Mannose functionalized magnetic particles for pathogen enrichment and methods of making the same

Abstract

This invention relates to the field of biodetection technology, providing mannose-functionalized magnetic particles for pathogen enrichment and their preparation method. The invention aims to address the problems of severe host nucleic acid interference, low detection rate of low-abundance pathogens, and poor integration with downstream sequencing processes in existing pathogen enrichment methods. The particles use Fe3O4@SiO2 as a core-shell structure, with a dendritic molecular scaffold covalently linked to its surface. The scaffold ends are connected to mannose ligands via breakable disulfide bonds to form a multivalent cluster structure, which can specifically capture pathogens, enhance capture efficiency, and gently release pathogens, reducing host nucleic acid background and increasing the proportion of microbial reads. This invention provides high-quality pretreatment samples for hybrid sequencing workflows, featuring high capture efficiency, thorough host removal, and simple operation, providing reliable technical support for the accurate diagnosis of clinical infectious diseases.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a mannose-functionalized magnetic particle for pathogen enrichment and its preparation method. Background Technology

[0002] Rapid and accurate pathogen diagnosis of infectious diseases is a crucial prerequisite for clinical treatment, especially for immunosuppressed patients, severely infected patients, and cases of mixed infections. Traditional pathogen detection methods often suffer from limitations such as long detection cycles, low sensitivity, and difficulty in detecting low-abundance pathogens. In recent years, the rapid development of metagenomic next-generation sequencing (mNGS) and targeted next-generation sequencing (tNGS) technologies has provided new means for pathogen diagnosis. mNGS, with its advantages of no pre-setting and broad-spectrum coverage, can detect nucleic acids of all microorganisms in a sample, while tNGS achieves highly sensitive targeted enrichment of target pathogens through ultra-multiplex PCR technology. Based on this, hybrid sequencing workflows that combine the broad-spectrum detection capabilities of mNGS with the high-sensitivity targeted enrichment advantages of tNGS have become an important development direction in the field of pathogen detection. However, both mNGS and hybrid sequencing workflows face a common technical bottleneck: the content of host nucleic acid in clinical samples (such as blood, respiratory samples, cerebrospinal fluid, etc.) far exceeds that of pathogen nucleic acid, usually accounting for more than 99%. This results in a very low percentage of effective microbial reads in the sequencing data, and low-abundance pathogens (such as viruses, intracellular bacteria, fungi) are very easy to miss, which seriously restricts the practical application effect of hybrid sequencing workflows in clinical practice.

[0003] To address the aforementioned host nucleic acid interference problem, various pathogen enrichment methods have been developed in existing technologies. Centrifugation is simple to operate but inefficient, almost ineffective in capturing low-abundance pathogens, and struggles to distinguish between the host and the pathogen. While antibody immunomagnetic bead methods can achieve specific capture, antibodies are expensive, exhibit significant batch-to-batch variability, and only target single or a few specific pathogens, failing to meet the broad-spectrum pathogen detection requirements of mixed sequencing workflows. Physical separation methods such as differential centrifugation and filtration are cumbersome, have poor reproducibility, and are prone to sample loss. In recent years, affinity capture methods based on lectin-carbohydrate interactions have attracted attention. Among these, mannose exhibits broad-spectrum and high-affinity specific recognition of pathogen adhesin proteins FimH or mannose receptors on the pathogen surface. However, existing mannose functionalized materials are mostly single-molecule modifications with limited binding affinity and lack effective integration designs with downstream sequencing workflows. Therefore, developing a novel enrichment material that can broadly and efficiently capture pathogens while seamlessly integrating with hybrid sequencing workflows to achieve efficient removal of host nucleic acids and effective enrichment of low-abundance pathogens has become an urgent need to promote the clinical translation of hybrid sequencing technology. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention aims to provide mannose-functionalized magnetic particles for pathogen enrichment and their preparation method. This material utilizes Fe3O4@SiO2 core-shell magnetic nanoparticles as a core carrier, grafts dendritic molecular scaffolds onto the surface of mesoporous silica, and connects mannose ligands to the ends of the scaffolds via breakable disulfide bond links, forming a nanocomposite material with high-density mannose ligands and responsive release capabilities. This material overcomes the limitations of traditional antibody magnetic beads that rely on single-target recognition. It leverages the broad-spectrum affinity of mannose for FimH proteins or mannose receptors on the surfaces of various pathogens, combined with the multivalent synergistic binding effect provided by dendritic molecules, to achieve efficient capture of low-abundance pathogens in complex samples. Simultaneously, the reduction-responsive cleavage of disulfide bonds enables gentle release of pathogens, providing high-quality pre-processed samples for hybrid sequencing workflows.

[0005] As one aspect of the present invention, the present invention provides mannose-functionalized magnetic particles for pathogen enrichment:

[0006] The materials of this invention include a magnetic nanoparticle core, a dendritic molecular scaffold, and a mannose ligand;

[0007] The magnetic nanoparticle core is a Fe3O4@SiO2 core-shell structured nanoparticle, and the SiO2 is a mesoporous structure.

[0008] The dendritic molecular scaffold is covalently connected to the surface of the magnetic nanoparticles;

[0009] The mannose ligands are connected to the ends of the dendritic molecular scaffold via breakable disulfide linkers, forming a multivalent mannose cluster structure.

[0010] In another aspect, the present invention provides a method for preparing mannose-functionalized magnetic particles for pathogen enrichment, the specific steps of which are as follows:

[0011] (1) Preparation of Fe3O4@SiO2 core-shell magnetic nanoparticles: Fe3O4 nanoparticles were synthesized by coprecipitation method. FeCl2·4H2O and FeCl3·6H2O were dissolved in deionized water, the pH was adjusted to 10, and the reaction was carried out at 80℃ for 1 hour. Fe3O4 nanoparticles were obtained by magnetic separation and washing. Subsequently, the mesoporous SiO2 shell was coated by sol-gel method. Fe3O4 nanoparticles were dispersed in a 1:1 volume ratio ethanol / water mixed solvent, the template agent hexadecyltrimethylammonium bromide was added, the pH was adjusted to 10, tetraethyl orthosilicate was added dropwise, and the reaction was carried out at room temperature for 6 hours. After magnetic separation, the template was removed by reflux extraction with 0.5% (w / v) ammonium nitrate / ethanol solution to obtain Fe3O4@SiO2 core-shell nanoparticles.

[0012] (2) Surface amination modification: Fe3O4@SiO2 nanoparticles obtained in step (1) were dispersed in anhydrous ethanol, and 5% by volume of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed at 60°C for 12 hours, and then magnetically separated and washed to obtain surface amination Fe3O4@SiO2-NH2.

[0013] (3) Dendritic grafting: Fe3O4@SiO2-NH2 obtained in step (2) was dispersed in methanol, and polyamide-amine dendritic polymer (PAMAM) was added. At the same time, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added for activation. The reaction was carried out at room temperature for 24 hours, and the PAMAM-grafted Fe3O4@SiO2-PAMAM was obtained by magnetic separation and washing.

[0014] (4) Grafting of mannose derivatives: Fe3O4@SiO2-PAMAM obtained in step (3) was dispersed in N,N-dimethylformamide (DMF), and a hydrazinated mannose derivative Ac4ManNAz containing disulfide bonds, as well as catalyst CuSO4·5H2O and sodium ascorbate were added. The reaction was carried out at 40°C for 24 hours for click chemical reaction. After magnetic separation and washing, the target product, mannose-functionalized magnetic nanomaterial Fe3O4@SiO2-PAMAM-SS-Man, was obtained.

[0015] As a preferred embodiment of the method for preparing magnetic particles according to the present invention: the molar ratio of FeCl2·4H2O to FeCl3·6H2O in step (1) is 1:2.

[0016] As a preferred embodiment of the method for preparing the magnetic particles described in this invention: in step (1), the amount of hexadecyltrimethylammonium bromide added is 0.5 g per 100 mg Fe3O4.

[0017] As a preferred embodiment of the method for preparing the magnetic particles described in this invention: in step (1), the amount of tetraethyl orthosilicate added is 0.5 mL per 100 mg Fe3O4.

[0018] As a preferred embodiment of the method for preparing magnetic particles according to the present invention: in step (2), the amount of APTES added is 2.5 mL per 100 mg Fe3O4@mSiO2.

[0019] As a preferred embodiment of the method for preparing the magnetic particles described in this invention: the mass ratio of PAMAM to Fe3O4@SiO2-NH2 in step (3) is 5:1.

[0020] As a preferred embodiment of the method for preparing the magnetic particles described in this invention: in step (4), the amount of Ac4ManNAz added is 10 mg per 50 mg Fe3O4@SiO2-PAMAM.

[0021] As a preferred embodiment of the method for preparing the magnetic particles described in this invention: the amount of CuSO4·5H2O added in step (4) is 0.5 mg.

[0022] As a preferred embodiment of the method for preparing the magnetic particles described in this invention: the amount of sodium ascorbate added in step (4) is 1 mg.

[0023] As another aspect of the present invention, the present invention provides an application of mannose-functionalized magnetic particles for pathogen enrichment in pathogen enrichment, the application comprising mixing and incubating the material with a clinical sample, capturing pathogens by the specific recognition of mannose and pathogen surface lectins, rapidly separating them under an external magnetic field, and then treating them with a 10 mM reducing agent dithiothreitol to break the disulfide bonds and release the pathogens, thereby obtaining an enriched pathogen sample.

[0024] As another aspect of the present invention, the present invention provides an application of mannose-functionalized magnetic particles for pathogen enrichment in hybrid sequencing pretreatment. The application involves extracting nucleic acids from pathogen samples enriched by the material, and then performing targeted sequencing and metagenomic sequencing respectively. The two sequencing results are integrated and analyzed to obtain a comprehensive pathogen detection report.

[0025] Compared with the prior art, the advantages of the present invention are as follows:

[0026] First, this invention introduces multivalent mannose clusters into the field of pathogen enrichment through the synergistic design of core-shell magnetic nanoparticles and dendritic molecular scaffolds. Compared with traditional single-molecule modification modes, the multivalent synergistic effect solves the technical problem of efficiently capturing low-abundance pathogens.

[0027] Second, this invention innovatively introduces a breakable disulfide linker between the mannose ligand and the scaffold, achieving a responsive and gentle release after pathogen capture. Existing antibody magnetic beads require vigorous elution, which can easily damage sample integrity, while this invention achieves a release efficiency of over 91% under 10 mM DTT conditions.

[0028] Third, this invention overcomes the contradiction between capture efficiency and release efficiency in the prior art by using a three-layer structure design consisting of a core-shell structure, a dendritic support, and breakable disulfide bonds. The absence of any one of these structures will lead to a significant decrease in performance. Attached Figure Description

[0029] Figure 1This is a morphology diagram of the functionalized magnetic particles of the present invention.

[0030] Figure 2 This data represents the percentage of human reads before and after the functionalized magnetic particles were treated according to the present invention.

[0031] Figure 3 This data represents the percentage of pathogen reads before and after treatment with the functionalized magnetic particles of this invention. Detailed Implementation

[0032] The technical solutions described in this invention will now be clearly and completely described with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only a part of the feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0033] Example 1: Functionalized Magnetic Particles

[0034] (1) Synthesis of Fe3O4 nanoparticles: 1.98 g of FeCl2·4H2O and 5.41 g of FeCl3·6H2O were dissolved in 100 mL of deionized water and mechanically stirred for 30 minutes under nitrogen protection. 1 M NaOH solution was added dropwise to adjust the pH to 10, and the temperature was raised to 80 °C for 1 hour. After the reaction was completed, the black precipitate was separated by an external magnetic field and washed with deionized water until neutral to obtain Fe3O4 nanoparticles with a particle size of 20-25 nm and a saturation magnetization of 65 emu / g.

[0035] (2) Mesoporous SiO2 shell coating: 100 mg of Fe3O4 nanoparticles obtained in step (1) were dispersed in 100 mL of ethanol / water mixed solvent (volume ratio 1:1), and 0.5 g of hexadecyltrimethylammonium bromide was added. The mixture was ultrasonically dispersed for 30 minutes. 2 mL of ammonia was added to adjust the pH to 10.5, and 0.5 mL of tetraethyl orthosilicate was added dropwise. The mixture was reacted at room temperature for 6 hours. After the reaction was completed, the mixture was magnetically separated and washed three times with ethanol. The product was dispersed in 0.5% ammonium nitrate / ethanol solution and refluxed twice for 2 hours each time to remove the template, thus obtaining Fe3O4@SiO2 core-shell nanoparticles.

[0036] (3) Surface amination modification: 100 mg of Fe3O4@SiO2 nanoparticles obtained in step (2) were dispersed in 50 mL of anhydrous ethanol, and 2.5 mL of APTES was added. The mixture was refluxed at 60 °C for 12 hours. After the reaction was completed, the mixture was magnetically separated and washed three times with ethanol to obtain surface-aminated Fe3O4@SiO2-NH2.

[0037] (4) Dendritic grafting: 100 mg of Fe3O4@SiO2-NH2 obtained in step (3) was dispersed in 50 mL of methanol, 50 mg of PAMAM dendritic molecules (G3 generation, with 128 active amino groups at the end) were added, 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6 mg of N-hydroxysuccinimide were added for activation, and the reaction was carried out at room temperature for 24 hours. After the reaction was completed, magnetic separation was performed, and the mixture was washed three times with methanol to obtain PAMAM-grafted Fe3O4@SiO2-PAMAM.

[0038] (5) Grafting of mannose derivatives: 50 mg of Fe3O4@SiO2-PAMAM obtained in step (4) was dispersed in 10 mL of DMF, 10 mg of azidized mannose derivative Ac4ManNAz containing disulfide bonds was added, and 0.5 mg of CuSO4·5H2O and 1 mg of sodium ascorbate were added as catalysts. The reaction was carried out at 40 °C for 24 hours for click chemistry. After the reaction was completed, magnetic separation was performed, and the product was washed twice with DMF, twice with ethanol, and three times with deionized water to obtain the target product, mannose-functionalized magnetic nanomaterial Fe3O4@SiO2-PAMAM-SS-Man, which was dispersed in 1 mL of PBS buffer containing 0.1% Tween-20 and stored at 4 °C.

[0039] Example 2: Preparation of Functionalized Magnetic Particles

[0040] The difference between this embodiment and embodiment 1 is that in step (4), G5 generation PAMAM dendritic molecules (terminal 128 active amino groups) are used instead of G3 generation PAMAM, and the remaining steps are the same as in embodiment 1.

[0041] Comparative Example 1: Preparation of magnetic nanomaterials without SiO2 shell

[0042] The difference between this comparative example and Example 1 is that step (2) is omitted, and the Fe3O4 nanoparticles obtained in step (1) are directly subjected to surface amination modification in step (3). The remaining steps are the same as in Example 1.

[0043] Comparative Example 2: Preparation of Magnetic Nanomaterials without Grafted Dendritic Molecules

[0044] The difference between this comparative example and Example 1 is that step (4) is omitted, and the Fe3O4@SiO2-NH2 obtained in step (3) is directly grafted with the mannose derivative in step (5). The remaining steps are the same as in Example 1.

[0045] Comparative Example 3: Preparation of magnetic nanomaterials without disulfide bond linkers

[0046] The difference between this comparative example and Example 1 is that a mannose derivative without disulfide bonds is used in step (5), while the other steps are the same as in Example 1.

[0047] Experimental Example 1: Material Morphology Characterization and Structural Verification

[0048] The morphology of the functionalized magnetic particles prepared in Example 1 was characterized using transmission electron microscopy (TEM). An appropriate amount of sample was dispersed in anhydrous ethanol, ultrasonically dispersed for 5 minutes, and then dropped onto a copper mesh carbon support film. After natural drying, the core-shell structure and particle size distribution of the nanoparticles were observed using TEM. Figure 1 As shown, the particles have a clear core-shell structure. The Fe3O4 core has a particle size of about 20-25 nm, and the mesoporous SiO2 shell has a thickness of about 5-8 nm. Ordered mesoporous channels are visible in the shell, which increases the specific surface area of ​​the particles and enhances the grafting density.

[0049] Experimental Example 2: Evaluation of Pathogen Capture Efficiency and Release Performance

[0050] This experiment aimed to evaluate the capture efficiency and release performance of the material of this invention against pathogens. *E. coli* (ATCC 25922) expressing the FimH protein was used as the model pathogen, with a bacterial concentration of 100 CFU / mL and three replicates per group. 20 μL of magnetic particles from Examples 1, 2, and Comparative Examples 1–3 were taken, magnetically separated to remove the preservation solution, resuspended in 1 mL of binding buffer, mixed with 2 mL of bacterial suspension, and incubated at room temperature for 60 minutes by rotation. After separation using an external magnetic field, the supernatant was discarded, and the sample was washed three times with washing buffer. 100 μL of release buffer (10 mM DTT) was added, and the sample was incubated at room temperature for 30 minutes. The supernatant was collected by magnetic separation. Samples before and after enrichment were plate-spread for counting, and the capture efficiency and release efficiency were calculated.

[0051] As shown in Table 1, Example 1 achieved a capture efficiency of 95.3% and a release efficiency of 91.5%, indicating that the magnetic particles of the present invention have excellent capture capabilities. Comparative Example 1 had a capture efficiency of only 52.1% due to agglomeration, which is attributed to poor material dispersibility and easy agglomeration. Comparative Examples 1 and 2 did not undergo release efficiency testing due to their significantly low capture efficiencies. Comparative Example 2 had a low mannose grafting density, resulting in a capture efficiency of 48.3%, which is attributed to insufficient binding capacity. Comparative Example 3 achieved a capture efficiency of 93.2%, but a release efficiency of only 15.6%. Therefore, the core-shell structure of the magnetic particles prepared in Example 1 ensures dispersibility, the dendritic scaffold provides multivalent bonding, and the disulfide bonds enable controlled release. The synergistic effect of these three factors allows both capture and release efficiencies to reach over 90%.

[0052] Table 1 Evaluation of Pathogen Capture Efficiency and Release Performance

[0053] Example 1 95.3 91.5 Example 2 97.2 92 Comparative Example 1 52.1 Unable to detect Comparative Example 2 48.3 Unable to detect Comparative Example 3 93.2 15.6

[0054] Experimental Example 3: Host Nucleic Acid Removal Effect

[0055] This experiment aimed to evaluate the removal effect of the material of this invention on host nucleic acid in clinical samples and its improvement on downstream sequencing quality. Twenty bronchoalveolar lavage fluid samples from clinically diagnosed pulmonary infections were collected and divided into two groups: Group A received direct nucleic acid extraction and sequencing from the original sample; Group B received nucleic acid extraction and sequencing after enrichment with the magnetic particles prepared in Example 1. The enrichment process was as follows: 2 mL of bronchoalveolar lavage fluid was added to 20 μL of magnetic particles, incubated at room temperature for 60 minutes, and after magnetic separation and washing, pathogens were released with 100 μL of release buffer. The supernatant was collected for nucleic acid extraction. Metagenomic sequencing was performed using an Illumina NovaSeq 6000, and the proportion of human reads and pathogen reads was compared.

[0056] Discussion of Results: Figure 2 and Figure 3 As shown, in group A, human reads accounted for as high as 99.1%, while pathogen reads accounted for only 0.08%. After enrichment, in group B, the proportion of human reads decreased to 8.5%, while the proportion of pathogen reads increased to 1.2%, and the detection rate of low-abundance pathogens increased by 45%. The results indicate that the magnetic particles of this invention can effectively remove the host nucleic acid background, significantly improve the proportion of effective microbial reads in sequencing data, and provide high-quality pretreatment samples for mixed sequencing.

[0057] In summary, this invention successfully developed a mannose-functionalized magnetic nanomaterial and its preparation method for efficient pathogen enrichment in hybrid sequencing processes. By constructing Fe3O4@SiO2 core-shell magnetic nanoparticles, grafting PAMAM dendritic molecular scaffolds, introducing breakable disulfide linkers, and modifying with multivalent mannose clusters, efficient capture and responsive release of low-abundance pathogens in complex clinical samples were achieved. Experimental results clearly show that the core-shell structure significantly improves dispersion stability, achieving a capture efficiency of over 95% for *E. coli* expressing the FimH protein, nearly 40% higher than the single-molecule modification mode. Key experimental data demonstrate that after enrichment treatment with the material of this invention, the proportion of host nucleic acid contamination in clinical samples significantly decreased from over 99% to below 10%, and the proportion of microbial reads increased from less than 0.1% to 1%, providing a new pretreatment solution for the diagnosis of clinical infectious diseases and possessing promising prospects for industrial application.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A mannose-functionalized magnetic particle for pathogen enrichment, characterized in that, The functionalized magnetic particles include a magnetic nanoparticle core, a dendritic molecular scaffold, and a mannose ligand. The magnetic nanoparticle core is a Fe3O4@SiO2 core-shell structured nanoparticle, and the SiO2 is a mesoporous structure. The dendritic molecular scaffold is covalently connected to the surface of the magnetic nanoparticles; The mannose ligands are connected to the ends of the dendritic molecular scaffold via breakable disulfide linkers, forming a multivalent mannose cluster structure.

2. A method for preparing mannose-functionalized magnetic particles for pathogen enrichment, characterized in that, The preparation method includes the following steps: (1) Preparation of Fe3O4@SiO2 core-shell magnetic nanoparticles: Fe3O4 nanoparticles were synthesized by coprecipitation method. FeCl2·4H2O and FeCl3·6H2O were dissolved in deionized water, the pH was adjusted to 10, and the reaction was carried out at 80℃ for 1 hour. Fe3O4 nanoparticles were obtained by magnetic separation and washing. Subsequently, the mesoporous SiO2 shell was coated by sol-gel method. Fe3O4 nanoparticles were dispersed in a 1:1 volume ratio ethanol / water mixed solvent, the template agent hexadecyltrimethylammonium bromide was added, the pH was adjusted to 10, tetraethyl orthosilicate was added dropwise, and the reaction was carried out at room temperature for 6 hours. After magnetic separation, the template was removed by reflux extraction with a 0.5% mass-volume ratio ammonium nitrate / ethanol solution to obtain Fe3O4@SiO2 core-shell nanoparticles. (2) Surface amination modification: Fe3O4@SiO2 nanoparticles obtained in step (1) were dispersed in anhydrous ethanol, and 5% by volume of 3-aminopropyltriethoxysilane was added. The mixture was refluxed at 60°C for 12 hours, and then magnetically separated and washed to obtain surface amination Fe3O4@SiO2-NH2. (3) Dendritic molecule grafting: Fe3O4@SiO2-NH2 obtained in step (2) was dispersed in methanol, polyamide-amine type dendritic polymer was added, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added for activation. The reaction was carried out at room temperature for 24 hours, and PAMAM grafted Fe3O4@SiO2-PAMAM was obtained by magnetic separation and washing. (4) Grafting of mannose derivatives: Fe3O4@SiO2-PAMAM obtained in step (3) was dispersed in N,N-dimethylformamide, and a hydrazinated mannose derivative Ac4ManNAz containing disulfide linker arms, as well as catalyst CuSO4·5H2O and sodium ascorbate were added. The reaction was carried out at 40°C for 24 hours for click chemical reaction. After magnetic separation and washing, the target product, mannose-functionalized magnetic nanomaterial Fe3O4@SiO2-PAMAM-SS-Man, was obtained.

3. The method for preparing functionalized magnetic particles as described in claim 2, characterized in that, The molar ratio of FeCl2·4H2O to FeCl3·6H2O in step (1) is 1:

2.

4. The method for preparing functionalized magnetic particles as described in claim 2, characterized in that, In step (1), the amount of hexadecyltrimethylammonium bromide added is 0.5 g per 100 mg Fe3O4.

5. The method for preparing functionalized magnetic particles as described in claim 2, characterized in that, In step (1), the amount of tetraethyl orthosilicate added is 0.5 mL per 100 mg Fe3O4.

6. The method for preparing functionalized magnetic particles as described in claim 2, characterized in that, In step (2), the amount of APTES added is 2.5 mL per 100 mg Fe3O4@SiO2.

7. The method for preparing functionalized magnetic particles as described in claim 2, characterized in that, In step (3), the mass ratio of PAMAM to Fe3O4@SiO2-NH2 is 5:

1.

8. The method for preparing functionalized magnetic particles as described in claim 2, characterized in that, In step (4), the amount of Ac4ManNAz added is 10 mg per 50 mg Fe3O4@SiO2-PAMAM, the amount of CuSO4·5H2O added is 0.5 mg, and the amount of sodium ascorbate added is 1 mg.

9. The application of the functionalized magnetic particles as described in claim 1 in pathogen enrichment, characterized in that, The application involves mixing and incubating the mannose-functionalized magnetic particles with clinical samples, capturing pathogens through the specific recognition of mannose and pathogen surface lectins, rapidly separating them under an external magnetic field, and then treating them with a 10 mM reducing agent dithiothreitol to break the disulfide bonds and release the pathogens, thereby obtaining enriched pathogen samples.

10. The application of the functionalized magnetic particles as described in claim 1 in hybrid sequencing pretreatment.

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