Porous filter membranes for efficient capture and immediate quantitative detection of airborne pathogens, their preparation methods and applications

Abstract

This invention discloses a porous filter membrane for efficient capture and real-time quantitative detection of airborne pathogens, its preparation method, and its applications, belonging to the field of filter membrane technology. The technical solution is as follows: the porous filter membrane has a micro-nano porous structure, enabling efficient filtration of air and capture and collection of airborne pathogens. It achieves a 99.4% rejection rate for bioaerosol particles and can disintegrate to release the captured airborne pathogens after capture. The porous filter membrane of this invention is prepared by a solvent exchange method. It not only efficiently filters air and captures and collects airborne pathogens, but its high water solubility allows for rapid disintegration after sampling, fully releasing the captured airborne pathogens. This significantly reduces sample loss during elution and extraction, improves the accuracy of subsequent sample analysis and detection, and enables real-time quantitative detection of airborne pathogens after sampling and enrichment. It has significant application prospects in bioaerosol sampling and real-time monitoring.

Description

Technical Field

[0001] This invention belongs to the field of filter membrane technology, specifically relating to a porous filter membrane for efficient capture and real-time quantitative detection of airborne pathogens, its preparation method, and its application. Background Technology

[0002] Ubiquitous airborne microorganisms in the environment can have numerous adverse effects on human health, such as allergies, inflammatory reactions, and infectious diseases. Pathogens hidden in bioaerosols are small and lightweight, easily spreading over large areas of the air, leading to large-scale transmission and infection. Quantitative detection of airborne pathogens is a crucial approach for early prediction and timely warning of aerosol-borne diseases. Traditional detection methods use gravity sedimentation to collect airborne pathogens onto agar plates and analyze bioaerosols in the environment through colony culture. However, this passive sampling and detection method is not only inefficient and time-consuming, often requiring several days of incubation to obtain microbial colonies, but also fails to accurately reflect the true state of bioaerosols in the environment because the proportion of culturable microorganisms in the overall microbial population is very small. Therefore, there is an urgent need to develop a more efficient and sensitive capture and detection system to track airborne pathogens.

[0003] Air filter membranes effectively intercept particulate matter and capture airborne microorganisms, playing a crucial role in preventing and controlling bioaerosol pollution and its spread. The porous filter media within these membranes can directly block the migration of microbial particles through various interaction forces, such as interception, sieving, diffusion, and inertial impaction, efficiently collecting bioaerosols across a wide particle size range. Generally, synthetic polymers and glass fibers are used as raw materials for preparing porous filter membranes. However, due to their insoluble and opaque nature, it is difficult to distinguish microbial colonies on culture dishes when using culturing methods to detect airborne microorganisms captured by these membranes. Especially when sampling in areas with high microbial contamination, colony overload often occurs on the filter membrane, hindering colony counting and identification. Although collected airborne microorganisms can be eluted from the filter media before analysis, insufficient elution and transfer often result in significant sample loss, greatly reducing the accuracy of subsequent detections. Furthermore, repeated washing and extraction of the filter membrane further increases the risk of sample contamination. In this context, although biomaterials such as gelatin, fructan, and chitosan hydrochloride have been used to develop soluble air filter membranes to replace traditional non-degradable filter membranes, their applications are currently limited to the capture and detection of viruses, and are constrained by high costs and complex manufacturing processes. Therefore, there is an urgent need to improve existing air filter membranes and expand their applications to better capture and detect airborne pathogens. Summary of the Invention

[0004] This invention provides a porous filter membrane for efficient capture and immediate quantitative detection of airborne pathogens, its preparation method, and its application. It aims to solve the problems of low sampling and capture efficiency, difficulty in sample transfer after sampling, limited detection methods, and inaccurate detection results associated with existing air filter membranes. The porous filter membrane of this invention can efficiently filter air and capture and collect airborne pathogens. Furthermore, its high water solubility allows it to quickly disintegrate after sampling, fully releasing the captured airborne pathogens. This significantly reduces sample loss during elution and extraction, improving the accuracy of subsequent sample analysis and detection.

[0005] The technical solution of this invention is as follows:

[0006] In one aspect, a porous filter membrane for efficient capture and immediate quantitative detection of airborne pathogens is disclosed. The porous filter membrane has a micro-nano porous structure, which can efficiently filter air and capture and collect airborne pathogens. It has a rejection rate of 99.4% for bioaerosol particles and can disintegrate and release the captured airborne pathogens after capture.

[0007] Preferably, the porous filter membrane automatically disintegrates upon contact with water. After contact with water, the cross-linked network of pores decomposes and collapses, and the three-dimensional structure disappears after collapse, restoring the membrane solution. The disintegration time is 2-3 minutes.

[0008] Preferably, the porous filter membrane is prepared by solvent exchange of a solute; the solute includes at least two of hydroxypropyl methylcellulose, pregelatinized starch, dextrin, xylan, tamarind gum, gelatin, sodium hyaluronate, and modified chitosan.

[0009] Secondly, a method for preparing the porous filter membrane for efficient capture and immediate quantitative detection of airborne pathogens is disclosed, specifically including the following steps:

[0010] 1) Preparation of initial membrane solution: Dissolve the solute in deionized water, heat and stir until dissolved, then add solvent A and continue stirring to obtain a stable and clear membrane solution;

[0011] 2) Solvent exchange: Pour the membrane solution into a petri dish, add undesirable solvent B to perform solvent exchange, and the solute in the membrane solution gradually dehydrates and precipitates out;

[0012] 3) Membrane formation: Add undesirable solvent C to continue inducing solute dehydration and precipitation until the membrane is fully formed;

[0013] 4) Drying: The formed filter membrane is vacuum dried to obtain the porous filter membrane.

[0014] Preferably, solvent A includes at least one of methanol, ethanol, glycerol, acetone, dimethyl sulfoxide, and dimethylformamide; the volume ratio of water to solvent A in the initial membrane solution is 1:(0.4-0.9); the initial membrane solution is prepared at a temperature of 40-100°C and the stirring time is 30-120 min.

[0015] Preferably, solvent B is at least one of ethyl acetate, methanol, methyl acetate, ethanol, acetone, dimethyl sulfoxide, dichloromethane, and petroleum ether, and the volume ratio of solvent B to membrane solution is 1-6:1-6. The solvent exchange temperature in step 2) is 10-50°C, and the time is 1-5 h.

[0016] Preferably, solvent C is at least one selected from methanol, ethanol, glycerol, n-butanol, tert-butanol, methyl acetate, ethyl acetate, acetone and cyclohexane, the volume ratio of solvent C to membrane solution is 1-4:1-4, the molding temperature is 10-50℃, and the molding time is 1-8h.

[0017] Preferably, the drying method is freeze drying, and the time is 1-8 hours.

[0018] Thirdly, the application of the porous filter membrane in air filtration, airborne pathogen capture, and bioaerosol monitoring is disclosed. The porous filter membrane is cut and placed on a suitable filter sampler. The sampling flow rate and sampling time are set to filter the air and perform sampling. After sampling, the filter membrane is removed, and the concentration of bioaerosols in the sampling environment is calculated by analyzing the airborne microorganisms trapped on the filter membrane.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. This invention prepares porous air filter membranes by solvent exchange, which is simple, effective, low-cost, highly practical, and environmentally friendly.

[0021] 2. The porous air filter membrane prepared by this invention can not only effectively control its micro-nano porous structure to efficiently filter air and capture and collect airborne pathogens, including Gram-positive bacteria, Gram-negative bacteria, and fungi, with a rejection rate of 99.4% for bioaerosol particles, but also has the property of being highly soluble in water, which allows it to quickly disintegrate after sampling, with a disintegration time of 2-3 minutes, fully releasing the captured airborne pathogens, greatly reducing sample loss during elution and extraction, and improving the accuracy of subsequent sample analysis and detection.

[0022] 3. After sampling, the collected airborne microbial samples are suitable for various detection methods. By effectively combining them with the ATP bioluminescence detection method, rapid quantitative detection of airborne microorganisms can be achieved. The detection time is only 10 minutes, which greatly improves the detection efficiency and has important application prospects in the fields of real-time monitoring of bioaerosols and prevention and control of environmental microbial pollution. Attached Figure Description

[0023] Figure 1 Photograph (A) of the porous filter membrane prepared in Example 1 of the present invention, and SEM images of its front (B), back (C) and cross-section (D) microstructures;

[0024] Figure 2 The following are SEM images showing the structural evolution of the porous filtration membrane provided by the present invention as the volume of undesirable solvent in the initial membrane solution increases; wherein (A) is the reverse side of the membrane material prepared in Comparative Example 1, (B) is the reverse side of the membrane material prepared in Example 2, and (C) is the reverse side of the membrane material prepared in Comparative Example 2.

[0025] Figure 3 This is a SEM image showing the microstructure changes of the porous filter membrane prepared in Example 2 of the present invention after it comes into contact with water.

[0026] Figure 4 This is a schematic diagram of the recyclable preparation process of the porous filter membrane prepared in Embodiment 2 of the present invention;

[0027] Figure 5 The effects of different concentrations (0-30 mg / mL) of the filter membrane prepared in Example 1 of this invention on the cell viability of Escherichia coli, Staphylococcus aureus and Candida albicans were investigated.

[0028] Figure 6 This is a SEM comparison image of the microstructure of the porous filter membrane prepared in Example 1 of the present invention before and after sampling;

[0029] Figure 7 The correspondence between the CFU value of different microbial colony counts and the corresponding RLU value of ATP bioluminescence intensity, with or without the presence of the porous filter membrane (HAFM) prepared in Example 2 of the present invention;

[0030] Figure 8 The relationship between the CFU value of mixed microbial colonies and the corresponding RLU value of ATP bioluminescence intensity, with or without the presence of the porous filter membrane (HAFM) prepared in Example 2 of the present invention;

[0031] Figure 9 The results of colony culture obtained by different sampling methods at the same bioaerosol concentration;

[0032] Figure 10 The colony culture results obtained by different sampling methods under high bioaerosol concentrations;

[0033] Figure 11 The results of colony culture of the remaining filter membrane (A) and the eluent (B) after washing and shaking following bioaerosol sampling of a commercial MCE filter membrane;

[0034] Figure 12 A comparison chart showing the number of microorganisms measured by colony culture and ATP luminescence method after sampling the porous filter membrane prepared in Example 2 of the present invention under different bioaerosols. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0036] Example 1

[0037] This embodiment provides a method for preparing a porous filter membrane for efficient capture and immediate quantitative detection of airborne pathogens, including the following steps:

[0038] 1) Preparation of initial membrane solution: Dissolve sodium hyaluronate (0.2g) and gelatin (0.4g) in deionized water (10.0mL) and mix thoroughly. Heat to 60℃ and stir for 30min. Add anhydrous ethanol (5mL) dropwise and continue stirring for 30min until the solution becomes clear to obtain a stable membrane solution.

[0039] 2) Solvent exchange: Pour 1 mL of membrane solution into a petri dish, add 2 mL of anhydrous ethanol for solvent exchange, at 20°C.

[0040] After standing at the temperature for 3 hours, the solute in the membrane solution gradually dehydrates and precipitates out;

[0041] 3) Membrane formation: After adding 2 mL of a mixed solvent of anhydrous ethanol and ethyl acetate in a volume ratio of 1:1, continue to induce solute dehydration at 20℃ for 4 h until the membrane solution is completely transformed into a solid membrane and the filter membrane is fully formed.

[0042] 4) Drying: The formed filter membrane is subjected to vacuum freeze drying for 5 hours to obtain the porous filter membrane.

[0043] Figure 1The images show SEM images of the dried porous filter membrane and its front, back, and cross-sectional microstructures. The images reveal that the porous filter membrane possesses a unique heterogeneous micro / nano porous structure, with countless interconnected network channels existing between the biomolecule particles. This porous network structure enables the filter membrane to efficiently trap and collect microbial particles during sampling.

[0044] Example 2

[0045] 1) Preparation of initial membrane solution: Dissolve sodium hyaluronate (0.2g) and gelatin (0.4g) in deionized water (10.0mL) and mix thoroughly. Heat to 60℃ and stir for 30min. Add anhydrous ethanol (4mL) dropwise and continue stirring for 30min until the solution becomes clear to obtain a stable membrane solution.

[0046] 2) Solvent exchange: Pour 1 mL of membrane solution into a petri dish, add 2 mL of anhydrous ethanol for solvent exchange, at 20°C.

[0047] After standing at the temperature for 3 hours, the solute in the membrane solution gradually dehydrates and precipitates out;

[0048] 3) Membrane formation: Replace solvent C, add 2 mL of a mixed solvent of anhydrous ethanol and ethyl acetate in a 1:1 ratio, and continue to induce solute dehydration at 20°C for 4 h until the membrane solution is completely transformed into a solid membrane and the filter membrane is fully formed.

[0049] 4) Drying: The formed filter membrane was subjected to vacuum freeze-drying for 5 hours to obtain the porous filter membrane. The SEM image of the reverse side of the prepared membrane material is shown below. Figure 2 As shown in (B).

[0050] Comparative Example 1

[0051] Unlike Example 2, in this comparative example, 0 mL of ethanol was added in step 1). The SEM image of the reverse side of the prepared membrane material is shown below. Figure 2 As shown in (A).

[0052] Comparative Example 2

[0053] Unlike Example 2, this comparative example modulates the structure by changing the volume ratio of anhydrous ethanol in the initial membrane solution. Specifically, in step 1), the amount of anhydrous ethanol added is 10.0 mL, and excess ethanol is added to the initial membrane solution. The rest is the same as in Example 2. The SEM image of the reverse side of the prepared filter membrane is shown below. Figure 2 (C).

[0054] Figure 2The image shows the evolution of the microstructure of the filter membrane as the volume of the undesirable solvent A in the initial membrane solution increases. It can be seen that the microstructure of the filter membrane changes significantly with the increase of the volume of undesirable solvent added. In the absence of undesirable solvent, the filter membrane exhibits a denser arrangement and a flatter stacking morphology. However, with the addition of excessive undesirable solvent, the filter membrane exhibits a looser arrangement and a coarser stacking morphology.

[0055] Comparative Example 3

[0056] Unlike Example 2, sodium hyaluronate was replaced with sodium alginate in step 1) of this comparative example; the rest was the same as in Example 2. In this case, the viscosity of the membrane solution increased significantly, preventing the solute from precipitating during subsequent solvent exchange and thus hindering membrane formation.

[0057] Comparative Example 4

[0058] Unlike Example 2, in step 1) of this comparative example, gelatin is replaced with agar, while the rest is the same as in Example 2. In this case, the membrane solution requires high-temperature heating to prepare, and the solute cannot precipitate during solvent exchange, preventing the filter membrane from forming.

[0059] Comparative Example 5

[0060] Unlike Example 2, in step 1) of this comparative example, anhydrous ethanol was replaced with ethyl acetate, while the rest was the same as in Example 2. Since ethyl acetate is insoluble in water and cannot be mixed with the membrane solution, a stable membrane solution could not be obtained.

[0061] Comparative Example 6

[0062] Unlike Example 2, in step 1) of this comparative example, the dissolution temperature was adjusted to 30°C, while the rest was the same as in Example 2. At this point, the solute could not be completely dissolved, resulting only in a viscous mixture, making further steps impossible.

[0063] Comparative Example 7

[0064] Unlike Example 2, in step 1) of this comparative example, the amounts of raw materials used were changed to sodium hyaluronate (0.01 g) and gelatin (0.4 g), while the rest remained the same as in Example 2. Although the solute could still precipitate after solvent exchange, the particles were loosely packed and could not aggregate into a cohesive mass, making it impossible to proceed to the next step.

[0065] Comparative Example 8

[0066] Unlike Example 2, only 0.2 mL of anhydrous ethanol was added in step 2) of this comparative example, while the rest was the same as in Example 2. In this case, the unsuitable solvent was severely insufficient and could not induce solute precipitation.

[0067] Comparative Example 9

[0068] Unlike Example 2, the solvent exchange time in step 2) of this comparative example is 0.5 h, while the rest is the same as in Example 2. In this case, the solvent exchange time is too short, and only a small amount of solute is precipitated, making it impossible to proceed to the next step.

[0069] Comparative Example 10

[0070] Unlike Example 2, in step 2) of this comparative example, the solvent exchange temperature is set to 60°C, while the rest is the same as in Example 2. At this temperature, a large amount of solvent will evaporate, affecting solute precipitation and preventing the filter membrane from forming.

[0071] Comparative Example 11

[0072] Unlike Example 2, in step 3) of this comparative example, only 0.2 mL of solvent C was added, while the rest was the same as in Example 2. At this point, the amount of solvent was severely insufficient, and the filter membrane could not be formed.

[0073] Comparative Example 12

[0074] Unlike Example 2, the membrane forming time in step 3) of this comparative example is 0.5 h, while the rest is the same as in Example 2. In this case, the membrane forming time is too short, and the precipitate remains viscous and cannot be dried.

[0075] Comparative Example 13

[0076] Unlike Example 2, in step 3) of this comparative example, the solvent (ethyl acetate and ethanol) was replaced with methanol; otherwise, it was the same as in Example 2. The membrane solution remained viscous after 4 hours of soaking, and the filter membrane could not form.

[0077] Comparative Example 14

[0078] Unlike Example 2, in step 4) of this comparative example, freeze drying is replaced with oven drying; the rest is the same as in Example 2. The white filter membrane collapses during the drying process and cannot maintain its original morphology.

[0079] Example 3: Verification of the water solubility of porous filter membranes

[0080] The porous filter membrane (30 mg) prepared in Example 2 was transferred to a centrifuge tube, 1.0 mL of deionized water was added, and the membrane was shaken before observing the changes. The hyaluronic acid component within the porous filter membrane imparts good water solubility to the membrane. Figure 3The diagram shows the microstructural changes of the porous filter membrane upon contact with water. It reveals that once the dried membrane comes into contact with water, its internal porous cross-linked network automatically decomposes within 3 minutes. The three-dimensional structure completely disappears after collapse, while the structure of the dried portion of the membrane remains stable. Semi-disintegrated biomolecular network chains can be directly observed at the interface between the dry and wet membranes. Furthermore, the porous cross-linked network structure within the filter membrane is guided by multiple non-covalent bonds between biomolecules, and the entire preparation process does not involve chemical reactions; the formation and disintegration of the filter membrane are reversible. Figure 4 This diagram illustrates the recyclable preparation process of the filter membrane. When the filter membrane is immersed in water, the white solid membrane rapidly absorbs the water, returning to its original membrane solution. After further solvent exchange and vacuum drying, the same porous filter membrane can be obtained again. The recyclable raw materials and green preparation process provide significant advantages for the large-scale production of this porous air filter membrane. Furthermore, the excellent water solubility of the filter membrane and its rapid structural disintegration upon contact with water greatly facilitate the transfer of captured microbial samples after filtration. Airborne microorganisms captured and collected on the filter membrane can be easily released and resuspended in water, effectively avoiding sample loss.

[0081] Example 4: Biocompatibility assessment of the porous filtration membrane

[0082] To further evaluate the biocompatibility of the porous filtration membrane, different microbial suspensions were mixed with membrane solutions of varying concentrations. First, the porous filtration membrane prepared in Example 1 was dissolved in culture medium to prepare a concentrated solution (60 mg / mL), which was then diluted at different ratios to prepare membrane solutions of different concentrations (0-30 mg / mL). Then, the membrane solutions of different concentrations were mixed with *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans*, respectively, to prepare bacterial suspensions of different concentrations. The mixed solutions were inoculated onto culture plates and incubated for 48 hours before colony counting (culture conditions: *Escherichia coli* and *Staphylococcus aureus* inoculated on LB agar medium, incubated at 37°C; *Candida albicans* inoculated on YPD agar medium, incubated at 30°C). Figure 5 The microbial activities of E. coli, S. aureus, and C. albicans treated with membrane solutions of different concentrations were compared. It can be seen that as the concentration of the membrane solution increases, the three microorganisms can still maintain high biological activity, indicating that the prepared porous filter membrane has good biocompatibility.

[0083] Example 5: Test of the trapping performance of the porous filtration membrane

[0084] A bioaerosol environment was simulated within a closed atomization chamber with a volume of 800mm × 600mm × 800mm to evaluate and test the bactericidal performance of the porous filter membrane prepared in Example 1 of this invention. First, different concentrations (10... 4 -10 7 A microbial suspension (E. coli, S. aureus, and C. albicans) at CFU / mL was dispersed in PBS and loaded into a nebulizer bottle. A bioaerosol generator (ZR-C03, Qingdao Zhongrui Intelligent Instrument Co., Ltd., Qingdao, China) was activated to atomize the microbial particles at a flow rate of 10 L / min for 2 min. A particle size analyzer (AQ-Guard, Palas, Germany) was used to monitor the number and diameter distribution of atomized particles, as well as temperature, pressure, and humidity in real time. The porous filter membrane was cut into 25 mm diameter circular pieces and placed on an air-filtering sampler (Sartorius MD8, Germany) for sampling to evaluate the capture performance of the porous filter membrane. After the atomized aerosol was loaded, the air sampler was activated to capture the atomized microbial particles (10 L / min) for 5 min.

[0085] Commercially available mixed cellulose (MCE) filter membranes, purchased from Shanghai Xinya Purification Equipment Factory, were used as a representative of non-degradable filter membranes for comparative capture experiments. The concentration of atomized particles upstream and downstream of the filter membrane was recorded using a particle size analyzer. The porous filter membrane was found to have a 99.4% rejection rate for bioaerosol particles, while the MCE filter membrane, under the same conditions, only achieved a 97.8% rejection rate. This indicates that the porous filter membrane exhibits superior microbial capture performance compared to the MCE filter membrane. The microbial capture performance of the filter membrane was further evaluated by comparing SEM images of the membrane surface before and after sampling. Figure 6 The SEM images of the filter membrane prepared in Example 1 of this invention before and after bioaerosol sampling show that the porous structure in the filter membrane is covered and filled by coarse particles after sampling, and the filter membrane does not undergo any deformation or cracks, indicating that the porous filter membrane can effectively capture particulate matter during air sampling and has good structural stability.

[0086] Example 6: Feasibility assessment of combining the porous filtration membrane with ATP bioluminescence detection method

[0087] Using *E. coli*, *S. aureus*, and *C. albicans* as representative Gram-negative bacteria, Gram-positive bacteria, and fungi, respectively, the feasibility of combining the porous filter membrane with ATP bioluminescence detection was evaluated. First, the correlation between the colony count (CFU) of *E. coli*, *S. aureus*, *C. albicans*, and a mixed suspension of the three microorganisms under different concentrations and the ATP bioluminescence intensity (RLU) was established. Specifically, the cultured microorganisms were diluted with PBS to prepare a series of concentrations (10... 3 -10 8 Microbial suspensions of different concentrations (10 μL) were prepared, and then CTAB lysis buffer (100 μL, 0.015%) was mixed to achieve microbial lysis. Luciferin / luciferase solution (100 μL) was then added, and RLU values ​​were read using a microplate reader. An equal volume of PBS was added as a blank control, and the RLU value was recorded as a background value. Mixed microbial samples were prepared by uniformly mixing suspensions of E. coli, S. aureus, and C. albicans at a 1:1:1 ratio, and then diluting them to a series of mixed microbial suspensions (10 CFU / mL). 3 -10 8 (CFU / mL) Figure 7 A shows the correspondence between the number of colonies (CFU) and the RLU values ​​of E. coli, S. aureus, and C. albicans under different concentration conditions. It can be found that the RLU value increases with the increase of the number of microorganisms, and a linear relationship with similar slope is shown in the three different types of microorganisms.

[0088] Subsequently, the correlation between CFU and RLU values ​​of E. coli, S. aureus, and C. albicans, as well as mixed bacterial suspensions of the three microorganisms, was established under different concentration conditions in the presence of the porous filter membrane prepared in Example 2. The combination of the filter membrane and microorganisms was achieved by adding 10 μL of each of the three types of microbial suspensions to the filter membrane (30 mg). The combined filter membrane rapidly disintegrated upon dissolution in water, and the added microorganisms dispersed in the water. The microorganisms could be enriched from the solution by centrifugation and ATP detection was performed. Figure 7 B shows the correlation between the CFU and RLU values ​​of E. coli, S. aureus, and C. albicans colonies at different concentrations in the presence of the porous filter membrane. It can be observed that the RLU values ​​measured by ATP bioluminescence increase linearly with the increase in the number of the three different microorganisms. Furthermore, by mixing microbial samples to simulate airborne pathogens collected from bioaerosols in a real environment, quantitative detection of the mixed microorganisms by ATP bioluminescence was performed.Figure 8 As shown, two almost overlapping linear relationships were found with or without the filter membrane. The linear relationship between the CFU and RLU values ​​remains unchanged, indicating that the porous filter membrane has a negligible effect on the ATP bioluminescence method. It can be well integrated with the ATP bioluminescence method to achieve real-time quantitative detection of captured airborne pathogens.

[0089] Example 7: Detection of the porous filter membrane after bioaerosol sampling

[0090] After the bacterial suspension is atomized to form a uniform bioaerosol, samples are collected using filtration, impaction, and natural sedimentation methods based on the filter membrane prepared in Example 2 of this invention, and a culture method is used for detection. When the bioaerosol concentration is low, the entire filter membrane can be directly transferred to an agar plate for colony culture after sampling. Figure 9 The results of colony culture obtained using different sampling methods show that, at the same bioaerosol concentration, the filter membrane of this invention collected the largest number of colonies, followed by impactor sampling and natural sedimentation sampling. When sampling the same volume of air, the filter membrane can capture more microorganisms, further demonstrating the high capture efficiency of the porous filter membrane.

[0091] Figure 10 It is at high bioaerosol concentrations (10) 7 Colony culture results obtained using different sampling methods (CFU / mL) showed that agar plates obtained by the impaction method had a large accumulation of colonies, making it impossible to accurately identify single colonies. However, culture plates based on the aforementioned filter membrane could still provide identifiable and countable colonies. This is mainly because the porous filter membrane can be smoothly converted into a membrane solution after sampling, allowing for selective partial culture without using the entire filter membrane for colony culture. This effectively avoids colony overload on the culture plate, thus providing accurate colony counting results.

[0092] Compared to commercially available MCE filter membranes, the porous filter membrane described in this invention requires repeated ultrasonic agitation and washing to elute the captured microbial samples from the membrane because MCE is insoluble in water. After sampling, the MCE filter membrane is cut into small pieces, thoroughly washed and agitated, and the remaining fragments are then plate-cultured. Figure 11 As shown in Figure A, a large number of bacteria remain on the MCE filter membrane fragment, and the colonies grow into patches along the filter membrane edge, making the colonies unidentifiable and uncountable. Furthermore, culturing the MCE eluent also reveals a low colony count. Figure 11As shown in Figure B, only a small fraction of microorganisms can be successfully eluted from the MCE filter membrane. Although commercially available MCE filter membranes can also capture airborne microorganisms, their water-insoluble nature severely affects sample transfer and subsequent detection. Compared to non-degradable commercial filter membranes, the porous filter membrane described in this invention can effectively capture airborne pathogens at various bioaerosol concentrations, is suitable for various processing methods after sampling, and facilitates accurate detection of bioaerosols.

[0093] Example 8: Feasibility assessment of combining the porous filter membrane with ATP bioluminescence detection in bioaerosol sampling

[0094] After bioaerosol sampling, the porous filter membrane prepared in Example 2 was transferred to a centrifuge tube and completely dissolved in water. The captured microorganisms were enriched by centrifugation, and then measured using ATP bioluminescence assay via cell lysis. Compared to the colony culture detection method, which takes 48 hours, the ATP bioluminescence assay can be completed in just 10 minutes. Figure 12 As shown in Figure A, with the increase of bioaerosol concentration, the number of microorganisms detected by both the colony culture method and the ATP method gradually increases. Comparing the quantitative relationship between the two methods, a linear correlation can be observed, both being directly proportional to the total number of bacteria captured by the filter membrane. However, the results measured by the ATP method are higher than those by the plate culture method. This is mainly due to the inherent limitations of the plate culture method, as not all microorganisms transferred into the petri dish can survive and grow uniformly into single colonies. This phenomenon is even more pronounced in the detection of mixed bacteria, such as... Figure 12 As shown in B, this is mainly because different types of microorganisms have different growth and reproduction conditions, and a single plate culture condition cannot meet the requirements for culturing multiple bacteria. Given that the proportion of culturable microorganisms in bioaerosols is very small, and all captured airborne microorganisms can be subjected to cell lysis and ATP detection, the ATP detection method based on the aforementioned filter membrane can provide more timely and accurate results for the quantitative detection of bioaerosols compared to colony culture detection methods.

[0095] This invention presents a porous filter membrane for efficient capture and immediate quantitative detection of airborne pathogens, based on a poor solvent separation strategy. Compared to non-degradable commercial filter membranes, the porous filter membrane prepared in this invention possesses a tunable micro / nanoporous structure, which not only facilitates efficient air filtration and microbial particle retention but also maximizes the transfer of microbial samples after filtration, minimizing sample loss. Compared to traditional impaction sampling methods, the filtration method based on this porous filter membrane can capture more airborne microorganisms, and the sampled microorganisms can be converted into a membrane solution for selective culture, avoiding colony overload on agar plates. Furthermore, the captured microorganisms can be directly enriched for various detection analyses. By combining with ATP bioluminescence, efficient sampling and immediate quantitative detection are effectively integrated.

[0096] Although the present invention has been described in detail by way of preferred embodiments, it is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A porous filter membrane for efficient capture and immediate quantitative detection of airborne pathogens, characterized in that, The porous filter membrane has a micro-nano porous structure, which can efficiently filter air and capture and collect airborne pathogens. It has a 99.4% rejection rate for bioaerosol particles and can disintegrate and release the captured airborne pathogens after capture. The porous filter membrane is prepared by solvent exchange of solutes; the solutes include at least two of the following: hydroxypropyl methylcellulose, pregelatinized starch, dextrin, xylan, tamarind gum, gelatin, sodium hyaluronate, and modified chitosan; The method for preparing the porous filter membrane for efficient capture and immediate quantitative detection of airborne pathogens specifically includes the following steps: 1) Preparation of initial membrane solution: Dissolve the solute in deionized water, heat and stir until dissolved, then add solvent A and continue stirring to obtain a stable and clear membrane solution; 2) Solvent exchange: Pour the membrane solution into a petri dish, add undesirable solvent B to perform solvent exchange, and the solute in the membrane solution will dehydrate and precipitate; 3) Membrane formation: Add undesirable solvent C to continue inducing solute dehydration and precipitation until the membrane is fully formed; 4) Drying: The formed filter membrane is vacuum dried to obtain the porous filter membrane; Solvent A includes at least one of methanol, ethanol, glycerol, acetone, dimethyl sulfoxide, and dimethylformamide. The volume ratio of water to solvent A in the initial membrane solution is 1:(0.4-0.9). The initial membrane solution is prepared at a temperature of 40-100℃ and a stirring time of 30-120 min. Solvent B is at least one of ethyl acetate, methanol, methyl acetate, ethanol, acetone, dimethyl sulfoxide, dichloromethane, and petroleum ether. The volume ratio of solvent B to membrane solution is 1-6:1-6. The solvent exchange temperature in step 2) is 10-50℃ and the time is 1-5h. Solvent C is at least one of methanol, ethanol, glycerol, n-butanol, tert-butanol, methyl acetate, ethyl acetate, acetone and cyclohexane. The volume ratio of solvent C to membrane solution is 1-4:1-4. The molding temperature is 10-50℃ and the molding time is 1-8h.

2. The porous filter membrane for efficient capture and immediate quantitative detection of airborne pathogens as described in claim 1, characterized in that, When the porous filter membrane comes into contact with water, the porous cross-linked network decomposes and collapses, the three-dimensional structure disappears, and it returns to the membrane solution. The disintegration time is 2-3 minutes.

3. The porous filter membrane for efficient capture and immediate quantitative detection of airborne pathogens as described in claim 1, characterized in that, The drying process is freeze drying, which takes 1-8 hours.

4. The application of the porous filter membrane as described in claim 1 in air filtration, airborne pathogen capture, and bioaerosol monitoring, characterized in that, After cutting the porous filter membrane, place it on a suitable filter sampler, set the sampling flow rate and sampling time, filter the air and take samples; after sampling, remove the filter membrane, and calculate the concentration of bioaerosols in the sampling environment by analyzing the air microorganisms trapped on the filter membrane.

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