Modified microporous filtration membranes for enriching bioparticles, methods of making and using the same

Abstract

The application discloses a modified microporous filter membrane for enriching biological particles and a preparation method and application thereof, and belongs to the technical field of biological separation, wherein the preparation method of the modified microporous filter membrane comprises the following steps: providing a mixed cellulose ester microporous filter membrane, and performing alkali activation treatment; placing the activated membrane in a weak alkaline buffer solution for balancing; in the weak alkaline buffer solution, the membrane surface is first contacted with a tannic acid solution and a first reaction is performed, then a polyethylene imine solution is directly added for a second reaction without changing the system, so that a phenol-amine crosslinking network is formed in situ on the membrane surface; sodium cyanoborohydride is continuously added for reduction stabilization treatment, and the modified microporous filter membrane is obtained. The modified microporous filter membrane prepared by modifying the microporous filter membrane by tannic acid and polyethylene imine can realize one-step synchronous enrichment of all negatively charged target objects, and problems such as target loss and efficiency loss caused by multi-step processes are avoided from the source.

Description

Technical Field

[0001] This invention relates to the field of bioseparation technology, specifically to a modified microporous filter membrane for enriching biological particles, its preparation method, and its application. Background Technology

[0002] Human papillomavirus (HPV) infection is a major causative factor for diseases such as cervical cancer and oropharyngeal cancer. Urine testing, as a completely non-invasive sampling method, has unique advantages in large-scale population screening, male infection surveillance, and treatment follow-up. However, the viral load of HPV in urine is much lower than in cervical samples, and complex background interference such as urea, salt, and cell debris poses a significant challenge to achieving high-sensitivity detection.

[0003] Urinary exosomes are ideal carriers of nucleic acids, and they themselves also carry HPV nucleic acids, making them highly valuable detection targets. Therefore, efficient enrichment of exosomes and HPV nucleic acids in urine is a prerequisite for accurate detection. Currently, the mainstream technical approaches to achieve this goal typically involve multi-step tandem operations, such as separating exosomes before extracting nucleic acids, or directly extracting total nucleic acids. These methods may lead to problems such as target loss, cumulative efficiency losses, or inhibitor interference.

[0004] Specifically, existing technologies mainly suffer from the following two paths that lead to incomplete or inefficient targets:

[0005] Pathway 1: Isolate exosomes first, then extract nucleic acids; Method: First, exosomes are separated from urine using methods such as ultracentrifugation, polymer precipitation, or membrane filtration. Then, the obtained exosome precipitate or concentrate is lysed and nucleic acids are extracted. This method has the following inherent drawbacks:

[0006] Incomplete target: This pathway mainly captures nucleic acids carried in exosomes, while a large amount of free HPV DNA and viral particles present in urine are discarded during the separation step, resulting in a serious loss of target information.

[0007] Efficiency bottleneck: Exosome separation itself has limited efficiency (e.g., ultracentrifugation recovery rate is usually only 30%-70%), and subsequent nucleic acid extraction also results in efficiency loss, forming a cascade of efficiency losses.

[0008] Pathway 2: Ignoring exosomes and directly extracting total nucleic acids; Method: Using a commercial nucleic acid extraction kit, total nucleic acids are directly extracted from urine precipitate or centrifuged supernatant. This method has the following inherent drawbacks:

[0009] Weak enrichment capacity: For targets with low concentrations, there is a lack of effective pre-enrichment methods, making it difficult to achieve the concentration required for high-sensitivity detection.

[0010] Inhibitor interference: Incomplete removal of PCR inhibitors in urine can severely inhibit downstream amplification reactions, leading to false negatives.

[0011] In addition, existing separation and enrichment technologies have the following limitations:

[0012] Ultracentrifugation: The equipment is expensive, the process is time-consuming, and the high-speed shearing force may damage the exosome structure, resulting in unstable recovery rates.

[0013] Polymer coprecipitation method: introduces high concentrations of polymer, which seriously affects downstream PCR detection and results in poor purity.

[0014] Size exclusion chromatography: processes small sample volumes, is time-consuming, and is not suitable for concentrated samples.

[0015] Commercial membrane filtration methods: Most rely solely on pore size for physical sieving, lack specific adsorption capacity, are prone to clogging, and have varying enrichment efficiencies for different forms of nucleic acid / virus particles.

[0016] In summary, existing technologies still have room for improvement in achieving efficient, simultaneous, and integrated enrichment of low-abundance nanoscale biological particles (such as exosomes and viral particles) and their carried nucleic acids in human body fluids (such as urine). Summary of the Invention

[0017] The purpose of this invention is to provide a method for preparing a modified microporous filter membrane for enriching biological particles, so as to solve the problems mentioned in the background art.

[0018] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0019] A method for preparing a modified microporous filter membrane for enriching biological particles includes the following steps:

[0020] A mixed cellulose ester microporous filter membrane is provided and activated by alkaline solution to obtain an activated membrane;

[0021] The activated membrane was equilibrated in a weakly alkaline buffer solution with a pH of 8.0-9.0.

[0022] In the weakly alkaline buffer solution, the membrane surface is first brought into contact with the tannic acid solution and a first reaction is carried out. Then, without changing the system, a polyethyleneimine solution is directly added to carry out a second reaction, thereby forming a phenol-amine crosslinking network in situ on the membrane surface.

[0023] Sodium cyanoborohydride was added to the weakly alkaline buffer solution for reduction and stabilization treatment to obtain a modified microporous filter membrane with a stable positive charge on its surface.

[0024] Furthermore, the alkaline solution is a NaOH solution with a concentration of 0.5-1.5 mM.

[0025] Furthermore, the buffer solution is a Tris-HCl buffer solution with a concentration of 8-12 mM and a pH value of 8-9.

[0026] Furthermore, the concentration of the tannic acid solution is 0.5-1.5 mg / mL.

[0027] Furthermore, the final concentration of the polyethyleneimine is 1.5-2.5 mg / mL.

[0028] Furthermore, the final concentration of the sodium cyanoborohydride is 10-20 mM.

[0029] Another objective of this invention is to provide a modified microporous filter membrane for enriching biological particles prepared by the above-described method, wherein the surface of the modified microporous filter membrane is cross-linked with tannic acid and polyethyleneimine to form a stable positively charged coating, the coating causing the modified microporous filter membrane to have a positive Zeta potential in an aqueous solution with a pH of 7.0, and the modified microporous filter membrane is capable of simultaneously adsorbing and capturing exosomes and nucleic acids from liquid samples.

[0030] Another objective of this invention is to provide the application of the modified microporous filter membrane described above for enriching biological particles in a preparation device or kit, wherein the device or kit is used to enrich pathogen nucleic acids from non-invasive liquid samples.

[0031] Another objective of this invention is to provide an apparatus for enriching pathogen nucleic acids from non-invasive liquid samples, comprising a filter and a multilayer membrane stack structure; the multilayer membrane stack structure comprises, from top to bottom, a pretreatment layer and a core capture layer; the pretreatment layer comprises a multilayer gradient pore size glass fiber membrane; the core capture layer is the aforementioned modified microporous filter membrane.

[0032] This invention modifies MCE membranes with tannic acid and polyethyleneimine to produce positively charged modified microporous membranes for enriching pathogen nucleic acids from non-invasive liquid samples. These modified microporous membranes can achieve one-step simultaneous enrichment of all negatively charged targets (including exosomes, viral particles, and free nucleic acids), fundamentally avoiding target loss and efficiency degradation associated with multi-step processes. The mechanism of this invention lies in controlling the sequential reaction of tannic acid and polyethyleneimine under weakly alkaline conditions to guide the formation of a stable, high-density positively charged three-dimensional nanonetwork structure on the surface of the microporous membrane. This structure not only strongly adsorbs negatively charged targets (such as exosome membranes, viral capsids, and nucleic acid phosphate backbones) through electrostatic interactions, but its multi-scale spatial network can also simultaneously and synergistically capture various targets with sizes ranging from tens of nanometers (exosomes / viruses) to linear molecules (nucleic acids), achieving true "one-step simultaneous enrichment" and overcoming the inherent efficiency loss problems in multi-step processes. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of a method for enriching pathogen nucleic acids from non-invasive liquid samples, provided in an embodiment of the present invention.

[0034] Figure 2 Electron micrograph of exosomes extracted from the MCE membrane before modification.

[0035] Figure 3 Electron micrograph of exosome extraction from the modified microporous filter membrane provided in the embodiments of the present invention.

[0036] Figure 4 This figure shows the comparison of DNA extraction concentrations of exosome components from urine and modified microporous membranes.

[0037] Figure 5 The image shows the results of HPV testing on a sample treated with a modified microporous filter membrane provided in an embodiment of the present invention. Detailed Implementation

[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] In one embodiment of the present invention, a modified microporous filter membrane for enriching nanoscale biological particles from biological samples is provided, the preparation method of which specifically includes the following steps:

[0040] S1. The mixed cellulose ester (MCE) membrane is cleaned by vacuum filtration with ultrapure water and then activated with NaOH solution to obtain the activated MCE membrane.

[0041] S2. After equilibrating the activated MCE membrane in a buffer solution, it is then reacted in a tannic acid solution. Polyethyleneimine and sodium cyanoborohydride are then added sequentially to obtain a modified microporous membrane. This modified microporous membrane is a positively charged membrane that can simultaneously adsorb exosomes and nucleic acids.

[0042] Specifically, the concentration of the NaOH solution is 0.5-1.5 mM; the buffer solution is Tris-HCl buffer with a concentration of 8-12 mM and a pH of 8-9; the concentration of the tannic acid solution is 0.5-1.5 mg / mL; the final concentration of the polyethyleneimine (PEI) is 1.5-2.5 mg / mL; and the final concentration of the sodium cyanoborohydride is 10-20 mM.

[0043] It should be noted that the MCE membrane is a microporous filter membrane made of a mixture of nitrocellulose and cellulose acetate, and has good hydrophilicity. Exosomes are extracellular vesicles with a diameter of approximately 30-150 nanometers, containing host genetic information, and can be used for disease diagnosis.

[0044] In this embodiment of the invention, polyphenolic substances such as tannic acid and amine polymers such as polyethyleneimine can undergo oxidative self-polymerization and crosslinking reactions under weakly alkaline conditions, which can be used to form a stable coating on the surface of various materials. Tannic acid acts as an anchoring and crosslinking platform, providing stable reaction sites for polyethyleneimine and achieving a uniform coating; polyethyleneimine acts as a positive power source and network framework, providing positive charges for capture and constructing a three-dimensional adsorption network with tannic acid; sodium cyanoborohydride acts as a system stabilizing reagent, transforming the above temporary connections into permanent connections, ensuring the long-term stability of the coating in complex urine environments.

[0045] The tannic acid-PEI modified microporous filter membrane prepared in this embodiment of the invention has a strong positive charge on its surface, which can simultaneously adsorb: negatively charged exosome membrane surface, negatively charged HPV virus particles, and free HPV DNA fragments through electrostatic interaction; moreover, the modified microporous filter membrane has a three-dimensional network structure: the modified layer forms a porous network, which simultaneously captures nanoscale exosomes and nucleic acid molecules.

[0046] In another embodiment of the present invention, an apparatus for enriching pathogen nucleic acids from non-invasive liquid samples is also provided, comprising a filter and a multilayer membrane stack structure; the multilayer membrane stack structure comprises, from top to bottom, a pretreatment layer and a core capture layer; the pretreatment layer comprises a multilayer gradient pore size glass fiber membrane; the core capture layer is the modified microporous filter membrane described above for enriching biological particles.

[0047] Specifically, the modified microporous filter membrane has a pore size of 0.22 μm to ensure fluid flux and provide enrichment function; the pretreatment layer includes three glass fiber membranes; the pore sizes of the three glass fiber membranes are 1.1-1.3 μm (for retaining epithelial cells and tissue debris), 0.8-1.0 μm (for removing bacteria and macromolecular aggregates), and 0.45-0.7 μm (for intercepting small particulate impurities).

[0048] In this embodiment of the invention, by setting a pretreatment layer: multiple layers of glass fiber membranes with different gradient pore sizes, impurities can be removed step by step; by setting a core capture zone: a tannic acid-PEI modified microporous filter membrane with a strong positive charge on its surface, it can simultaneously capture exosomes (with negatively charged membrane structure), HPV virus particles and free nucleic acids in urine.

[0049] like Figure 1 As shown, in another embodiment of the present invention, a method for enriching pathogen nucleic acids from non-invasive liquid samples is also provided, which is implemented using the above-described apparatus and includes the following steps:

[0050] S1. Centrifuge 10-50 mL of urine sample to remove cell precipitate and collect the supernatant.

[0051] S2. The supernatant is added to the filter and a negative pressure (-0.08 to -0.1 MPa) is applied for filtration, so that the supernatant first passes through multiple layers of the glass fiber membrane for gradient impurity removal, and then passes through the modified microporous filter membrane to enrich exosomes and nucleic acids;

[0052] S3. Elute the modified microporous membrane enriched with exosomes and nucleic acids, and collect the eluent. Specifically, use Tris-HCl or PBS buffer containing 1-2M NaCl as the eluent. Vortex the membrane to desorb the exosomes and nucleic acids, and collect the eluent to obtain high-purity exosomes and nucleic acids. The eluent can then be used for subsequent detection, such as real-time quantitative PCR, digital PCR, and isothermal amplification.

[0053] This method achieves integrated operation from sample processing to target enrichment, effectively solving the problems of target loss and low efficiency caused by multi-step processes.

[0054] The embodiments of the present invention solve the following core problems existing in the current urine HPV detection technology:

[0055] Severe target loss: Existing technologies treat exosome isolation and nucleic acid extraction as separate steps, resulting in significant loss of viral particles and nucleic acids carried by exosomes during multiple transfer operations.

[0056] Low detection sensitivity: Due to insufficient enrichment efficiency, low concentrations of HPV nucleic acid in urine are difficult to reach a reliable detection threshold, and the detection limit of existing methods is usually higher than 1000 copies / mL.

[0057] The process is cumbersome and time-consuming: it requires sequential separation of exosomes, extraction of nucleic acids, and detection and analysis, and the whole process takes 4-6 hours, which cannot meet the needs of rapid screening.

[0058] High cost: It relies on expensive equipment and reagents such as ultracentrifugation and commercial nucleic acid extraction kits, making it difficult to popularize in primary healthcare institutions.

[0059] This invention successfully established an integrated technology platform for the simultaneous enrichment of exosomes and HPV nucleic acids in urine. This platform enables single-step enrichment of the target analytes, greatly simplifying the operational process and significantly improving detection sensitivity, raising the detection limit to below 100 copies / mL. This invention also develops a low-cost detection solution suitable for primary healthcare and large-scale screening. Specifically, it can be used for non-invasive cervical cancer screening, dynamic monitoring of treatment efficacy, HPV infection screening, large-scale population screening, epidemiological surveys, and vaccine efficacy evaluation. Furthermore, it can be extended to non-invasive diagnosis of other viral diseases, tumor marker detection, and liquid biopsy technology platforms.

[0060] Example 1: This example provides a modified microporous filter membrane for enriching biological particles, the preparation method of which specifically includes the following steps:

[0061] S1. After vacuum filtration and cleaning the MCE membrane with ultrapure water, it is activated by vacuum filtration with 1mM NaOH solution, and then washed with ultrapure water until neutral to obtain the activated MCE membrane.

[0062] S2. After equilibration in Tris-HCl buffer (10 mM, pH 8.5), the activated MCE membrane is placed in a 1 mg / mL tannic acid solution for 1 hour. Then, polyethyleneimine with a final concentration of 2 mg / mL is added and the reaction continues for 2 hours. Sodium cyanoborohydride with a final concentration of 15 mM is added for reduction and stabilization for 3 hours. Finally, the membrane is thoroughly washed with ultrapure water to obtain the modified microporous filter membrane.

[0063] Example 2: This example provides a modified microporous filter membrane for enriching biological particles, the preparation method of which specifically includes the following steps:

[0064] S1. After vacuum filtration and cleaning the MCE membrane with ultrapure water, it is activated by vacuum filtration with 0.5mM NaOH solution, and then washed with ultrapure water until neutral to obtain the activated MCE membrane.

[0065] S2. After equilibration in Tris-HCl buffer (10 mM, pH 8.5), the activated MCE membrane is placed in 0.5 mg / mL tannic acid solution for 1 hour. Then, polyethyleneimine with a final concentration of 1.5 mg / mL is added and the reaction continues for 2 hours. Sodium cyanoborohydride with a final concentration of 10 mM is added for reduction and stabilization for 3 hours. Finally, the membrane is thoroughly washed with ultrapure water to obtain the modified microporous filter membrane.

[0066] Example 3: This example provides a modified microporous filter membrane for enriching biological particles, the preparation method of which specifically includes the following steps:

[0067] S1. After vacuum filtration and cleaning the MCE membrane with ultrapure water, it is activated by vacuum filtration with 1.5mM NaOH solution, and then washed with ultrapure water until neutral to obtain the activated MCE membrane.

[0068] S2. After equilibration in Tris-HCl buffer (10 mM, pH 8.5), the activated MCE membrane is placed in 1.5 mg / mL tannic acid solution for 1 hour. Then, polyethyleneimine with a final concentration of 2.5 mg / mL is added and the reaction continues for 2 hours. Sodium cyanoborohydride with a final concentration of 20 mM is added for reduction and stabilization for 3 hours. Finally, the membrane is thoroughly washed with ultrapure water to obtain the modified microporous filter membrane.

[0069] Example 4: This example provides a device for enriching pathogen nucleic acids from non-invasive liquid samples, including a filter and a multilayer membrane stack structure. The filter is an existing 47mm diameter sand core filter, which includes a base, a funnel, and a vacuum filtration device. A modified microporous membrane with a pore size of 0.22μm, prepared in Example 1, is placed on the base. On the modified microporous membrane, a layer of 0.45μm, a layer of 0.8μm, and a layer of 1.2μm glass fiber membrane are stacked sequentially. The membranes are in close contact to form a gradient structure with decreasing pore size from top to bottom. Then, the funnel is installed and fixed with clamps to obtain a complete device for enriching pathogen nucleic acids from non-invasive liquid samples.

[0070] Example 5: This example provides a device for enriching pathogen nucleic acids from non-invasive liquid samples, including a filter and a multilayer membrane stack structure. The filter is an existing 47mm diameter sand core filter, which includes a base, a funnel, and a vacuum filtration device. A modified microporous membrane with a pore size of 0.22μm, prepared in Example 1, is placed on the base. On the modified microporous membrane, a layer of 0.7μm, a layer of 1.0μm, and a layer of 1.2μm glass fiber membrane are stacked sequentially. The membranes are in close contact to form a gradient structure with decreasing pore size from top to bottom. Then, the funnel is installed and fixed with clamps to obtain a complete device for enriching pathogen nucleic acids from non-invasive liquid samples.

[0071] Example 6: This example provides a method for enriching pathogen nucleic acids from non-invasive liquid samples, implemented using the apparatus provided in Example 4 above, and includes the following steps:

[0072] S1. Centrifuge 10 mL of morning urine sample at 2000 g for 10 minutes to remove cell precipitate and collect supernatant;

[0073] S2. Add the supernatant to the filter funnel, turn on the vacuum pump to apply a negative pressure of -0.1MPa for filtration, so that the supernatant first passes through a multilayer glass fiber membrane for gradient impurity removal, and then the modified microporous filter membrane enriches exosomes and nucleic acids.

[0074] S3. After filtration, disassemble the device, discard the upper glass fiber membrane, take out the bottom modified microporous filter membrane, put it into a centrifuge tube, add 500 μL of Tris-HCl buffer (pH 8.0) containing 1.5 M NaCl as the elution solution, and then vortex for 5 minutes to elute.

[0075] S4. The eluent was collected, and nanoparticle tracking analysis showed that the exosome recovery rate was extremely high, and electron microscopy revealed that the exosomes remained morphologically intact. Compared with the traditional ultracentrifugation method, this method reduces the operation time by more than 90%.

[0076] Comparative Example 1: This comparative example provides a device for enriching pathogen nucleic acids from non-invasive liquid samples, including a filter and a multilayer membrane stack structure; wherein the filter is an existing 47mm diameter sand core filter, which includes a base, a funnel, and a vacuum filtration device; an unmodified MCE membrane is placed on the base; a modified microporous filter membrane is stacked sequentially on top of the modified microporous filter membrane: a 0.7μm layer, a 1.0μm layer, and a 1.2μm layer of glass fiber membrane; the membrane layers are in close contact to form a gradient structure with decreasing pore size from top to bottom; then the funnel is installed and fixed with clamps to obtain a complete device for enriching pathogen nucleic acids from non-invasive liquid samples.

[0077] Comparative Example 2: This comparative example provides a method for enriching pathogen nucleic acids from non-invasive liquid samples. The only difference between this method and Example 6 is that the apparatus of Comparative Example 1 is used instead of the apparatus of Example 4 for enrichment. All other aspects are the same as those in Example 6.

[0078] Experimental testing: 1. Surface position detection was performed on the modified microporous filter membrane and the unmodified MCE membrane prepared in Example 1 above. The results are shown in Table 1.

[0079] Table 1 Surface potential of MCE films before and after modification

[0080]

[0081] As can be seen from Table 1, the surface potential of the modified microporous filter membrane prepared in the embodiments of the present invention has been significantly improved.

[0082] II. The exosome extraction efficiency of the methods provided in Example 6 and Comparative Example 2 was tested, and the results are shown in Table 2.

[0083] Table 2

[0084]

[0085] As can be seen from Table 2, using the modified microporous membrane provided in the embodiments of the present invention for exosome enrichment can significantly improve the exosome extraction efficiency, which is about 5 times higher than that of the unmodified MCE membrane.

[0086] In addition, electron micrographs of the exosomes enriched in Comparative Example 2 and Example 6 are shown below. Figure 2 and Figure 3 As shown in the figure, compared with the unmodified microporous filter membrane used in Comparative Example 2, the modified microporous filter membrane used in this embodiment of the invention extracts more exosomes smaller than 200 nm and with more uniform particle size. This indicates that the modified microporous filter membrane provided in this embodiment of the invention can effectively enrich exosomes in urine, and has higher purity and greater quantity.

[0087] III. To evaluate the impact of different sample types on nucleic acid extraction efficiency, the concentration of DNA extracted from exosomes enriched in Example 6 and raw urine samples was measured respectively. The results are as follows: Figure 4 As shown; Figure 4In the figure, the bar chart shows the DNA extraction concentration distribution (unit: ng / μL) of urine (light blue bar on the left, n=6) and urine exosomes (dark blue bar on the right, n=10) using two methods; the error bars represent the standard deviation (SD) (urine group: ±5.34 ng / μL; exosome group: ±5.37 ng / μL), and the scatter plots above the bars represent the original values ​​of each sample; *P<0.05 indicates that the difference is statistically significant. The results show that the DNA concentration in the exosomes enriched in Example 6 was significantly higher than the DNA concentration directly extracted from the original urine sample (mean concentration: 23.49±5.37 ng / μL vs. 17.38±5.34 ng / μL, P=0.04, t-test), indicating that the modified microporous membrane exosome enrichment process helps improve the efficiency of urine DNA acquisition. This result supports the potential application value of exosomes as a non-invasive sample type in HPV detection.

[0088] IV. Six HPV-negative and five HPV-positive urine samples, confirmed using the clinical gold standard detection method, were processed according to the method provided in Example 6 above. qPCR experiments were performed using HPV L1 gene (MY9 / 11) primers and GAPDH internal reference primers. In this embodiment, the relative expression level of HPV was calculated using the 2^(-ΔΔCt) method. The specific calculation process is as follows: First, the data were normalized to the internal reference gene using ΔCt = Ct(MY) - Ct(GAPDH). Then, the expression level of each sample was compared with the baseline expression level of the negative group using ΔΔCt = ΔCt(sample) - ΔCt(negative group average). Finally, the relative expression fold was obtained using the formula 2^(-ΔΔCt). Figure 5 As shown, based on this calculation method, the relative expression level of HPV in positive urine samples was significantly higher than that in the negative group. The expression level of positive samples had a wider distribution range and a significantly upward shift in the median, indicating a high heterogeneity in viral load among HPV-infected individuals; while the expression level in negative samples was concentrated around the baseline level (relative expression level ≈ 1). Statistical analysis showed a significant difference between the two groups (p < 0.05). The results indicate that the method provided in this embodiment of the invention has the same accuracy as the gold standard method.

[0089] V. To verify the detection performance of the above-described embodiments in real clinical scenarios, the present invention conducted the following large-scale clinical sample verification experiment. Urine was processed using the method provided in Embodiment 6 above, and then urine HPV nucleic acid detection was performed. The sample sources were: paired urine and cervical samples from 89 female patients diagnosed with HPV using the gold standard method (cervical swab); positive group: 57 cases (samples diagnosed as HPV positive by the gold standard); negative group: 32 cases (samples diagnosed as HPV negative by the gold standard); detection method: urine samples were enriched using the method provided in Embodiment 6 above and then subjected to qPCR detection; cervical samples were extracted using traditional DNA methods and then subjected to qPCR detection; the detection results of the urine samples were compared with those of the cervical swab method, as shown in Table 3.

[0090] Table 3

[0091]

[0092] As can be seen from Table 3, the method provided in this embodiment of the invention, when applied to urine HPV nucleic acid detection, achieves an overall concordance rate of 95.5% with the clinical gold standard detection results.

[0093] In summary, the technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0094] 1. Improved technical performance:

[0095] Enrichment efficiency: Exosome recovery rate >85%, nucleic acid recovery rate >90%.

[0096] Detection sensitivity: The detection limit for HPV DNA is 50 copies / mL, which is 20 times higher than existing methods;

[0097] Operation time: From sample preparation to elution buffer preparation, it only takes 30 minutes, which is 80% shorter than traditional methods;

[0098] Sample adaptability: It maintains stable performance for urine samples with different characteristics.

[0099] 2. Advantages in clinical application:

[0100] Consistency of detection: The concordance rate with cervical sample test results reached 95.2%;

[0101] Multiplex detection: can simultaneously detect exosome markers and HPV nucleic acid;

[0102] Early diagnosis: Excellent detection rate for early infection and low viral load samples;

[0103] 3. Economic benefits:

[0104] Cost reduction: The cost of a single detection material is only 1 / 5 of that of a commercial kit;

[0105] Simplified equipment: No need for expensive equipment such as high-speed centrifuges;

[0106] Increased throughput: Enables batch processing of samples, suitable for large-scale screening.

[0107] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A modified microporous filter membrane for enriching biological particles, characterized in that, The surface of the modified microporous filter membrane is cross-linked with tannic acid and polyethyleneimine to form a stable positively charged coating. This coating results in a positive Zeta potential for the modified microporous filter membrane in an aqueous solution with a pH of 7.0, and the modified microporous filter membrane can simultaneously adsorb and capture exosomes and nucleic acids from liquid samples. The preparation method of the modified microporous filter membrane includes the following steps: A mixed cellulose ester microporous filter membrane is provided and activated by alkaline solution to obtain an activated membrane; The activated membrane was equilibrated in a weakly alkaline buffer solution with a pH of 8.0-9.

0. In the weakly alkaline buffer solution, the membrane surface is first brought into contact with the tannic acid solution and a first reaction is carried out. Then, without changing the system, a polyethyleneimine solution is directly added to carry out a second reaction, thereby forming a phenol-amine crosslinking network in situ on the membrane surface. Sodium cyanoborohydride was added to the weakly alkaline buffer solution for reduction and stabilization treatment to obtain a modified microporous filter membrane with a stable positive charge on its surface. The concentration of the tannic acid solution is 0.5-1.5 mg / mL; the final concentration of the polyethyleneimine is 1.5-2.5 mg / mL; and the final concentration of the sodium cyanoborohydride is 10-20 mM.

2. The modified microporous filter membrane for enriching biological particles according to claim 1, characterized in that, The alkaline solution is a NaOH solution with a concentration of 0.5-1.5 mM.

3. The modified microporous filter membrane for enriching biological particles according to claim 1, characterized in that, The buffer solution is a Tris-HCl buffer solution with a concentration of 8-12 mM and a pH value of 8-9.

4. The application of a modified microporous filter membrane for enriching biological particles as described in any one of claims 1-3 in a preparation apparatus or kit, characterized in that, The device or kit is used to enrich pathogen nucleic acids from non-invasive liquid samples.

5. An apparatus for enriching pathogenic nucleic acids from non-invasive liquid samples, comprising a filter, characterized in that, It also includes a multilayer membrane stacked structure; the multilayer membrane stacked structure includes a pretreatment layer and a core capture layer from top to bottom; the pretreatment layer includes a multilayer glass fiber membrane with gradient pore size; the core capture layer is a modified microporous filter membrane according to any one of claims 1-3.

Images