Polypeptide magnetic beads for capturing pathogenic bacteria and outer vesicles thereof, and preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, and more particularly to a polypeptide magnetic ball for capturing pathogenic bacteria and outer vesicles thereof, a preparation method and application thereof. A magnetic microsphere carrier is prepared from a soluble iron salt, and then a silica functional layer magnetic ball is prepared by coating the magnetic microsphere carrier with an alkoxysilane. An amine functionalized magnetic ball is obtained by modifying the silica functional layer magnetic ball with an amino silane coupling agent. Next, the amine functionalized magnetic ball is subjected to an acid anhydride treatment to obtain a carboxyl functionalized magnetic ball, and streptavidin is covalently coupled to the surface of the carboxyl functionalized magnetic ball to obtain a streptavidin magnetic ball. After a biotin-maleimide conjugate is connected to the surface of the magnetic ball, a polypeptide is coupled to the maleimide-biotin functionalized magnetic ball to obtain the polypeptide magnetic ball for capturing pathogenic bacteria and outer vesicles thereof. The polypeptide magnetic ball comprises a magnetic microsphere carrier core and a polypeptide functional layer coated on the surface of the core, and can efficiently enrich various bacteria and outer vesicles thereof, and has good broad-spectrum capturing capacity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to polypeptide magnetic beads for capturing pathogens and their outer vesicles, their preparation methods, and applications. Background Technology

[0002] Bacterial infections and related diseases (including those related to gut microbiota imbalance) play a crucial role in clinical diagnosis, treatment monitoring, and public health control. Besides directly detecting the bacteria themselves, bacteria release bacterial extracellular vesicles (BEVs) during their growth and metabolism. BEVs carry characteristic molecules such as lipopolysaccharide (LPS) and outer membrane proteins, serving as potential information carriers reflecting bacterial status and host-microbe interactions in complex biological samples. Simultaneously, host cells also release extracellular vesicles (EVs), and both often coexist in complex matrices such as blood and feces. Therefore, when conducting pathogen and microecological related detection, challenges often arise such as low levels of target bacteria and BEVs, strong matrix interference, and the difficulty in distinguishing between BEVs and host EVs.

[0003] Current bacterial detection methods largely rely on culture and identification, molecular detection (PCR / sequencing, etc.), or immunological detection. At the sample pretreatment level, common capture or enrichment methods to improve detection sensitivity include: First, immunomagnetic beads or antibody capture, which utilizes specific antibodies or surface antigens to achieve magnetic separation. While this method offers high selectivity, it suffers from high antibody costs, stringent storage conditions, limited coverage of bacterial species or serotypes, and false negatives due to antigenic variation. Second, affinity capture using aptamers, lectins, or receptor proteins. While this method can replace some antibodies, it also suffers from target dependence, limited coverage, and stability. Third, capture based on non-specific adsorption, surface charge, or hydrophobic interactions. This method is simple to implement, but suffers from high background adsorption, poor selectivity, and insufficient enrichment purity in complex samples.

[0004] Current common methods for vesicle enrichment include ultracentrifugation, density gradient centrifugation, polymer precipitation, membrane filtration / ultrafiltration, and size exclusion chromatography. These methods mostly rely on size / density as the separation criterion and generally suffer from time-consuming operation, strong dependence on equipment, significant operator-influenced recovery rates and batch-to-batch reproducibility, and the tendency to introduce co-precipitated impurities in high-background samples such as feces. Furthermore, existing separation methods face the challenge of balancing yield and purity, and are prone to co-precipitation / co-retention of non-vesicle proteins and lipoproteins, resulting in significant differences in reproducibility between different methods. Since BEVs and host EVs highly overlap in particle size and density ranges, physical separation alone is insufficient for thorough source-level differentiation. Therefore, typical procedures for preparing BEVs from samples such as feces often require a complex, time-consuming, and highly dependent process involving multiple orthogonal steps such as filtration / SEC and density gradient ultracentrifugation, which are highly dependent on centrifugation equipment and operational consistency.

[0005] In summary, although existing technologies have methods for bacterial capture or vesicle separation, there are still technical problems that need to be solved in order to simultaneously cover multiple bacterial species and BEVs on the same platform, achieve rapid magnetic enrichment in complex samples, and minimize interference from host EVs. Summary of the Invention

[0006] The present invention aims to at least solve one of the technical problems existing in the related art. Therefore, the first objective of the present invention is to provide a method for preparing polypeptide magnetic spheres for capturing pathogens and their outer vesicles; the second objective of the present invention is to provide a polypeptide magnetic sphere for capturing pathogens and their outer vesicles; and the third objective of the present invention is to provide an application of the polypeptide magnetic sphere for capturing pathogens and their outer vesicles.

[0007] To achieve the first objective, the technical solution adopted by this invention is as follows: A method for preparing polypeptide magnetic beads that capture pathogens and their outer vesicles includes: Magnetic microsphere carriers were prepared using soluble iron salts as raw materials via a solvothermal method. Magnetic spheres coated with a silica functional layer were prepared by surface modification of the magnetic microsphere carrier with alkoxysilane. Amino-functionalized magnetic spheres were prepared by modifying the magnetic spheres coated with the silica functional layer using an aminosilane coupling agent. The amino-functionalized magnetic spheres were subjected to anhydride treatment to prepare carboxyl-functionalized magnetic spheres; Streptavidin magnetic spheres were prepared by coupling streptavidin to the surface of the carboxyl-functionalized magnetic spheres using a carbodiimide coupling agent. A biotin-maleimide conjugate was attached to the surface of the streptavidin magnetic spheres via the specific binding of biotin to streptavidin to prepare maleimide-biotin-functionalized magnetic spheres. A polypeptide containing cysteine ​​is coupled to the surface of the maleimide-biotin-functionalized magnetic sphere to prepare a polypeptide magnetic sphere for capturing pathogens and their exovesicles. The cysteine-containing polypeptide is selected from any of the following structural formulas: , , , , , , , .

[0008] Preferably, the soluble iron salt is selected from any one of FeCl3·6H2O, Fe(NO3)3·9H2O, FeCl2·4H2O, and FeSO4·7H2O.

[0009] Furthermore, the reaction temperature of the solvothermal method is 210–230°C, the reaction time is 10–14 h, and the reaction system includes sodium acetate, sodium citrate, and ethylene glycol.

[0010] Furthermore, the alkoxysilane is selected from one of tetraethyl orthosilicate, methyl orthosilicate, propyl orthosilicate, isopropyl orthosilicate, butyl orthosilicate, and pentyl orthosilicate; The aminosilane coupling agent is selected from 3-aminopropyltriethoxysilane and 3-aminopropyltrimethoxysilane.

[0011] Furthermore, the carbodiimide coupling agent is selected from one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, dicyclohexylcarbodiimide, and diphenylcarbodiimide.

[0012] To achieve the second objective, the technical solution adopted by this invention is as follows: The polypeptide magnetic spheres that capture pathogens and their outer vesicles are prepared using the method described above. The polypeptide magnetic spheres include a magnetic microsphere carrier core and a polypeptide functional layer coating the outer surface of the core.

[0013] To achieve the third objective, the technical solution adopted by this invention is as follows: Application of peptide magnetic spheres for capturing pathogens and their outer vesicles, wherein the application involves constructing a capture platform using the peptide magnetic spheres for capturing pathogens and their outer vesicles, the capture platform being used to capture one or more of the following targets: Gram-positive bacteria, Gram-negative bacteria, Candida albicans, Gram-positive bacterial vesicles, Gram-negative bacterial vesicles, and Candida albicans vesicles.

[0014] Furthermore, the Gram-positive bacterium is Staphylococcus aureus; The Gram-negative bacteria are any one of Escherichia coli, Salmonella enterica, Shigella dysenteriae, Citrobacter freundii, Morganella morganii, and Proteus vulgaris.

[0015] Furthermore, the capture time of the capture platform is 30 to 150 minutes.

[0016] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: This invention provides polypeptide magnetic spheres for capturing pathogens and their exovesicles, their preparation method, and applications. Using soluble iron salts as raw materials, magnetic microsphere carriers are prepared via a solvothermal method. Subsequently, alkoxysilanes are used to coat these carriers to prepare silica-functionalized magnetic spheres, which are further modified with aminosilane coupling agents to obtain amino-functionalized magnetic spheres. Next, the amino-functionalized magnetic spheres are anhydride-treated to convert them into carboxyl-functionalized magnetic spheres. Streptavidin is covalently coupled to the surface of these spheres using a carbodiimide coupling agent to obtain streptavidin magnetic spheres. Finally, utilizing the specific binding of biotin to streptavidin, a biotin-maleimide conjugate is attached to the surface of the magnetic spheres. A cysteine-containing polypeptide is then coupled to the maleimide-biotin-functionalized magnetic spheres, ultimately yielding polypeptide magnetic spheres for capturing pathogens and their exovesicles. These polypeptide magnetic spheres comprise a magnetic microsphere carrier core and a polypeptide functional layer coating the outer surface of the core. The polypeptide magnetic beads provided by this invention were used in capture experiments on various bacteria. The results showed that in pure bacterial solutions, the polypeptide magnetic beads could achieve efficient enrichment of various bacteria, with capture rates exceeding 90%, indicating that the polypeptide magnetic beads have good broad-spectrum bacterial capture ability. Furthermore, in an artificially mixed bacterial solution of 100 CFU / mL, the polypeptide magnetic beads still achieved a capture rate of approximately 90%, indicating that the polypeptide magnetic beads are suitable for enriching low-abundance target bacteria. To evaluate its resistance to interference in complex backgrounds, this invention further constructed an artificial simulated blood system; even in the presence of blood components, the polypeptide magnetic beads maintained a bacterial capture rate of over 90%, proving that the polypeptide magnetic beads can work stably in the complex blood matrix and can serve as a universal magnetic separation platform for bacterial detection, enrichment, or pretreatment.

[0017] This invention, building upon the proven efficiency of peptide magnetic beads in capturing pathogenic bacterial cells, further investigated and verified the capture performance of these peptide magnetic beads on bacterial extracellular vesicles (BEVs). Experimental results showed that the BEV capture signal reached its optimal level at an incubation time of approximately 90 minutes. This time window balances BEV capture efficiency with experimental operational efficiency, making it suitable for constructing standardized BEV capture procedures.

[0018] Building upon this foundation, this invention further validates the selective enrichment ability of peptide magnetic beads for BEVs in a complex background of host-derived extracellular vesicles (EVs). This invention constructs a mixed vesicle model, using *E. coli*-derived BEVs to simulate bacterial vesicles and human 5637 cell line-derived EVs to simulate host-derived vesicles. The capture effect is qualitatively and quantitatively evaluated using the bacterial BEV-specific marker LPS and the host EV-specific marker CD9, respectively. In a 1:1 ratio of BEVs to host EVs, the LPS-positive signal of the peptide magnetic bead capture product is significantly stronger, confirming that it preferentially enriches bacterial BEVs, rather than indiscriminately co-capturing host EVs. To further simulate the differences in BEV abundance in different clinical samples, this invention adjusts the ratio of BEVs to host EVs from 1:99 to 99:1 in a multi-gradient capture experiment. The results show that when the proportion of BEVs in the system is not less than 30:70, the peptide magnetic beads still stably exhibit a selective enrichment advantage for BEVs. The above experiments demonstrate that the polypeptide magnetic spheres can not only achieve efficient capture of BEVs in a single system, but also specifically enrich bacterial-derived vesicles in a complex context where a large number of host EVs coexist, effectively reducing the risk of source confusion caused by non-specific co-capture of host EVs.

[0019] Finally, this invention employs fecal samples from healthy adults for validation experiments to evaluate the applicability and anti-interference capability of the capture platform constructed using peptide magnetic spheres in real, complex biological samples. Fecal samples are extremely complex, containing various interfering components such as food residue, host cell debris, gut microbiota, host-derived EVs, and bacterial-derived BEVs, thus fully validating the adaptability of the capture platform to complex biological matrices. This invention utilizes peptide magnetic spheres to enrich and capture vesicle components in healthy adult fecal samples. The differences in sample components before and after capture are compared and analyzed using various characterization techniques, including transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Experimental results confirm that the capture platform can stably and preferentially enrich bacterial-derived BEVs in complex fecal matrices. In summary, the capture platform constructed using peptide magnetic spheres is not only suitable for capturing BEVs in artificially simulated systems but also enables efficient pretreatment and enrichment of BEVs in real clinical fecal samples, providing reliable sample pretreatment technology support for gut-related gut microbiota research and downstream molecular detection.

[0020] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0021] Figure 1 The Fourier transform infrared (FTIR) spectra of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-NH2, Fe3O4@SiO2-NH2-COOH and Fe3O4@SiO2-NH2-COOH@SA provided in Example 1 of this invention are shown.

[0022] Figure 2 These are the FTIR images of MB, MB-Biotin-Mal, and different MB-Biotin-Mal-Pep provided in Embodiment 1 of the present invention.

[0023] Figure 3 The diagrams provided in Embodiment 1 of this invention show the particle size and potential of MB, MB-Biotin-Mal, and different MB-Biotin-Mal-Pep particles; wherein, Figure A is a bar chart of particle size of different magnetic spheres, and Figure B is a bar chart of potential of different magnetic spheres.

[0024] Figure 4The figures show the physicochemical characterization of MB, MB-Biotin-Mal, and MB-Biotin-Mal-Pep provided in Embodiment 1 of the present invention; wherein, Figure A shows the hysteresis loops of different magnetic spheres, Figure B shows the differential scanning calorimetry (DSC) of different magnetic spheres, Figure C shows the thermogravimetric analysis (TGA) of different magnetic spheres, and Figure D shows the X-ray diffraction (XRD) of different magnetic spheres.

[0025] Figure 5 This is a high-performance liquid chromatogram of the synthetic polypeptide provided in Example 1 of the present invention.

[0026] Figure 6 This is the mass spectrum of the synthetic polypeptide provided in Example 1 of the present invention.

[0027] Figure 7 These are confocal images of the interaction between the FITC-modified peptide provided in Example 1 of this invention and bacteria and cells, respectively; wherein, Image A is a confocal image of peptide Spep4 after co-incubation with bacteria, and Image B is a confocal image of peptide Spep4 after co-incubation with cells.

[0028] Figure 8 This invention demonstrates the capture of bacteria and fungi by the polypeptide magnetic beads provided in Example 2 at different times; wherein, Figure A is a plate colony imaging diagram, and Figure B is a quantitative bar chart corresponding to Figure A.

[0029] Figure 9 This invention verifies the capture of Escherichia coli, Staphylococcus aureus, and Candida albicans by MB and MB-Biotin-Mal-SPep4 provided in Example 2 within 30 minutes; wherein, Figure A is a plate colony imaging image, and Figure B is a quantitative bar chart corresponding to Figure A.

[0030] Figure 10 This invention provides an example of how magnetic beads modified with different polypeptides captured low and high concentrations of Escherichia coli, Staphylococcus aureus, and Candida albicans within 30 minutes. Figure A shows the low-concentration plate colony image, and Figure B shows the high-concentration plate colony image.

[0031] Figure 11 This invention provides Example 4 of the test on the capture of low and high concentrations of *Escherichia coli*, *Citrobacter freundii*, *Morganella morganii*, and *Proteus vulgaris* by magnetic balls modified with different polypeptides within 30 minutes. Figure A shows the capture of low concentrations of *Escherichia coli*, *Citrobacter freundii*, *Morganella morganii*, and *Proteus vulgaris* by the polypeptide magnetic balls, while Figure B shows the capture of high concentrations of *Escherichia coli*, *Citrobacter freundii*, *Morganella morganii*, and *Proteus vulgaris* by the polypeptide magnetic balls.

[0032] Figure 12 This is an example of the binding of the polypeptide magnetic beads provided in Example 4 of this invention to different bacteria.

[0033] Figure 13 This is a gel electrophoresis (SDS-PAGE) image of MB-Biotin-Mal-SPep4 capturing Escherichia coli, Citrobacter freundii, Salmonella, and Proteus vulgaris, provided in Test Example 4 of this invention.

[0034] Figure 14 This is the case of MB-Biotin-Mal-LPep4 provided in Test Example 5 of the present invention capturing pathogens in a simulated blood environment.

[0035] Figure 15 This is the original titration curve of heat power-time provided in Test Example 6 of the present invention.

[0036] Figure 16 This is the enthalpy change (ΔH)-molar ratio fitting curve provided in Example 6 of the present invention.

[0037] Figure 17 This is a confocal image of the interaction between bacterial exovesicles and FITC-modified polypeptides provided in Example 6 of this invention.

[0038] Figure 18 This is a characterization of the combination of MB-Biotin-Mal-Spep4 and BEVs provided in Example 6 of this invention; Figure A is a TEM image of MB-Biotin-Mal-Spep4 and MB-Biotin-Mal-Spep4-BEVs; Figure B is a SEM image of MB-Biotin-Mal-Spep4 and MB-Biotin-Mal-Spep4-BEVs; Figure C is a SEM-EDS elemental distribution map of MB-Biotin-Mal-Spep4-BEVs; Figure D is a co-localization and confocal image of MB-Biotin-Mal-Spep4-BEVs; Figure E is a potential map of MB, MB-Biotin-Mal-Spep4 and MB-Biotin-Mal-Spep4-BEVs; Figure F is a dot pattern of MB, MB-Biotin-Mal-Spep4 and MB-Biotin-Mal-Spep4-BEVs. Figure G shows the Western blot results of MB, MB-Biotin-Mal-Spep4 and MB-Biotin-Mal-Spep4-BEVs, and Figure H shows the nanoparticle tracking analysis (NTA) results of MB-Biotin-Mal-Spep4 and MB-Biotin-Mal-Spep4-BEVs.

[0039] Figure 19This is an SDS-PAGE image of MB-Biotin-Mal-Spep4 binding to Escherichia coli and Staphylococcus aureus provided in Test Example 7 of this invention.

[0040] Figure 20 This is a statistical table of quantitative data for MB-Biotin-Mal-Spep4-BEVs protein blots and dot blots provided in Example 8 of this invention; wherein, Figure A shows the quantitative data results of concentration-optimized Dot Blot, Figure B shows the quantitative data results of time-optimized Western Blot, and Figure C shows the quantitative data results of concentration-optimized Western Blot.

[0041] Figure 21 The results of selective quantitative analysis of magnetic sphere capture in the equal proportion BEVs / host EVs mixed system provided in Example 9 of this invention are shown in Figure A, which shows the gray values ​​of BEVs marker LPS and host EVs marker CD9 and the corresponding capture rates after capture by 50–200 μL gradient volumes of MB-Biotin-Mal-SPep4 magnetic spheres. Figure B shows the gray values ​​of LPS and CD9 and the capture rate of the parallel validation group with a concentration of 200 μL magnetic spheres.

[0042] Figure 22 This is a bar chart showing the peptide magnetic ball capture efficiency under different particle ratios of BEVs / host EVs mixed systems provided in Example 10 of this invention.

[0043] Figure 23 The above are the characterization results of the crude extract of total fecal vesicles provided in Example 11 of the present invention; wherein, Figure A is a TEM image of the outer vesicles in the fecal sample, Figure B is an NTA image of the outer vesicles in the fecal sample, and Figure C is a Western blotting image of BEVs and EVs in the fecal sample.

[0044] Figure 24 This invention demonstrates the specific capture effect of the polypeptide magnetic ball provided in Example 11 on BEVs and host EVs in fecal samples; wherein, Figure A is a protein imprint and gray quantification before and after polypeptide magnetic ball capture, and Figure B is a scatter bar chart of polypeptide magnetic ball capture rate. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0046] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0047] Example 1 The preparation of polypeptide magnetic beads for capturing pathogens and their outer vesicles includes the following steps: I. Magnetic microsphere carriers (denoted as Fe3O4) were prepared using soluble iron salts as raw materials via a solvothermal method. The specific process is as follows: Take a round-bottom flask, add 60 mL of ethylene glycol, then add 2.2 g of FeCl3·6H2O, 3.6 g of sodium acetate, and 0.5 g of anhydrous sodium citrate to the round-mouth flask and stir to dissolve, obtaining a uniform yellow solution; transfer the solution to a high-pressure reactor and react at 220 °C for 12 h. After the reaction is complete, cool naturally to room temperature to obtain a black solution; separate the black solution using a magnetic separator, wash it 6 times alternately with ethanol and ultrapure water, and then uniformly disperse it in 10 mL of anhydrous ethanol to obtain an ethanol dispersion of Fe3O4. Take 1 mL of the dispersion and dry it to obtain Fe3O4 (84 mg).

[0048] II. Surface modification of Fe3O4 with a silica-coated magnetic sphere (denoted as Fe3O4@SiO2) was performed using alkoxysilanes via the sol-gel method. The specific process is as follows: Take 75 μL of Fe3O4 (containing approximately 40 mg) ethanol dispersion and add it to a three-necked flask. Then add 6 mL of concentrated ammonia, 80 mL of H2O, and 320 mL of ethanol. After the addition is complete, sonicate for 10 min to ensure uniform dispersion of Fe3O4. After sonication, add 288 μL of tetraethyl orthosilicate (TEOS) dropwise. After the addition is complete, stir the mixture magnetically at 50 °C and 500 rpm for 12 h. Collect the product using a magnetic separator. Wash the collected product 6 times with anhydrous ethanol and disperse it in 10 mL of anhydrous ethanol to obtain Fe3O4@SiO2 ethanol dispersion.

[0049] III. Amine-functionalized magnetic spheres (denoted as Fe3O4@SiO2-NH2) were prepared by modifying Fe3O4@SiO2 with an aminosilane coupling agent. The specific process is as follows: The Fe3O4@SiO2 ethanol dispersion was added to a three-necked flask, and the supernatant was removed after magnetic separation. Then, 32 mL of a mixed solvent of acetone and water (volume ratio 1:1) was added, and the pH was adjusted to 3-4 by adding 0.5 mol / L HCl solution dropwise. The mixture was then activated by magnetic stirring at 50°C and 500 rpm for 1 h. Under an argon atmosphere, 1.35 mL of 3-aminopropyltriethoxysilane (APTES) was added, and the mixture was reacted by magnetic stirring at 50°C for 18 h. After the reaction was completed, the supernatant was discarded after magnetic separation, and the product was washed 6 times with anhydrous ethanol and dispersed in 10 mL of anhydrous ethanol to obtain the Fe3O4@SiO2-NH2 ethanol dispersion.

[0050] IV. Anhydride treatment of Fe3O4@SiO2-NH2 was performed to prepare carboxyl-functionalized magnetic spheres (denoted as Fe3O4@SiO2-NH2-COOH). The specific process is as follows: The Fe3O4@SiO2-NH2 ethanol dispersion was solvent-replaced to remove ethanol and then re-dispersed ultrasonically in 32 mL of anhydrous N,N-dimethylformamide (DMF). This dispersion was then mixed with 8 mL of anhydrous DMF solution containing 2 g of succinic anhydride. After ultrasonic treatment for 10 min, the mixture was magnetically stirred at 50 °C and 500 rpm for 24 h. After the reaction, the product was washed alternately with anhydrous DMF and ultrapure water for a total of 6 washes. After washing, the magnetic particles were dispersed in 5 mL of ultrapure water to obtain a Fe3O4@SiO2-NH2-COOH dispersion. 1 mL of this dispersion was dried in an oven to obtain solid Fe3O4@SiO2-NH2-COOH.

[0051] V. Streptavidin (SA) was coupled to the surface of Fe3O4@SiO2-NH2-COOH using a carbodiimide coupling agent to prepare streptavidin magnetic spheres (denoted as Fe3O4@SiO2-NH2-COOH@SA, abbreviated as MB). The specific process is as follows: Take 1.2 mg Fe3O4@SiO2-NH2-COOH and place it in a 1.5 mL centrifuge tube. Wash four times with 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 5.5) (500 μL each time, magnetic separation, water removal). After washing, add 500 μL of MES buffer containing 22.5 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 13.5 mg N-hydroxysuccinimide (NHS) to the collected precipitate. Shake at 25 °C and 300 rpm for 15 min (activate carboxyl groups). After shaking, remove the activation solution after magnetic separation, add 250 μL of MES buffer containing 25 μg SA, and react at 28 °C and 200 rpm for 6 h. Then, add 500 μL of 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Tris-HCl (pH 8.0) was shaken for 30 min to block unreacted active esters. The particles were washed three times with washing buffer (W-buffer) and once with H-buffer (H-buffer) (500 μL each time for magnetic separation). Finally, the magnetic particles were resuspended in 1 mL of H-buffer to obtain the MB dispersion.

[0052] The composition of H-buffer: using ultrapure water as solvent, containing 20mM Tris-HCl, 750mM NaCl, 5mM KCl, and 5mM MgCl2; The composition of W-buffer is as follows: ultrapure water is used as the solvent, containing 7mM Tris-HCl, 5mM KCl, 5mM MgCl2, and 0.05% Tween-20.

[0053] FTIR spectra of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-NH2, Fe3O4@SiO2-NH2-COOH, and Fe3O4@SiO2-NH2-COOH@SA, as shown Figure 1 As shown.

[0054] VI. A maleimide-biotin functionalized magnetic bead (denoted as MB-Biotin-Mal) was prepared by attaching a biotin-maleimide conjugate (Biotin-Mal) to the surface of MB via the specific binding of biotin to streptavidin. The specific process is as follows: Take 1 mL of MB magnetic beads with a concentration of 10 mg / mL, wash the surface of the magnetic beads with PBS buffer solution (pH 7.2) to obtain pretreated MB magnetic beads; weigh 1 mg of Biotin-Mal, dissolve it completely in 100 μL of dimethyl sulfoxide (DMSO), then add 2 mL of PBS buffer solution (pH 7.2), vortex to mix, and obtain Biotin-Mal working solution. Add the working solution to the aforementioned pretreated MB magnetic beads, place in a constant temperature incubator, and shake at 37℃ and 900 rpm for 1.5 h to allow Biotin to interact and bind with SA, thus preparing magnetic microspheres with maleimide active sites on the surface, namely MB-Biotin-Mal.

[0055] 7. A polypeptide containing cysteine ​​is coupled to the surface of MB-Biotin-Mal magnetic beads to prepare polypeptide magnetic beads (denoted as MB-Biotin-Mal-Pep). The specific process is as follows: Take 1 mL of MB-Biotin-Mal magnetic microspheres with a concentration of 10 mg / mL and wash the surface of the magnetic microspheres with PBS buffer (pH 7.4). Weigh 3 mg of different cysteine-containing peptides as shown in Table 1, dissolve them in 3 mL of PBS buffer solution (pH 7.4) containing 2 mM ethylenediaminetetraacetic acid (EDTA), mix thoroughly, and then add the previously washed MB-Biotin-Mal magnetic microspheres. React at room temperature for 3 h (or react overnight at 4 °C). After the reaction is completed, perform magnetic separation, wash and collect the magnetic microspheres to obtain MB-Biotin-Mal-Pep.

[0056] Table 1. Names and structural formulas of different cysteine-containing polypeptides.

[0057] The MB-Biotin-Mal-Pep prepared using Spep1, Spep2, Spep3, Spep4, Lpep1, Lpep2, Lpep3, and Lpep4 are respectively denoted as: MB-Biotin-Mal-SPep1, MB-Biotin-Mal-SPep2, MB-Biotin-Mal-SPep3, MB-Biotin-Mal-SPep4, MB-Biotin-Mal-LPep1, MB-Biotin-Mal-LPep2, MB-Biotin-Mal-LPep3, MB-Biotin-Mal-LPep4.

[0058] FTIR plots of MB, MB-Biotin-Mal, and different MB-Biotin-Mal-Pep, such as Figure 2 As shown.

[0059] The particle size and potential of MB, MB-Biotin-Mal, and different MB-Biotin-Mal-Pep particles were determined using a nanoparticle size analyzer. The results are as follows: Figure 3 As shown in the figure; where Figure A is a bar chart of particle size for different magnetic spheres, and Figure B is a bar chart of potential for different magnetic spheres.

[0060] Physicochemical characterization of MB, MB-Biotin-Mal, and MB-Biotin-Mal-Pep (MB-Biotin-Mal-SPep4), such as Figure 4 As shown in the figure; where A is the hysteresis loop of different magnetic spheres, B is the DSC diagram of different magnetic spheres, C is the TGA diagram of different magnetic spheres, and D is the XRD diagram of different magnetic spheres.

[0061] Taking the synthesis of peptide Spep2 as an example, this paper illustrates the solid-phase synthesis process of cysteine-containing peptides under Fmoc protection. Rink MBHA resin was used, following the principle of stepwise condensation from the C-terminus to the N-terminus, including resin pretreatment, deprotection, amino acid coupling, detection, cleavage, and purification steps. Details are as follows: Resin swelling pretreatment: 0.6 g of Rink MBHA resin (loading 0.352 mmol / g, Fmoc protection) was placed in a solid-phase synthesis tube with a sand core; 8-10 mL of anhydrous dichloromethane (DCM) was added to the synthesis tube to ensure that the liquid surface completely submerged the resin, and the polytetrafluoroethylene (PTFE) stopper was sealed. After swelling at 25°C and 300 rpm for 30 min, the swollen resin was obtained.

[0062] Fmoc group deprotection: Open the bottom valve of the synthesis tube, connect the vacuum pump, and filter to remove the DCM solvent; then, add 5 mL of 30% (v / v) piperidine DMF deprotection solution to the synthesis tube, seal the PTFE stopper, and shake at 25℃ and 300 rpm for 15 min (to remove the resin-attached Fmoc protecting groups). After the deprotection reaction is completed, filter and discard the solution, then wash three times with anhydrous DMF (5 mL each time, shake for 1 min and then filter), and then wash three times with anhydrous DCM (5 mL each time, shake for 1 min and then filter).

[0063] Qualitative detection of ninhydrin (to check if deprotection is complete): Dissolve 20g of phenol in 5mL of anhydrous ethanol to obtain solution A; dissolve 1g of ninhydrin in 20mL of anhydrous ethanol to obtain solution B; then, take a 500μL EP tube, add 1 drop of solution A and 1 drop of solution B respectively, and use a spoon to take a small amount of resin (about the size of a grain of rice) and put it into the EP tube. After standing in a sand bath at 120℃ for 1 minute, if the solution turns purple, it indicates that the amino group of the resin has been exposed (deprotection is complete), and the next coupling step can be carried out; if the solution is colorless or light yellow, it indicates that deprotection is incomplete, and the Fmoc deprotection-washing-detection process needs to be repeated until the solution turns purple.

[0064] Amino acid coupling: The polypeptide synthesis sequence from C-terminus to N-terminus consists of phenylalanine (Phe), diaminobutyric acid (Dab), isoleucine (Ile), diaminobutyric acid (Dab), isoleucine (Ile), diaminobutyric acid (Dab), and cysteine ​​(Cys). Taking the coupling of the first amino acid at the C-terminus, Fmoc-Phe, as an example, the subsequent amino acids (Dab→Ile→Dab→Ile→Dab→Cys) repeat this step: In a 50 mL centrifuge tube, add Fmoc-Phe (0.8 mmol, approximately 309 mg), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) (0.8 mmol, approximately 303 mg), and 6–7 mL of anhydrous DMF. Seal the tube cap and vortex for 1 min (if insoluble, sonicate for 30 s to aid dissolution). Then add 150 μL of N,N-diisopropylethylamine (DIEA), vortex again, and transfer the mixture to a synthesis tube. Seal the tube with a PTFE stopper and incubate at 25 °C and 300 rpm for 1.5–2 h. Repeat the above coupling procedure for all subsequent amino acids, including Dab, Ile, and Cys.

[0065] Washing and detection after coupling: The reaction solution in the synthesis tube was removed by filtration, and the tube was washed three times each with anhydrous DMF and anhydrous DCM (5 mL each time, shaken for 1 min and then filtered). After washing, the ninhydrin qualitative detection procedure described above was performed.

[0066] Peptide resin cleavage and dissociation: After the last amino acid (Cys) is coupled and tested to be qualified, the Fmoc deprotection, washing, and ninhydrin detection process is performed again to ensure that the N-terminal amino group of the peptide is completely deprotected. Finally, the system is washed three times alternately with anhydrous DMF and anhydrous DCM to remove residual impurities.

[0067] Preparation of peptide cleavage solution: Add 9.5 mL of trifluoroacetic acid (TFA), 250 μL of triisopropylsilane (TIPS), and 250 μL of ultrapure water to a 50 mL centrifuge tube, shake to mix well, and the peptide cleavage solution is obtained. Transfer the peptide cleavage solution to a synthesis tube, and shake at 25 °C and 300 rpm for 3 h. Filter and collect the filtrate (containing crude peptides). Place the filtrate in a fume hood and concentrate it at low temperature using a rotary evaporator to concentrate the filtrate to 1-2 mL to obtain the peptide concentrate.

[0068] Precipitate the peptides and wash and dry: Slowly add 20 mL of pre-cooled ice-cold ether (-20°C) to the peptide concentrate, vortex to mix, and then add 10,000 mL of water. g After centrifugation at 4℃ for 3 min, the supernatant of diethyl ether is discarded, and the resulting white precipitate is the crude polypeptide. Repeat the ice-cold diethyl ether precipitation-centrifugation and supernatant discarding process three times to thoroughly remove residual TFA, organic solvents, and small molecule impurities. The purified polypeptide precipitate is then dried in a 40℃ forced-air drying oven for 30 min to completely remove any remaining diethyl ether, finally yielding crude polypeptide powder.

[0069] HPLC purification: Take crude peptide powder and dissolve it in ultrapure water (if the solubility is poor, slowly adjust the pH of the system to 7-8 with dilute NaOH aqueous solution to promote complete dissolution of peptide) to prepare a peptide aqueous solution with a concentration of 1 mg / mL; filter through a 0.45 μm aqueous phase filter membrane, transfer to an HPLC injection needle, collect the fraction corresponding to the target peak, combine the collected fractions, and freeze dry them in a freeze dryer to obtain high-purity peptide Spep2 powder.

[0070] The high-performance liquid chromatography and mass spectrometry (HPLC) of Spep1, Spep2, Spep3, Spep4, Lpep1, Lpep2, Lpep3, and Lpep4 prepared according to the above method are shown in the figures below. Figure 5 and Figure 6 As shown, the above-mentioned polypeptides have been successfully synthesized.

[0071] Test Example 1 The selective recognition of bacterial membrane structures by peptides was investigated as follows: The FITC-labeled peptide Spep4 was incubated with bacterial and mammalian cells, respectively. After washing with PBS, the cells were observed using a confocal microscope. The results are as follows: Figure 7 As shown in the figure, the peptide Spep4 tends to bind to bacterial membrane-related components, laying the molecular recognition foundation for the subsequent immobilization of the peptide on the surface of the magnetic bead and the efficient capture of bacteria and bacterial extravesicles (BEVs).

[0072] Test Example 2 The capture of bacteria and fungi by polypeptide magnetic beads was investigated as follows: Using MB-Biotin-Mal-SPep4 as a representative polypeptide magnetic bead, suspensions of *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans* were incubated for 10, 20, and 30 min, respectively. After magnetic separation, plate counting was performed on the original solution and the supernatant after capture. The results are as follows: Figure 8 As shown in the figure, it can be seen that the number of residual colonies in the supernatant gradually decreases with the extension of incubation time. Quantitative statistical results confirm that the microbial capture efficiency continues to improve. The figure, represented by SPep4 peptide-modified magnetic beads, confirms that the peptide magnetic bead capture platform has excellent rapid capture performance.

[0073] A blank control group consisting of bare magnetic beads (MB) and a group of magnetic beads modified with MB-Biotin-Mal-SPep4 peptide were set up. Under the same incubation conditions, *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans* were captured, respectively. After magnetic separation, plate counting was performed on the original solution and the supernatant after capture. The results are as follows: Figure 9 As shown in the figure, the number of live bacteria in the supernatant after capture by the peptide-modified magnetic sphere group was much lower than that of the bare magnetic sphere control group. This confirms that the enhanced microbial capture ability of this system comes from the peptide recognition structure on the surface of the magnetic spheres. The magnetic sphere substrate material alone only has weak non-specific adsorption and cannot achieve efficient capture.

[0074] Test Example 3 The capture performance of modified magnetic spheres prepared using eight different peptides (Spep1, Spep2, Spep3, Spep4, Lpep1, Lpep2, Lpep3, and Lpep4) – namely MB-Biotin-Mal-SPep1, MB-Biotin-Mal-SPep2, MB-Biotin-Mal-SPep3, MB-Biotin-Mal-SPep4, MB-Biotin-Mal-LPep1, MB-Biotin-Mal-LPep2, MB-Biotin-Mal-LPep3, and MB-Biotin-Mal-LPep4) – against low (100 CFU / mL) and high (10000 CFU / mL) *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans* was investigated within 30 min. Colony changes before and after capture were compared. The results are as follows: Figure 10 As shown, the above-mentioned polypeptide magnetic beads all have excellent bacterial capture ability, with a capture rate of over 90% in pure bacterial solutions. The polypeptide magnetic beads modified with SPep3, LPep3, and LPep4 have the best capture performance.

[0075] Test Example 4 MB-Biotin-Mal-SPep4 magnetic beads, which exhibited the best capture performance among polypeptide magnetic beads, were selected to investigate their capture performance against various intestinal-associated bacteria, including low-concentration (100 CFU / mL), high-concentration (10000 CFU / mL) Citrobacter freundii, Morganella morganii, and Proteus vulgaris, within 30 minutes. Escherichia coli was used as a control. The results are as follows: Figure 11 As shown in the figure, the supernatant colonies were significantly reduced after capture. This result indicates that the peptide magnetic beads are not only applicable to a single bacterial species, but can also be extended to a variety of Gram-negative enteroassociated bacteria.

[0076] MB-Biotin-Mal-SPep4 was co-incubated with *Escherichia coli*, *Citrobacter freundii*, *Morganella morganii*, and *Proteus vulgaris*, respectively. After magnetic separation, the samples were observed using scanning electron microscopy (SEM) to further investigate the binding interaction between the peptide magnetic beads and different bacteria. The results are as follows: Figure 12 As shown in the figure, the peptide magnetic beads can adsorb and aggregate on the surface of bacterial cells. This morphological feature directly confirms the capture and binding mechanism of the peptide magnetic beads on bacteria, providing direct visual evidence for the bacterial capture effect of the peptide magnetic beads. SDS-PAGE analysis was performed on the bacterial stock solution and the peptide magnetic bead capture products, with parallel capture replicates set up. The results are shown below. Figure 13 As shown in the figure (the first column of each figure is the untreated bacterial stock solution control, and the second to fourth columns are the samples captured by MB-Biotin-Mal-SPep4 peptide magnetic beads in three parallel steps), it can be seen from the figure that the protein band characteristics of the captured product are consistent with those of the bacterial stock solution, proving that the sample obtained by magnetic separation is a bacterial component; the high overlap of the parallel sample bands indicates that the peptide magnetic bead capture system has good reproducibility.

[0077] Test Example 5 To investigate the anti-interference capture performance of the peptide magnetic beads in complex biological matrices, an artificial blood system was constructed for bacterial capture validation experiments. High concentrations (10000 CFU / mL) and low concentrations (100 CFU / mL) of bacteria were added to the simulated blood, respectively. MB-Biotin-Mal-LPep4 peptide magnetic beads were used for bacterial enrichment and capture. The capture effect was evaluated using a plate count method. The results are as follows: Figure 14As shown, under both high and low bacterial concentration conditions, the original bacterial solution plates were covered with colonies. The number of colonies in the supernatant plates after capture by the peptide magnetic beads was significantly reduced, while a large number of target colonies were observed in the captured magnetic bead sample plates collected after elution. Quantitative statistical results indicate that even under complex background interference from coexisting blood components, the peptide magnetic beads still achieve a bacterial capture efficiency of over 90%, confirming that the blood environment does not significantly inhibit the bacterial enrichment ability of the peptide magnetic beads. These results demonstrate that the peptide magnetic beads are not only suitable for simple clean systems but also can stably achieve interference-resistant bacterial capture in complex samples containing blood matrices, providing strong experimental support for the pretreatment of clinical infectious biological samples and the enrichment detection of trace amounts of low-abundance pathogenic bacteria.

[0078] Test Example 6 The preparation of BEVs is as follows: Target bacteria are inoculated into liquid culture medium and cultured at 37°C with shaking until mid-log growth (OD2). 600 After the concentration is 0.6~1.0, continue culturing for 4~6 hours. After culturing, pass through 4℃ and 4000× temperature zones sequentially. g Centrifuge for 30 min to remove intact bacterial cells, 4℃, 8000× g Centrifuge for 30 min to remove bacterial debris and large molecular aggregates. Filter the supernatant through a 0.22 μm filter membrane to remove residual bacteria, then incubate at 4℃ and 150,000 × 10⁻⁶. g After ultracentrifugation for 90 min, the BEVs precipitate was collected and resuspended in sterile PBS to obtain the BEVs suspension.

[0079] The molecular binding affinity of MB-Biotin-Mal-LPep4 peptide to four Gram-negative bacterial extracellular organisms (BEVs): *Escherichia coli*, *Salmonella enterica*, *Proteus vulgaris*, and *Shigella dysenteriae* was quantitatively analyzed using isothermal titration calorimetry (ITC). The results are as follows: Figure 15 and Figure 16 As shown, from Figure 15 It can be seen that the original titration curves of each group showed a continuous and stable thermal signal, indicating that the polypeptide can specifically bind to the BEVs of the four bacteria; from Figure 16 The dissociation constant obtained from the fitting analysis revealed that the peptide exhibits the best affinity for BEVs of *E. coli*, followed by *Salmonella*, while the affinity for BEVs of *Proteus vulgaris* and *Shigella dysenteriae* gradually decreases. This thermodynamic result confirms that the peptide possesses broad-spectrum binding activity against the BEVs of various enteropathogenic bacteria, providing direct experimental evidence at the molecular level for the broad-spectrum capture of multiple types of bacteria by peptide magnetic beads.

[0080] SPep4 peptide was fluorescently labeled with FITC, and LPS, a characteristic lipopolysaccharide on the surface of BEVs membranes, was fluorescently labeled with AF647. After co-incubating the SPep4-FITC and AF647-LPS-labeled BEVs system, the spatial distribution of the two fluorescence types was observed using a laser confocal microscope. The imaging results are shown below. Figure 17 As shown in the figure, the fluorescence of the peptide and the fluorescence of BEVs show significant co-localization, with their signal distribution areas highly overlapping. This result confirms that the SPep4 peptide can directly bind to BEV particles and their membrane structures, which is consistent with the quantitative thermodynamic data from ITC mentioned earlier. This further supports the feasibility of using peptide-modified magnetic spheres for the enrichment and separation of BEVs from the perspective of microscopic imaging.

[0081] MB-Biotin-Mal-SPep4 was co-incubated with BEVs and then subjected to magnetic separation. Multidimensional characterization and validation were performed using transmission electron microscopy (TEM), scanning electron microscopy (SEM), EDS elemental distribution mapping, laser confocal microscopy, zeta potential, dot blot, Western blot, and nanoparticle tracking analysis (NTA). The results are as follows: Figure 18 As shown, electron microscopy morphology images intuitively demonstrate that peptide-modified magnetic spheres can stably bind to BEV vesicles; EDS elemental distribution maps and laser confocal fluorescence co-localization results further confirm that a large number of BEV vesicles are enriched and distributed on the surface of the magnetic spheres. The Zeta potential of the magnetic spheres and BEVs shifted significantly before and after incubation, directly reflecting the surface adsorption and binding between the two. The results of Dot Blot, Western Blot specific detection and NTA particle quantitative analysis jointly confirm that peptide magnetic spheres can efficiently capture BEVs in the system and achieve vesicle signal enrichment.

[0082] Test Example 7 The broad-spectrum capture ability of MB-Biotin-Mal-SPep4 magnetic beads for extracellular vesicles from different bacterial species was investigated. Capture experiments were conducted using BEVs secreted by Gram-negative *Escherichia coli* and Gram-positive *Staphylococcus aureus*. SDS-PAGE protein electrophoresis analysis was performed on three groups of samples: the source bacteria, the original vesicle fluid, and the magnetic bead capture products. The electrophoresis results are shown below. Figure 19 As shown in the figure, the capture magnetic bead lanes for *Escherichia coli* and *Staphylococcus aureus* both exhibit characteristic protein bands matching their respective original BEV solutions, completely reproducing the protein profiles of the two strains' BEVs. This demonstrates that the peptide-modified magnetic beads can effectively enrich extracellular vesicles secreted by two different types of bacteria, Gram-positive and Gram-negative. These results indicate that this peptide magnetic bead capture platform is not only applicable to BEVs from a single bacterial species but also possesses the potential for broad-spectrum enrichment of BEVs across Gram-negative / positive bacterial communities.

[0083] Test Example 8 To determine the optimal process parameters for capturing bioelectrical energy particles (BEVs) using MB-Biotin-Mal-SPep4 magnetic spheres, capture experiments were conducted with two sets of variables: gradient magnetic sphere dosage and gradient incubation time. The capture signal intensity of BEVs was evaluated based on quantitative data from Western blotting and dot blotting strip grayscale / integrated optical density. The results are as follows: Figure 20 As shown in the figure, the capture of BEVs by peptide magnetic beads is not a random physical mixing and adsorption. The capture efficiency changes regularly with the amount of magnetic beads and the incubation time. There is a process parameter window that can be precisely controlled and standardized. The optimal BEV capture effect can be achieved with an incubation time of 90 min and a magnetic bead amount of 300 μL.

[0084] Test Example 9 This study investigated the selective capture of bioactive vesicles (BEVs) by MB-Biotin-Mal-SPep4 magnetic beads in a hybrid vesicle system. A coexistence model was constructed by mixing *E. coli* BEVs and host EVs derived from 5637 cells at a 1:1 particle ratio. Capture experiments were conducted using gradient amounts of the peptide magnetic beads. The grayscale values ​​of LPS (a BEV biomarker) and CD9 (an EV biomarker) were quantitatively detected by Western blotting, and the capture efficiency was calculated. The results are as follows: Figure 21 As shown in the figure, the peptide-modified magnetic beads can efficiently enrich *E. coli* BEVs in a mixed system, but their ability to capture host-derived eukaryotic EVs is significantly weaker. This result demonstrates that the capture platform constructed using peptide magnetic beads exhibits excellent targeting selectivity in mixed samples where BEVs and host EVs coexist in equal proportions, significantly reducing background interference caused by non-specific co-capture of host EVs and achieving specific separation of bacterial vesicles.

[0085] Test Example 10 To simulate the complex background of fluctuating bacterial vesicle abundance and the coexistence of numerous host cell vesicles in actual clinical samples, *E. coli* BEVs and human 5637 cell-secreted EVs were mixed in gradient ratios of 1:99, 10:90, 20:80, 30:70, 40:60, 60:40, 70:30, 80:20, 90:10, and 99:1 to construct multiple mixed vesicle models. Western blotting experiments were performed, and the results were quantified. The quantification results are shown below. Figure 22As shown in the figure, when the proportion of BEVs in the mixed system is not less than 30:70, the LPS signal representing BEVs is significantly enriched in the capture product of MB-Biotin-Mal-SPep4 magnetic beads, while the CD9 signal representing host EVs is significantly suppressed. These results confirm that the peptide-modified magnetic beads have a preferential capture ability for bacterial BEVs, maintaining excellent species-selective enrichment even with background interference from a large number of host EVs.

[0086] Test Example 11 The capture and enrichment of BEVs by the peptide magnetic beads (MB-Biotin-Mal-SPep4) in actual fecal samples were investigated as follows: Sample source and processing: Fresh stool samples were collected from 12 healthy adults. All samples were donated by volunteers in our laboratory, and all volunteers signed informed consent forms. None of the subjects had taken antibiotics or probiotics within 2 weeks prior to sampling. Each stool sample was aliquoted immediately after collection, and all processing was completed within 6 hours of sampling. The aliquoted samples were then frozen at -80°C for later use.

[0087] Fecal sample pretreatment and crude extraction of total vesicles (ultracentrifugation): Take 10.0 g of each fecal sample, add 50 mL of pre-cooled PBS containing a mixture of protease inhibitors, and vortex for 5 min to fully disperse the sample; then, purify by stepwise differential centrifugation at 4℃: centrifuge at 1000×g for 10 min to remove large particles and collect the supernatant; centrifuge at 3000×g for 10 min to remove large particles and collect the supernatant; g Centrifuge for 20 minutes to remove fine impurities and precipitate, then collect the supernatant; 10000× g Centrifuge for 30 min to remove cell debris and bacterial cells, and collect the supernatant; finally, centrifuge at 100,000 × 100000. g Centrifuge at ultracentrifugation for 90 min, discard the supernatant, gently resuspend the precipitate in 200 μL PBS to obtain crude total vesicle extract (EVs), aliquot and store at -80℃.

[0088] Each fecal vesicle crude extract was divided into two portions, 20 μL each, for processing into a stock solution group and a capture group. The stock solution group crude extract was directly aliquoted and stored without magnetic bead capture, serving as a control sample before enrichment. The capture group crude extract was specifically targeted and captured using MB-Biotin-Mal-SPep4 peptide magnetic beads, and the peptide magnetic beads binding the vesicles were collected as the test sample after enrichment. Twelve fecal samples from different volunteers were used in the experiment, independently. Each case had its own paired control: a stock solution group before capture and a capture group after capture. Subsequent detection and analysis were conducted based on this paired sample system for comparative studies.

[0089] First, the crude extract of total fecal vesicles prepared by ultracentrifugation was characterized using TEM, NTA, and Western blotting techniques. The results are as follows: Figure 23 As shown in the figure, it can be seen that BEVs and host EVs coexist in the actual fecal sample. BEVs can express characteristic markers such as lipopolysaccharide (LPS) and outer membrane protein C (OmpC), while host EVs can express markers such as CD9, CD63 and ALIX.

[0090] Based on the aforementioned results, an experimental design using the same sample paired with other samples was employed to compare the enrichment effect of BEVs before and after peptide-modified magnetic bead trapping treatment. The results are as follows: Figure 24 As shown in the figure, compared to the untreated stock solution group, the band signal of the host vesicle marker CD9 was significantly weakened in the magnetic bead capture group; even with a significant reduction in the host vesicle background signal, the bacterial vesicle marker LPS... + LPS can still be detected stably. + The signal ratio to CD9 increased significantly. These results confirm that the polypeptide magnetic bead capture system constructed in this invention can preferentially and specifically enrich BEVs in a complex matrix composed of fecal proteins, cell debris, and host extracellular vesicles, while effectively reducing background interference from host EVs.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing polypeptide magnetic beads that capture pathogens and their outer vesicles, characterized in that, include: Magnetic microsphere carriers were prepared using soluble iron salts as raw materials via a solvothermal method. Magnetic spheres coated with a silica functional layer were prepared by surface modification of the magnetic microsphere carrier with alkoxysilane. Amino-functionalized magnetic spheres were prepared by modifying the magnetic spheres coated with the silica functional layer using an aminosilane coupling agent. The amino-functionalized magnetic spheres were subjected to anhydride treatment to prepare carboxyl-functionalized magnetic spheres; Streptavidin magnetic spheres were prepared by coupling streptavidin to the surface of the carboxyl-functionalized magnetic spheres using a carbodiimide coupling agent. A biotin-maleimide conjugate was attached to the surface of the streptavidin magnetic spheres via the specific binding of biotin to streptavidin to prepare maleimide-biotin-functionalized magnetic spheres. A polypeptide containing cysteine ​​is coupled to the surface of the maleimide-biotin-functionalized magnetic sphere to prepare a polypeptide magnetic sphere for capturing pathogens and their exovesicles. The cysteine-containing polypeptide is selected from any of the following structural formulas: 、 、 、 、 、 、 、 。 2. The method for preparing polypeptide magnetic beads for capturing pathogens and their outer vesicles as described in claim 1, characterized in that, The soluble iron salt is selected from any one of FeCl3·6H2O, Fe(NO3)3·9H2O, FeCl2·4H2O, and FeSO4·7H2O.

3. The method for preparing polypeptide magnetic beads for capturing pathogens and their outer vesicles as described in claim 1, characterized in that, The reaction temperature of the solvothermal method is 210-230℃, the reaction time is 10-14h, and the reaction system includes sodium acetate, sodium citrate, and ethylene glycol.

4. The method for preparing polypeptide magnetic beads for capturing pathogens and their outer vesicles as described in claim 1, characterized in that, The alkoxysilane is selected from one of tetraethyl orthosilicate, methyl orthosilicate, propyl orthosilicate, isopropyl orthosilicate, butyl orthosilicate, and pentyl orthosilicate; The aminosilane coupling agent is selected from 3-aminopropyltriethoxysilane and 3-aminopropyltrimethoxysilane.

5. The method for preparing polypeptide magnetic beads for capturing pathogens and their outer vesicles as described in claim 1, characterized in that, The carbodiimide coupling agent is selected from one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, dicyclohexylcarbodiimide, and diphenylcarbodiimide.

6. A polypeptide magnetic ball for capturing pathogens and their outer vesicles, characterized in that, The polypeptide magnetic spheres are prepared using the method for preparing pathogenic bacteria and their outer vesicles as described in any one of claims 1 to 5. The polypeptide magnetic spheres include a magnetic microsphere carrier core and a polypeptide functional layer coating the outer surface of the core.

7. The application of polypeptide magnetic beads for capturing pathogens and their outer vesicles, characterized in that, The application involves constructing a capture platform using the polypeptide magnetic spheres for capturing pathogens and their outer vesicles as described in claim 6, wherein the capture platform is used to capture one or more of the following targets: Gram-positive bacteria, Gram-negative bacteria, Candida albicans, Gram-positive bacterial vesicles, Gram-negative bacterial vesicles, and Candida albicans vesicles.

8. The application of the polypeptide magnetic beads for capturing pathogens and their outer vesicles as described in claim 7, characterized in that, The Gram-positive bacteria is Staphylococcus aureus; The Gram-negative bacteria are any one of Escherichia coli, Salmonella enterica, Shigella dysenteriae, Citrobacter freundii, Morganella morganii, and Proteus vulgaris.

9. The application of the polypeptide magnetic beads for capturing pathogens and their outer vesicles as described in claim 7, characterized in that, The capture time of the capture platform is 30 to 150 minutes.

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