.

[0004] Peroxide-like nanozymes, as a class of inorganic nanomaterials with enzyme-mimicking catalytic functions, catalyze the interaction between substrates and chromogenic agents by mimicking the reaction mechanism of natural horseradish peroxidase (HRP), generating measurable colorimetric signals. Among various developed peroxide-like nanozymes, Prussian blue (PB) nanoparticles, with their tunable structure formed by the coordination of iron ions and cyano groups, as well as their good biocompatibility and high catalytic activity, are considered ideal colorimetric signal materials. However, PB nanozymes suffer from poor selectivity in complex systems due to the lack of specific substrate-recognizing binding sites.

[0005] Molecular imprinting (MIT) is an effective strategy for constructing three-dimensional cavities in polymer matrices that are highly complementary to template molecules in terms of size, shape, and functional groups. It features high recognition specificity, good stability, and wide applicability, and has been widely used in separation analysis, sensing and detection, and other fields.

[0006] Currently, there are no reports on the detection of small extracellular vesicles by combining PB nanozymes and molecular imprinting technology. Summary of the Invention

[0007] Objectives of this invention: The first objective is to provide a Prussian blue nanozyme based on molecular imprinting technology capable of simultaneously detecting multiple proteins on small extracellular vesicles. The second objective is to provide its preparation method and applications. The third objective is to provide a specific method for detecting proteins on small extracellular vesicles.

[0008] Technical solution: The Prussian blue nanozyme based on molecular imprinting technology described in this invention is PB-MIP-PEG, which is obtained by first constructing a molecularly imprinted polymer layer on the surface of Prussian blue PB nanoparticles and then modifying it with silane.

[0009] The method for preparing Prussian blue nanozyme according to the present invention includes the following steps: (1) PB nanoparticles were dispersed in an ethanol-water mixed solution, and 3,3',5,5'-tetramethylbenzidine was added and stirred in the dark. Then (3-aminopropyl)triethoxysilane and ethyl silicate were added and stirred. Finally, ammonia water was added and stirred to complete the polymerization and molecular imprinting process. The product was then washed with methanol-acetic acid solution until no TMB signal was detected by UV-Vis spectroscopy. The product was then washed with ethanol and deionized water to remove residual methanol and acetic acid. Finally, it was dried in vacuum to obtain PB-MIP. (2) Dissolve PB-MIP in ethanol-water solvent, add silane PEG-COOH, heat and stir; then centrifuge, collect PB-MIP-PEG product, and wash three times with deionized water to remove excess silane PEG-COOH, and finally obtain PB-MIP-PEG.

[0010] Specifically, (1) PB nanoparticles were dispersed in 5 mL of ethanol-water (V:V=49:1) mixed solution, and 5 mmol of 3,3',5,5'-tetramethylbenzidine (TMB) was added in the dark and stirred for 40 min. Then, 16 μL of (3-aminopropyl)triethoxysilane (APTES) and 8 μL of tetraethyl orthosilicate (TEOS) were added and stirred for 2 h. Finally, 8 μL of ammonia water (NH3·H2O) was added and stirred for 12 h to complete the polymerization and molecular imprinting process. The product was then washed with a methanol-acetic acid solution with a volume ratio of 9:1 until no TMB signal was detected by UV-Vis spectroscopy. The product was then washed with ethanol and deionized water to remove residual methanol and acetic acid. Finally, the product was dried in vacuum at 60 °C for 12 h to obtain PB-MIP. (2) Dissolve 20 mg of PB-MIP in 30 mL of ethanol-water (V:V = 1:1) solvent, add 40 mg of silane PEG-COOH (MW 5000 Da), stir at 55℃ for 12 h; then centrifuge at 12,000 rpm for 30 min, collect the PB-MIP-PEG product, and wash it three times with deionized water to remove excess silane PEG-COOH, and finally obtain PB-MIP-PEG.

[0011] The application of the Prussian blue nanozyme described in this invention in the preparation of reagents for detecting proteins on small extracellular vesicles.

[0012] The method for detecting proteins on small extracellular vesicles according to the present invention includes the following steps: (1) Fe3O4@TiO2 magnetic beads were co-incubated with small extracellular vesicles of different concentrations, washed, and Fe3O4@TiO2 magnetic beads / sEVs complex was obtained by magnetic separation; (2) The PB-MIP-PEG of claim 1 is coupled with the antibody of the protein to be tested, blocked, washed, and resuspended to obtain the signal tag; (3) The Fe3O4@TiO2 magnetic beads / sEVs complex was mixed with the signal tag and incubated. After washing, 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide solution were added. The absorbance value at 652 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and a standard curve between the concentration C of small extracellular vesicles and the signal value Y was plotted. (4) Incubate Fe3O4@TiO2 magnetic beads with the sample to be tested to prepare Fe3O4@TiO2 magnetic beads / sEVs complex. Then mix and incubate with signal tag, wash, place in NaAc-HAc buffer, add 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide solution, measure absorbance at 652 nm using microplate reader, and input into the standard curve in step (3) to obtain the concentration of the protein to be tested.

[0013] Furthermore, the amount of Fe3O4@TiO2 magnetic beads used in steps (1) and (4) is 0.9-1.9 mg, and the incubation time is 4-12 min.

[0014] Further, the protein to be tested in step (2) includes one or more of CD20, CD19 or PD-L1.

[0015] Furthermore, the concentration of the signal tag mentioned in steps (3) and (4) is 1-3 mg / mL.

[0016] Furthermore, when the signal tag is coupled to a CD20 antibody, the linear relationship between the concentration C of small extracellular vesicles and the signal value Y is: Y = 0.117 * LogC sEVs -0.322.

[0017] Furthermore, when the signal tag is coupled to a CD19 antibody, the linear relationship between the concentration C of small extracellular vesicles and the signal value Y is: Y = 0.174 * LogC sEVs -0.518.

[0018] Furthermore, when the signal tag is coupled to a PD-L1 antibody, the linear relationship between the concentration C of small extracellular vesicles and the signal value Y is: Y = 0.204 * LogC sEVs -0.609.

[0019] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention discloses a molecularly imprinted PB-MIP-PEG nanozyme, which can effectively enrich sEVs and simultaneously detect three proteins: CD20, CD19, and PD-L1. Fe3O4@TiO2 magnetic beads bind to the phosphate groups in the phospholipid bilayer of sEVs, achieving effective separation from complex samples under the action of an external magnetic field. The molecularly imprinted nanozyme PB-MIP-PEG possesses excellent peroxidase activity, exhibiting a specific binding ability to TMB, thereby enhancing catalytic efficiency and reducing non-specific adsorption. Attached Figure Description

[0020] Figure 1This is a flowchart illustrating the process principle of the present invention, wherein: A) signal tag synthesis; B) sensor assembly and working principle.

[0021] Figure 2 This is a characterization diagram of the morphology and size of sEVs observed under a transmission electron microscope.

[0022] Figure 3 The experimental results are shown in the figure to optimize the amount of Fe3O4@TiO2 magnetic beads used in the sEVs separation process.

[0023] Figure 4 The experimental results are shown in the figure, which are used to optimize the incubation time during the separation of sEVs.

[0024] Figure 5 The image shows the morphology and size of PB-MIP-PEG as observed under a transmission electron microscope.

[0025] Figure 6 Characterization diagram of the elemental composition of PB-MIP-PEG analyzed by energy dispersive spectroscopy.

[0026] Figure 7 For PB, PB-MIP and PB-MIP-PEG a nano Value comparison chart.

[0027] Figure 8 The figure shows the experimental results of TMB adsorption capacity of PB, PB-MIP and PB-MIP-PEG.

[0028] Figure 9 Comparison diagrams of colorimetric signals when PB, PB-MIP, and PB-MIP-PEG are used as signal labels.

[0029] Figure 10 To prepare Prussian blue nanozymes based on molecular imprinting technology and to detect them in small extracellular vesicles, a standard curve of CD20 on sEVs was obtained by colorimetric analysis.

[0030] Figure 11 To prepare Prussian blue nanozymes based on molecular imprinting technology and to detect them in small extracellular vesicles, a standard curve of CD19 on sEVs was obtained by colorimetric analysis.

[0031] Figure 12 To prepare Prussian blue nanozymes based on molecular imprinting technology and to apply them to the detection of small extracellular vesicles, a standard curve of PD-L1 on sEVs was obtained by colorimetric analysis.

[0032] Figure 13Figure 1 shows the results of CD20, CD19 and PD-L1 analysis on plasma sEVs from healthy individuals and lymphoma patients to illustrate the preparation of Prussian blue nanozymes based on molecular imprinting technology and their application in the detection of small extracellular vesicles. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0034] Example 1: Optimization of conditions for capturing extracellular vesicles with Fe3O4@TiO2 magnetic beads 1. Reagents and equipment Fe3O4@TiO2 magnetic beads (thick-shelled, concentration 25 mg / mL) were purchased from Nanjing Dongna Biotechnology Co., Ltd. PKH26 dye, centrifuge, ultra-high speed centrifuge, and magnetic rack were also provided.

[0035] 2. Implementation Methods (1) Obtaining small extracellular vesicles: The collected Farage cell culture medium was centrifuged sequentially at 4℃. The specific steps are as follows: First, centrifuge at 300 g for 10 min to remove cells; then centrifuge at 2000 g for 10 min to remove cell debris; then centrifuge at 10,000 g for 30 min to further remove impurities; then centrifuge at 100,000 g for 70 min at 4℃, collect the precipitate, wash the precipitate with PBS buffer, and centrifuge again at 100,000 g for 70 min; finally, disperse the obtained sEVs in 500 μL PBS buffer and store them as sEVs standards at -80℃ for subsequent experiments.

[0036] (2) The process of Fe3O4@TiO2 magnetic beads capturing small extracellular vesicles: Two samples with a concentration of 1.8×10⁻⁶ were prepared. 7 For parallel samples of sEVs (sEVs per μL), 10 μL of each sample was labeled with PKH26 dye. For sample I, the fluorescence signal intensity of the labeled sEVs was directly measured and recorded as F0. For sample II, the labeled sEVs were mixed with 1.5 mg of Fe3O4@TiO2 magnetic beads and incubated at 37℃ for 9 min. After magnetic separation, the fluorescence signal intensity of the supernatant was measured and recorded as F1. The capture efficiency was calculated by the difference in fluorescence signals between sample I and sample II, and was directly proportional to the difference. The calculation formula is as follows:

[0037] (3) Condition optimization: The effects of the amount of Fe3O4@TiO2 magnetic beads (0.1-1.9 mg) and the incubation time (1-12 min) on the capture efficiency were also studied.

[0038] 3. Implementation Results like Figure 2 As shown, extracellular vesicles observed by transmission electron microscopy exhibit a typical disc-shaped morphology, with a diameter ranging from 50 to 200 nm. Figure 2 To optimize capture efficiency, the amount of Fe3O4@TiO2 magnetic beads was determined by staining with PKH26. Figure 3 ) and incubation time ( Figure 4 The results showed that the Fe3O4@TiO2 magnetic beads achieved the highest capture efficiency for sEVs when the amount of Fe3O4@TiO2 magnetic beads was 1.5 mg and the incubation time was 9 min.

[0039] Example 2: Preparation and specific activity measurement of PB, PB-MIP and PB-MIP-PEG nanozymes ( a nano ) 1. Reagents and equipment Polyvinylpyrrolidone (PVP), potassium ferricyanide (K3[Fe(CN)6]), hydrochloric acid (HCl), (3-aminopropyl)triethoxysilane (APTES), 3,3',5,5'-tetramethylbenzidine (TMB), ethyl silicate (TEOS), ammonia (NH3·H2O), methanol, acetic acid, hydrogen peroxide (H2O2), stirrer, centrifuge, vacuum drying oven, microplate reader, water bath.

[0040] 2. Implementation Methods (1) Preparation of PB: 3 g PVP and 131.9 mg K3[Fe(CN)6] were added to 40 mL of 0.01 M HCl solution and stirred continuously until the solution was clear and transparent; then the reaction system was placed under constant temperature of 80℃ and stirred for 20 h; after the reaction was completed, the blue solid product was collected by centrifugation at 12,000 rpm for 30 min, the solid was washed several times with anhydrous ethanol and deionized water, and finally dried under vacuum at 60℃ for 12 h to obtain PB.

[0041] (2) Preparation process of PB-MIP: PB-MIP was prepared by sol-gel method. First, PB was dispersed in 5 mL of ethanol-water (V:V=49:1) mixed solvent; then 5 mmol TMB was added under light-protected conditions and stirred for 40 min; then 16 μL APTES and 8 μL TEOS were added and stirred for 2 h; then 8 μL NH3·H2O was added and stirred for 12 h to complete the polymerization and molecular imprinting process; then the product was washed with methanol-acetic acid solution with a volume ratio of 9:1 until no TMB signal was detected by UV-Vis spectrum, and then washed with ethanol and deionized water to remove residual methanol and acetic acid. Finally, it was dried in vacuum at 60°C for 12 h to obtain PB-MIP.

[0042] (3) Preparation process of PB-MIP-PEG: 20 mg of PB-MIP was dissolved in 30 mL of ethanol-water (V:V = 1:1) solvent, and 40 mg of silane PEG-COOH (MW 5000 Da) was added. The mixture was stirred at 55℃ for 12 h. Then, the product was centrifuged at 12,000 rpm for 30 min, and the PB-MIP-PEG product was collected. The product was washed three times with deionized water to remove excess silane PEG-COOH.

[0043] (4) Determination of specific activity of nanozymes: 0.0001 mg, 0.0002 mg, 0.00025 mg, 0.00035 mg and 0.0005 mg of PB, PB-MIP and PB-MIP-PEG nanozyme samples were weighed and mixed with 10 μL of 10 mg / mL TMB solution, 20 μL of 30% H2O2 solution and 190 μL of 0.2 M NaAc-HAc buffer solution at pH 3.6. The absorbance of the system at 652 nm was then measured to calculate the specific activity.

[0044] 3. Implementation Results like Figure 5 As shown, transmission electron microscopy revealed that PB-MIP-PEG exhibits a cubic structure with a rough surface. Energy dispersive spectroscopy analysis confirmed that the PB-MIP-PEG material contains C, N, O, Fe, and Si elements, with Si uniformly coated on the PB surface, forming a thin SiO2 layer. Figure 6 The activity results of nanozymes are as follows: Figure 7 As shown, PB, PB-MIP, and PB-MIP-PEG a nano The values ​​were 10.73, 16.46, and 12.92 U·mg, respectively. -1 This indicates that molecular imprinting technology significantly improves the catalytic activity of PB-MIP. Notably, silane PEG-COOH modification reduces the catalytic activity of PB-MIP-PEG, which may be due to the steric hindrance effect of the PEG molecular chain leading to a decrease in catalytic efficiency.

[0045] Example 3: Comparative test of the adsorption performance of PB, PB-MIP and PB-MIP-PEG nanozymes for TMB 1. Reagents and equipment NaAc-HAc buffer (pH 3.6), TMB, UV spectrophotometer, centrifuge.

[0046] 2. Implementation Methods TMB (100 μM) and different nanozymes (0.4 mg·mL) were added. –1 The sample was incubated in NaAc-HAc buffer (0.2 M, pH 3.6). Samples were taken at 0, 1, 5, 10, 15, 20, 40, 60, 80, 100, and 120 min, and then centrifuged (12000 rpm, 10 min) to obtain the supernatant. The UV-Vis absorption spectrum of TMB in the supernatant was measured to determine the adsorption amount. The adsorption amount of TMB was calculated according to the following formula:

[0047] Among them, Q t (mg·g -1 ) represents the adsorption amount of TMB at time t, C0 (mg·g) -1 C represents the concentration of TMB at time 0. t (mg·g -1 ) represents the concentration of TMB at time t, V (L) represents the solution volume, and m (g) represents the mass of the nanozyme.

[0048] 3. Implementation Results Compared to PB, PB-MIP's adsorption capacity for TMB increases over time, reaching 95% of its equilibrium adsorption capacity (Qe) within 40 minutes. Figure 8 During the rapid adsorption phase, the abundant specific recognition sites in PB-MIP promote the rapid binding of TMB, while these sites gradually become saturated during the slow adsorption phase. The Qe of PB-MIP modified with silane PEG-COOH is reduced, possibly due to the steric hindrance effect between PEG molecular chains, thus leading to a decrease in catalytic efficiency.

[0049] Example 4: Comparison of colorimetric signals when PB, PB-MIP, and PB-MIP-PEG nanozymes are used as signal tags. 1. Reagents and equipment NaAc-HAc buffer (pH 6.0), CD20 antibody purchased from Xinda Biopharmaceutical (Suzhou) Co., Ltd., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), bovine serum albumin (BSA), CD19 antibody purchased from Sinocare Biotechnology (Beijing) Co., Ltd., and PD-L1 antibody purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0050] 2. Implementation Methods (1) Preparation method of signal tags: PB, PB-MIP, and PB-MIP-PEG were conjugated with CD20 antibody / CD19 antibody / PD-L1 antibody using the EDC / NHS chemical cross-linking method. The simplified steps are as follows: 2.6 mg of PB, PB-MIP, or PB-MIP-PEG was dispersed in 500 μL of deionized water, and 100 μL of EDC / NHS (0.1 mol / L, V:V=1:4) was added. The mixture was stirred at room temperature for 30 min. Then, 500 μL of CD20 antibody, CD19 antibody, or PD-L1 antibody with a concentration of 52 μg / mL was added, and the mixture was stirred at 4℃ for 12 h to complete covalent conjugation. Then, 300 μL of BSA (0.5 wt%) was added to the system, and the mixture was stirred at 4℃ for 2 h to block non-specific binding sites. Finally, the obtained signal tags were centrifuged and resuspended in 500 μL of NaAc-HAc buffer (pH 6.0) and stored at 4℃ for later use.

[0051] (2) Comparison of colorimetric signals generated by three signal tags: 1.5 mg Fe3O4@TiO2 magnetic beads were mixed with 10 μL of 1.8×10⁻⁶ magnetite. 7 The Fe3O4@TiO2 magnetic bead / sEVs standard solution was mixed and incubated at 37°C for 9 min, with gentle shaking to ensure complete binding. The Fe3O4@TiO2 magnetic bead / sEVs complex was then magnetically separated and enriched using a magnetic rack, and washed with 0.1 M NaAc-HAc buffer (pH 6.0) to remove unbound sEVs. Next, 20 μL of signal tags conjugated with different antibodies (i.e., the PB, PB-MIP, or PB-MIP-PEG signal tags conjugated with CD20, CD19, or PD-L1 antibodies prepared above) were added to the resulting complex, and incubated at 37°C for 35 min. After incubation, the Fe3O4@TiO2 magnetic bead / sEVs / signal tag complex was separated using a magnetic rack, and washed with NaAc-HAc buffer to remove unbound signal tags. The complex was then placed in a NaAc-HAc buffer solution at pH 4.8, and TMB (10 mg / mL, 10 μL) substrate and H2O2 (30%, 20 μL) were added. After reacting for 10 min, the absorbance at 652 nm was measured using a microplate reader.

[0052] 3. Implementation Results Colorimetric analysis results ( Figure 9The results show that, compared to unmodified PB and PB-MIP, PB-MIP-PEG exhibits a significantly enhanced characteristic absorption peak intensity as a signal tag. This enhancement stems from the fundamental difference in their surface functional groups: the carboxyl group at the end of PB-MIP-PEG, after activation by EDC / NHS, can form a stable amide bond with the primary amino group of the antibody molecule, thereby achieving directional and robust antibody coupling. In contrast, the PB surface is dominated by ferricyanide ions and lacks active functional groups that can efficiently bind to antibodies. The physical adsorption of PVP on its surface results in weak binding force, easy detachment, and easy masking of the antibody-antigen binding domain during random adsorption, leading to signal inactivation and instability. Studies also indicate that the PB-MIP surface similarly lacks the necessary functional groups for efficient coupling, and alternative coupling strategies have inherent limitations, making it difficult to achieve stable antibody immobilization. In summary, PB-MIP-PEG demonstrates superior signal intensity and stability in colorimetric detection, reflecting its significant advantages as a signal tag.

[0053] Example 5: The analytical performance of this biosensor was evaluated by using a Prussian blue nanozyme based on molecular imprinting technology, its preparation method, and its application in the detection of small extracellular vesicles.

[0054] 1. Reagents and equipment Fe3O4@TiO2 magnetic beads were purchased from Nanjing Dongna Biotechnology Co., Ltd., along with NaAc-HAc buffer (pH 6.0), extracellular vesicles, an ELISA reader, and a magnetic rack.

[0055] 2. Implementation Methods (1) Construction of biosensors: 1.5 mg of Fe3O4@TiO2 magnetic beads were mixed with sEVs diluted to different concentrations and incubated at 37°C for 9 min, with gentle shaking to ensure full binding. The Fe3O4@TiO2 magnetic beads / sEVs complex was then magnetically separated and enriched using a magnetic rack, and washed with 0.1 M NaAc-HAc buffer (pH 6.0) to remove unbound sEVs. Next, 20 μL of a CD20-conjugated PB-MIP-PEG signal tag at a concentration of 2 mg / mL was added to the resulting complex and incubated at 37°C for 35 min. After incubation, the Fe3O4@TiO2 magnetic beads / sEVs / signal tag complex was separated using a magnetic rack and washed with NaAc-HAc buffer to remove unbound signal tags. The complex was then placed in a NaAc-HAc buffer solution at pH 4.8, and TMB (10 mg / mL, 10 μL) substrate and H2O2 (30%, 20 μL) were added. After reacting for 10 min, the absorbance at 652 nm was measured using a microplate reader. For the detection of CD19 and PD-L1, the sensor assembly process was the same as described above, except that a PB-MIP-PEG signal tag coupled to CD19 or PD-L1 was used instead of the CD20 signal tag.

[0056] (2) Performance evaluation: Under optimal conditions, the performance of the biosensor was evaluated; as the concentration of sEVs increased, PB-MIP-PEG exhibited excellent peroxidase-like activity, catalyzing the generation of TMBox from TMB, which led to changes in absorbance value; the signal data was read using an enzyme-linked immunosorbent assay (ELISA) reader, and a standard curve between the concentration of extracellular vesicles C and the signal value Y was plotted.

[0057] 3. Implementation Results like Figure 10 As shown, when PB-MIP-PEG modified with CD20 antibody is used as a signal tag, the concentration of sEVs is 9 × 10⁻⁶. 3 ~1.8×10 7 At a concentration of 1000 sEVs / μL, the absorbance value showed a good linear relationship with the sEV concentration, and the linear relationship equation was Y = 0.117 * LogC. sEVs -0.322.

[0058] like Figure 11 As shown, when PB-MIP-PEG modified with CD19 antibody is used as a signal tag, the concentration of sEVs is 4.5 × 10⁻⁶. 3 ~1.8×10 7 At a concentration of 1000 sEVs / μL, the absorbance value showed a good linear relationship with the sEV concentration, and the linear relationship equation was Y = 0.174 * LogC. sEVs-0.518.

[0059] like Figure 12 As shown, when PB-MIP-PEG modified with PD-L1 antibody is used as a signal tag, the concentration of sEVs is 3×10⁻⁶. 3 ~1.8×10 7 At a concentration of 1000 sEVs / μL, the absorbance value showed a good linear relationship with the sEV concentration, and the linear relationship equation was Y = 0.204 * LogC. sEVs -0.609.

[0060] Example 6: Verification of the detection capability of a Prussian blue nanozyme based on molecular imprinting technology, its preparation method, and its application in detecting small extracellular vesicles in actual samples.

[0061] 1. Reagents and equipment Fe3O4@TiO2 magnetic beads were purchased from Nanjing Dongna Biotechnology Co., Ltd., along with NaAc-HAc buffer (pH 6.0), plasma samples, an ELISA reader, and a magnetic rack.

[0062] 2. Implementation Methods (1) Processing of plasma samples: Plasma samples were obtained from healthy donors and lymphoma patients at Zhongda Hospital Affiliated to Southeast University. Cells and cell debris were first removed by low-speed centrifugation, and then larger extracellular vesicles were filtered out using a 0.22 μm filter.

[0063] (2) Capture and detection of sEVs: 1.5 mg Fe3O4@TiO2 magnetic beads were mixed with 20 μL of filtered plasma sample and incubated in a 37℃ constant temperature shaker for 9 min to capture sEVs in the plasma. The mixture was then transferred to a magnetic separation device to achieve solid-liquid separation, and the supernatant was discarded. NaAc-HAc buffer was added and washed twice to remove unbound impurities. 20 μL of signal tag (i.e., the PB-MIP-PEG signal tag prepared for detecting the three proteins as described in Example 4) was added to the Fe3O4@TiO2 magnetic bead / sEVs complex and incubated in a 37℃ constant temperature shaker for 35 min to form the Fe3O4@TiO2 magnetic bead / sEVs / signal tag complex. The complex was then washed twice with NaAc-HAc buffer to remove unbound signal tags. The complex was then placed in a pH 4.8 NaAc-HAc buffer solution, and 10 μL of 0.1 mg / mL TMB substrate solution and 20 μL of 30% TMB substrate solution were added. H2O2 solution. After reacting for 10 min, the colorimetric signal intensity was detected using an ELISA reader, and the concentration of small extracellular vesicles in the sample was quantitatively analyzed using the standard curve in Example 5.

[0064] 3. Implementation Results Clinical plasma samples were analyzed using this biosensor. Figure 13 As shown, CD20 ( ) on sEVs in the lymphoma group Figure 13 A), CD19 ( Figure 13 B) and PD-L1 ( Figure 13 C) The colorimetric signal intensity corresponding to the protein was generally higher than that of the healthy group (P<0.001), indicating that the sensor can effectively distinguish between lymphoma patients and healthy individuals.

Claims

1. A Prussian blue nanozyme based on molecular imprinting technology, characterized in that, The Prussian blue nanozyme is PB-MIP-PEG, which is obtained by first constructing a molecularly imprinted polymer layer on the surface of Prussian blue nanoparticles and then modifying it with silane.

2. The method for preparing Prussian blue nanozyme according to claim 1, characterized in that, Includes the following steps: (1) PB nanoparticles were dispersed in an ethanol-water mixed solution, and 3,3',5,5'-tetramethylbenzidine was added and stirred in the dark. Then (3-aminopropyl)triethoxysilane and ethyl silicate were added and stirred. Finally, ammonia water was added and stirred to complete the polymerization and molecular imprinting process. The product was then washed with methanol-acetic acid solution until no TMB signal was detected by UV-Vis spectroscopy. The product was then washed with ethanol and deionized water to remove residual methanol and acetic acid. Finally, it was dried in vacuum to obtain PB-MIP. (2) Dissolve PB-MIP in an ethanol-water solvent, add silane PEG-COOH, and heat and stir; The product was then centrifuged, collected, and washed three times with deionized water to remove excess silane PEG-COOH, thus obtaining PB-MIP-PEG.

3. The use of the Prussian blue nanozyme according to claim 1 in the preparation of reagents for detecting proteins on small extracellular vesicles.

4. A method for detecting proteins on small extracellular vesicles, characterized in that, Includes the following steps: (1) Fe3O4@TiO2 magnetic beads were co-incubated with small extracellular vesicles of different concentrations, washed, and Fe3O4@TiO2 magnetic beads / sEVs complex was obtained by magnetic separation; (2) The PB-MIP-PEG of claim 1 is coupled with the antibody of the protein to be tested, blocked, washed, and resuspended to obtain the signal tag; (3) The Fe3O4@TiO2 magnetic beads / sEVs complex was mixed with the signal tag and incubated. After washing, it was placed in NaAc-HAc buffer, and 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide solution were added. The absorbance value at 652 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and a standard curve between the concentration C of small extracellular vesicles and the signal value Y was plotted. (4) Incubate Fe3O4@TiO2 magnetic beads with the sample to be tested to prepare Fe3O4@TiO2 magnetic beads / sEVs complex. Then mix and incubate with signal tag, wash, add 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide solution, measure absorbance at 652 nm using microplate reader, and input into the standard curve in step (3) to obtain the concentration of the protein to be tested.

5. The method according to claim 4, characterized in that, The amount of Fe3O4@TiO2 magnetic beads used in steps (1) and (4) is 0.9-1.9 mg, and the incubation time is 4-12 min.

6. The method according to claim 4, characterized in that, The protein to be tested in step (2) includes one or more of CD20, CD19 or PD-L1.

7. The method according to claim 4, characterized in that, The concentration of the signal tag mentioned in steps (3) and (4) is 1-3 mg / mL.

8. The method according to claim 4, characterized in that, When the signal tag is coupled to a CD20 antibody, the linear relationship between the concentration C of small extracellular vesicles and the signal value Y is: Y = 0.117 * LogC sEVs -0.

322.

9. The method according to claim 4, characterized in that, When the signal tag is coupled to a CD19 antibody, the linear relationship between the concentration C of small extracellular vesicles and the signal value Y is: Y = 0.174 * LogC sEVs -0.

518.

10. The method according to claim 4, characterized in that, When the signal tag is coupled to a PD-L1 antibody, the linear relationship between the concentration C of small extracellular vesicles and the signal value Y is: Y = 0.204 * LogC sEVs -0.609.

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