Integrated method for sampling-capturing microorganisms in aerosols using an integrated sampling enrichment apparatus and immunomagnetic beads incubation technique

Abstract

This invention discloses an integrated method for sampling and capturing microorganisms in aerosols using an integrated sampling and enrichment device and immunomagnetic bead incubation technology. The integrated sampling and enrichment device, equipped with immunomagnetic beads, collects and incubates microorganisms in aerosols; a nanochannel membrane is used to retain large-sized magnetic bead-microorganism conjugates; and an electrochemical sensing platform is used to characterize the sampling and capture effects. This invention achieves highly efficient enrichment of aerosol microorganisms through the synergistic use of immunomagnetic beads and the integrated sampling and enrichment device. The integrated sampling and enrichment device of this invention highly integrates microbial pretreatment and detection modules, achieving both rapid isolation and highly sensitive detection of microorganisms. Furthermore, the integrated sampling device is simple to manufacture and easy to mass-produce.

Description

Technical Field

[0001] This invention belongs to the field of nanochannel electrochemical analysis and sensing technology, specifically relating to an integrated method for sampling and capturing microorganisms in aerosols using an integrated sampling and enrichment device and immunomagnetic bead incubation technology. Background Technology

[0002] Aerosol pathogens mainly include viruses and bacteria (bioaerosols). Due to their small particle size, aerosols are often suspended in the air, making them prone to causing large-scale outbreaks of respiratory infectious diseases. Therefore, numerous sampling and detection methods have been developed for monitoring and preventing avian influenza. Bioaerosol sampling is particularly important for the control of infectious diseases. Currently, traditional bioaerosol sampling methods include natural sedimentation, impaction, percussion, and sampling membrane methods. Commonly used detection methods for bioaerosol pathogens include cell culture colony counting, immunoassay, and nucleic acid detection. Cell culture colony counting requires manual operation, is time-consuming and labor-intensive, and cell culture can exceed 24 hours, making automated rapid detection impossible. PCR methods require sophisticated instruments to precisely control temperature changes and must be operated and analyzed by professional technicians, significantly limiting their application in field testing with limited resources. Traditional immunoassay methods offer relatively simple sample processing, but cross-contamination can occur with complex samples. Several techniques, such as bioaerosol mass spectrometry (BAMS), surface-enhanced Raman spectroscopy (SERS), flow cytometry with fluorescent dyes, and other fluorescence-based techniques, such as ultraviolet aerodynamic particle size analyzer (UVAPS), have been studied or used for the real-time detection of potential airborne biological agents.

[0003] Unfortunately, most of these technologies are unable to perform species-level identification and / or have high false alarm rates. While advanced bioaerosol sampling systems, such as qPCR, PCR, and RT-PCR, can identify species and improve the detection limits of biological reagents in air samples, they are difficult to automate as unattended bioaerosol sensing systems. For example, DNA sample preparation is a labor-intensive and cumbersome process, with detection times potentially reaching several hours. This falls far short of the target "detected warning" time span, typically considered to be one minute, to allow for timely response or rescue in the event of human biological error. Furthermore, these technologies cannot distinguish between dead and live cells. UVAPS can generate the total active bioaerosol concentration in real time based on the fluorescence emitted by reduced pyridine nucleotides (e.g., NAD(P)H) and riboflavin) associated with active particles, but its main drawback is the inability to perform species-level differentiation. For bioaerosol mass spectrometry, background noise and analysis of multiple spectral peaks contribute to high false alarm rates, affecting its practical application. Clearly, there is an urgent need for updated or novel technologies for the enrichment and detection of microorganisms in real-time bioaerosols.

[0004] Enrichment sampling equipment uses a virtual impactor that splits incoming aerosol particles into two channels, primary and secondary, depending on their size. Smaller particles follow the diverted flow into the higher channel (primary), while larger particles, due to their greater inertia, enter directly into the smaller channel. The smaller flow typically accounts for about 10% of the inlet flow rate, while the larger flow accounts for the other inlet flow rate. Most of the larger particles can be diverted into the secondary flow to increase the concentration of airborne particles in the secondary channel, typically using two stages to increase it by about 100 times or more.

[0005] Immunomagnetic beads are prepared by covalently binding carrier microspheres and immunoligands. The principle involves introducing metal molecules onto tiny spheres with diameters of several to tens of micrometers, allowing the microspheres to be attracted by a magnetic field. Under the influence of this magnetic field, the magnetic beads, which are bound to antigen-antibody complexes, move, thus separating the antigen (or antibody) specifically bound to the magnetic beads from other substances, achieving the effect of separating antigens (or antibodies). Immunomagnetic separation can rapidly separate target analytes, preserve their biological activity, effectively eliminate substances affecting nucleic acid amplification, and improve detection sensitivity. The process is simple and easy to use, requiring no expensive centrifugation equipment, and is a potentially effective method for concentrating and separating microorganisms.

[0006] Biosensors are sensors that use bioactive substances as recognition elements. After specific recognition, the resulting complex is converted into optical or electrical signals by a signal converter, thus achieving analytical detection. Based on the signal conversion method, biosensors can be classified into optical biosensors, electrochemical biosensors, and mass biosensors. Among these, electrochemical biosensors use electrical signals as the final detection signal, possessing advantages such as high sensitivity, label-free operation, and short detection time. Nanochannels, used for nanofiltration to detect changes in the resistance of the target substance and generate electrical signals, can be used to detect specific microorganisms. Summary of the Invention

[0007] To address the problems mentioned in the background, the present invention aims to provide an integrated method for sampling and capturing avian influenza virus in aerosols using an integrated sampling and enrichment device and immunomagnetic beads incubation. This invention combines an impactor aerosol sampler with immunomagnetic beads to construct a virus sampling device that integrates sampling and enrichment functions. This invention combines impactor aerosol sampling with nanochannel electrodes to achieve rapid, efficient, sensitive, and low-cost detection of avian influenza virus in aerosols. Anodized aluminum oxide is selected as the substrate, and an integrated and miniaturized analytical detection platform is constructed using an electrochemical sensor.

[0008] The main steps of the method of the present invention are as follows: selecting liquid containing target microorganisms as aerosol or closed air as the detection object; using sampling equipment combined with immunomagnetic beads to collect microorganisms in aerosols to achieve microbial enrichment and purification; aspirating virus liquid onto the surface of nanoelectrode, filtering and then detecting the virus; and finally, quantitative detection of target microorganisms by detecting changes in impedance intensity.

[0009] The innovation of this invention lies in the clever combination of sampling, enrichment and separation by using a designed sampler and immunomagnetic beads, and further integrating it with the signal amplification of nanochannel nanofiltration. This achieves a high degree of integration of the three modules of separation and enrichment, signal generation and amplification and high-sensitivity signal acquisition, resulting in efficient operation, portable equipment and high versatility.

[0010] The technical solution adopted in this invention is as follows:

[0011] I. An integrated sampling and enrichment device

[0012] The device is an integrated sampling enrichment tube consisting of an upper tube and a lower tube connected vertically. The upper and lower tubes are detachable. An air inlet pipe and an air outlet pipe are integrated on the upper tube. The air inlet pipe is inverted L-shaped, with the horizontal section located outside the tube and the bottom of the vertical section extending from the top of the upper tube into the tube and extending to the bottom of the lower tube. The air outlet pipe is horizontally located on the top side of the upper tube and communicates with the inside of the tube.

[0013] The outlet pipe is connected to an impact-type aerosol sampling pump to assist the incubation process of microorganisms and immunomagnetic beads. The height of the outlet pipe is higher than the bottom of the inlet pipe, and the aerosol is directly delivered to the bottom of the lower pipe through an inverted L-shaped inlet pipe.

[0014] The materials used in the integrated sampling enrichment tube include, but are not limited to, quartz glass and silicone.

[0015] II. An integrated method for sampling and capturing microorganisms in aerosols using an integrated sampling and enrichment device and immunomagnetic bead incubation technology.

[0016] Includes the following steps:

[0017] Step 1) Fill the bottom of the integrated sampling enrichment tube with sample collection liquid, connect the outlet tube to the impact aerosol sampling pump, and then introduce the target gas into the bottom of the integrated sampling enrichment tube through the inlet tube.

[0018] Step 2) Sampling and enrichment of potential target microorganisms in aerosols:

[0019] When the impact aerosol sampling pump is turned on, if the target microorganism is present in the aerosol, the impact aerosol sampling pump will sample and enrich the microorganism in the aerosol for a certain period of time, so that the microorganism in the target gas is dispersed into the sample collection liquid, and the target microorganism is specially bound to the functional magnetic beads in the sample collection liquid.

[0020] Step 3) Process the collected liquid after sampling and enrichment based on the magnetic separation method, remove the waste liquid to obtain the enriched liquid, and sonicate the enriched liquid to redisperse the magnetic beads in the enriched liquid.

[0021] Step 4) Take the dispersed test droplet from Step 3) onto the surface of the nanochannel electrode chip, filter the electrode chip until the surface of the electrode chip dries, and use the EIS impedance change to characterize the conductivity of the electrode chip, thereby completing the detection of the presence and concentration of microorganisms in the target gas.

[0022] In step 1), the sample collection solution contains functional substances that specifically recognize target microorganisms. These functional substances include, but are not limited to, magnetic beads that modify recognition elements such as antibodies, aptamers, and nucleic acids. The magnetic beads have a diameter of 20 nm, the BSA concentration used to prepare the magnetic beads is 1%, and the blocking time with BSA during the preparation of the magnetic beads is 30 min.

[0023] In step 1), the target gas source includes aerosols formed by the vaporization of space gas or liquid, and the liquid includes allantoic fluid, blood, saliva, and swab diluent.

[0024] In step 2), the sampling enrichment time using an impact aerosol sampling pump is 10-30 min, and the sampling flow rate of the impact aerosol sampling pump is 5-15 lpm.

[0025] The nanochannel electrode chip in step 4):

[0026] The membrane pore size of nanochannels ranges from 0.01 to 10 μm, including single nanopores or nanochannel arrays;

[0027] Nanochannel materials include porous anodic aluminum oxide (AAO) membranes, block copolymer self-assembled membranes, silicon nitride porous membranes, and carbon nanotube membranes.

[0028] The conductive layer materials on the surface of nanochannels include, but are not limited to, silver, gold, copper, and carbon.

[0029] The nanochannel electrode uses a dual-electrode connection method.

[0030] In step 4):

[0031] The EIS impedance changes of the nanochannel electrode chip were characterized before and after filtration.

[0032] If the impedance changes before and after filtration, it indicates that there are microorganisms in the target gas. The measured impedance change is compared with the standard curve model established by fitting the impedance change and the target microorganism concentration obtained by pre-calibration to obtain the corresponding microorganism concentration result.

[0033] If the impedance does not change before and after filtration, it indicates that there are no microorganisms in the target gas.

[0034] The beneficial effects of this invention are:

[0035] This invention combines an impaction sampling system with immunomagnetic beads to capture, enrich, and separate microorganisms from aerosols. Free magnetic beads are rapidly separated by nanofiltration (nanochannels) in the electrode chip, while the bead-microorganism complex is retained. Subsequently, a simple and highly sensitive detection of microorganisms is achieved based on a dual-electrode system platform. This invention provides an integrated method for sampling and capturing microorganisms from aerosols using an integrated sampling and enrichment device and immunomagnetic bead incubation technology. Its advantages over existing methods are:

[0036] (1) The method of this invention can collect representative biological particles of various sizes within a specific space without losing the microbial state and the physical collection of microorganisms. The sampler should also be compatible with subsequent microbial analysis in terms of speed (or high sampling flow rate), portability, and collection capability;

[0037] (2) The method of the present invention is based on the high integration of sampling equipment and detection equipment with capture, enrichment, separation and detection modules, which can realize the integrated sampling, separation and detection of viruses. It is simple and efficient to operate, the equipment is portable and inexpensive and easy to prepare, avoiding cumbersome and time-consuming processing steps and the use of a variety of expensive instruments.

[0038] (3) The method of the present invention combines the high efficiency of immunomagnetic bead incubation with the chemical signal generated by the retention of antigen-antibody complex by nanochannel electrodes, which can significantly improve the sensitivity of microbial detection and achieve highly sensitive detection of trace target bacteria in aerosols.

[0039] (4) The method of the present invention has good universality. Different microorganisms can be detected by changing the antibody. The sampling equipment and the detection equipment can also be separated. The enriched microorganisms can be used for other virus research after simple magnetic separation - that is, after purification.

[0040] (5) The method of the present invention is expected to be combined with a portable electrochemical workstation, which has the potential to rapidly analyze the presence of aerosol microorganisms on site, showing good application prospects. Attached Figure Description

[0041] Figure 1 The diagram shows the design of this invention. a is an integrated sampling enrichment tube, b is a magnetic separation method, and c is a nanochannel-electrode chip.

[0042] Figure 2 This is a detailed diagram of an integrated sampling enrichment tube. In the diagram: 1. Inlet pipe, 2. Outlet pipe, 3. Upper fitting, 4. Thread, 5. Lower fitting.

[0043] Figure 3 The images are SEM images of the electrodes, where (a) is a surface image of the bare electrode (500 nm pore size); (b) is a morphology of the virus trapped on the electrode (20-30 nm pore size); and (c) is a morphology of the immunomagnetic bead-virus complex trapped on the electrode (500 nm pore size).

[0044] Figure 4 The resistance changes of different substances after filtration onto the same electrode.

[0045] Figure 5 The resistance changes after sampling and testing of avian influenza viruses of different subtypes and titers.

[0046] Figure 6 The resistance changes of different substances after filtration at the same electrode (EIS plot).

[0047] Figure 7 EIS diagram of the same subtype of avian influenza virus after filtration. Detailed Implementation

[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the method provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0049] like Figure 1 and Figure 2 As shown, the integrated enrichment sampling tube consists of an upper fitting and a lower fitting connected vertically, and the upper fitting 3 and the lower fitting 5 are detachable. The upper fitting and the lower fitting are sealed together by a thread 4. The upper fitting is compatible with a 15mL spiral-jointed lower fitting, and can be designed to a larger diameter according to actual needs.

[0050] The upper fitting 3 is integrated with an inlet pipe 1 and an outlet pipe 2. The outlet pipe opening is located at the top to avoid backflow of the sampling liquid; the outlet pipe is higher than the bottom of the inlet pipe, so that the aerosol is delivered directly into the bottom of the centrifuge tube in an inverted "L" shape, which assists the incubation process of microorganisms and immunomagnetic beads through impact.

[0051] The horizontal section of the inlet pipe 1 has an inner diameter of 0.5 cm and an outer diameter of 0.8 cm; the vertical section of the inlet pipe 1 has an inner diameter of 0.3 cm and an outer diameter of 0.7 cm; the outlet pipe 2 has an inner diameter of 0.4 cm and an outer diameter of 0.6 cm. 100 μL of immunomagnetic bead solution is placed at the bottom of the lower fitting 5.

[0052] The embodiments of the present invention are as follows:

[0053] (1) Preparation of MNPs-Ab

[0054] Carboxylated MNPs were prepared according to previously reported methods. MNPs were centrifuged with MEST buffer (10 min, 12000 rpm, hereinafter the same) and the supernatant was removed. This process was repeated three times. The MNPs were then resuspended in MEST buffer containing 10 mM EDC and 15 mM NHSS and incubated at room temperature for 30 min to activate the carboxyl groups on the MNP surface. Next, the MNPs were centrifuged with BST buffer and the supernatant was removed (same as above). After washing three times, the MNPs were resuspended in BST buffer containing 17 μg mL Anti-St and incubated at room temperature for 2.5 h to conjugate the antibody. The antibody-modified MNPs were incubated with PBST buffer containing 20 mg mL-1 BSA for 1 h to block residual reaction sites. The MNPs were then centrifuged with PBST buffer and the supernatant was removed. After washing three times, the MNPs were finally dispersed in PBST buffer to obtain MNPs-Ab.

[0055] (2) Aerosol virus sampling and enrichment

[0056] The virus in aerosol is generated using a TK-3 microbial aerosol generator. First, insert the nebulizer reservoir into the ring of the tripod. Then, connect the air outlet of the main unit to the air inlet of the nebulizer reservoir using an air hose. Add 2 mL of AIV allantoic fluid to the reservoir; gradient dilution can be performed as needed for subsequent testing. Add 0.5% (w / v) BSA. (The TCID50 of the virus solution for each test should be determined using a hemagglutination test before each sample addition).

[0057] An impact-type aerosol sampler with sampling and enrichment functions was constructed. To maximize enrichment, a 15 mL centrifuge tube was used as the collection bottle, and 100 μL of immunomagnetic bead solution was added for virus capture and enrichment. A sampling tube with the same thread was fitted at the top to ensure airtightness. Rubber tubing was used to connect the nebulizer reservoir outlet to the sampling tube inlet and the sampling tube outlet to the impact-type aerosol sampling pump. The aerosol generator and aerosol sampler were simultaneously turned on. The sampling flow rate was set to 20 L / min, and sampling was performed for 10 min for rapid virus capture from the aerosol. The flow rate was then adjusted to 5 L / min for 20 min to capture remaining viruses and incubate the immunomagnetic beads with the virus, obtaining an MNPs-Ab-AIV suspension. The MNPs-Ab-AIV suspension was magnetically separated, the supernatant was discarded, and the suspension was resuspended in 200 μL PBST. After three rounds of the above washing steps, the final precipitate was dispersed in 50 μL PBST. Using MNPs-Ab suspension as a control, after each sampling, the virus aerosol was first generated with 0.01% sodium dodecyl sulfate (SDS) for 5 minutes to flush away the virus aerosol. Then, the storage tank, rubber tubing, and sampling tube were rinsed with deionized water for 25 minutes to flush away SDS residue.

[0058] (3) Electrochemical detection of viruses

[0059] 100 μL of MNPs-Ab was mixed with 1 mL of viral allantoic fluid (10⁻¹⁰⁻³ TCID₅₀) and incubated at room temperature for 45 min to capture avian influenza virus using antigen-antibody specificity, yielding a complex of MNPs, Ab, and St (MNPs-Ab-St). The MNPs-Ab-AIV suspension was magnetically separated, the supernatant was discarded, and the suspension was resuspended in 200 μL of PBST. After three rounds of the above washing steps, the final precipitate was dispersed in 50 μL of PBST. Using the MNPs-Ab suspension as a control, 10 μL of the MNPs-Ab-AIV suspension was used for filtration and electrochemical testing, and the EIS curve was collected. The signal of the target analyte is the difference in charge transfer resistance (Rct) between the virus sample and the control group in the EIS test results.

[0060] The method of this invention has high sensitivity to avian influenza virus in aerosols, which is mainly due to two factors: (1) The self-designed aerosol sampling equipment is based on the impact-type aerosol sampling principle, which has strong sampling ability and will not damage the virus structure. At the same time, the use of immunomagnetic beads can capture and enrich more viruses. (2) The particle size of avian influenza virus is about 100nm, while the particle size of immunomagnetic beads is 20nm and the AAO pore size is 500nm. Although the immunomagnetic beads have a small particle size, they will hydrate and aggregate, and can easily pass through the AAO channel. After binding with the virus, they will also aggregate, and the particle size will be larger, which will be trapped on the electrode surface. The subtle changes bring about changes in impedance, thereby achieving sensitive detection.

[0061] Viruses typically have particle sizes in the nanometer range. The detection target chosen for this invention—avian influenza virus—can be extended to other viruses that can be transmitted via aerosols, such as infectious bronchitis virus and infectious bursal disease virus. Simply replace the antibodies on the immunomagnetic beads with other antibodies.

[0062] As demonstrated by the above implementation examples, the integrated sampling and enrichment device and immunomagnetic bead incubation technology proposed in this invention, which uses an integrated sampling and enrichment device and immunomagnetic bead incubation technology to capture microorganisms in aerosols, enriches avian influenza virus in the air through the sampling device; captures avian influenza virus through immunomagnetic beads, while simultaneously achieving rapid magnetic separation and purification of the virus; rapidly separates interfering magnetic beads using nanochannel "nanofiltration" to enrich signal molecules; and simultaneously outputs readable electrochemical signals using an electrode chip. The entire process takes only about 1 hour. This invention's method is simple to operate, rapid, highly specific, sensitive, and versatile. Furthermore, the sampling device, functional magnetic beads, and integrated electrode are simple to prepare and easy to mass-produce. Therefore, this invention's method is expected to become a rapid on-site detection method, showing promising development prospects.

[0063] like Figure 3 As shown, a is a SEM image of the surface pore size of a 500nm nanochannel electrode chip, b shows a SEM image of the virus morphology trapped on the surface of a 20nm pore size nanochannel electrode chip, and c is a SEM image of the surface trapping state after filtering immune complexes from the nanochannel electrode chip.

[0064] like Figure 4 As shown, the same electrode was subjected to (1) BSA blocking; (2) small molecule filtration; (3) immunomagnetic bead filtration; and (4) immunomagnetic bead-virus conjugate filtration. EIS detection was then performed to record the resistance changes before and after filtration. It can be seen that the Rct value changed the most after immunomagnetic bead-virus conjugate filtration.

[0065] like Figure 5 As shown, the resistance of avian influenza viruses of different subtypes and titers changed after sampling and testing, indicating that the immunomagnetic beads can recognize multiple viral subtypes and have a broad spectrum.

[0066] like Figure 6 As shown, the same electrode was subjected to BSA blocking, immunomagnetic bead filtration, and immune complex filtration sequentially. The initial EIS resistance and the EIS resistance after each of the above steps were measured. The EIS curves basically overlapped after BSA blocking and immunomagnetic bead filtration, and the semicircle of the curve increased after immune complex filtration.

[0067] like Figure 7 The image shows samples collected using an aerosol sampler at different concentrations (from ×1 to ×10). -2The H5N8 virus solution was magnetically separated and then filtered using a nanochannel electrode chip. The resulting EIS changes showed a detection limit as low as ×10⁻¹⁰. -2 concentration.

[0068] 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 modifications without departing from the method of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An integrated sampling and enrichment device and an integrated method for sampling and capturing microorganisms in aerosols using immunomagnetic bead incubation technology. The method uses an integrated sampling enrichment device, which is an integrated sampling enrichment tube composed of an upper tube and a lower tube connected together. The upper tube (3) and the lower tube (5) are detachable. An air inlet pipe (1) and an air outlet pipe (2) are integrated on the upper pipe fitting (3); the air inlet pipe (1) is inverted L-shaped, with the horizontal section located outside the pipe fitting, and the bottom of the vertical section extending from the top of the upper pipe fitting (3) into the pipe fitting and extending to the bottom of the lower pipe fitting (5); the air outlet pipe (2) is horizontally set on the top side of the upper pipe fitting (3) and communicates with the inside of the pipe fitting; the air outlet pipe (2) is connected to an impact-type aerosol sampling pump to assist the incubation process of microorganisms and immunomagnetic beads; the height of the air outlet pipe (2) is higher than the bottom height of the air inlet pipe (1), and the aerosol is directly sent into the bottom of the lower pipe fitting through the inverted L-shaped air inlet pipe; the material of the integrated sampling enrichment tube includes, but is not limited to, quartz glass and silicone. Its features are, Includes the following steps: Step 1) Fill the bottom of the integrated sampling enrichment tube with sample collection liquid, connect the outlet tube (2) to the impact aerosol sampling pump, and then introduce the target gas into the bottom of the integrated sampling enrichment tube through the inlet tube (1). Step 2) Sampling and enrichment of potential target microorganisms in aerosols: When the impact aerosol sampling pump is turned on, if the target microorganism is present in the aerosol, the impact aerosol sampling pump will sample and enrich the microorganism present in the aerosol, so that the microorganism in the target gas is dispersed into the sample collection liquid, and the target microorganism present binds to the functional magnetic beads in the sample collection liquid. Step 3) Process the collected liquid after sampling and enrichment based on the magnetic separation method, remove the waste liquid to obtain the enriched liquid, and sonicate the enriched liquid thoroughly to redisperse the magnetic beads in the enriched liquid. Step 4) Take the dispersed test droplet from Step 3) onto the surface of the nanochannel electrode chip, filter the electrode chip until the surface of the electrode chip dries, and use the EIS impedance change to characterize the conductivity of the electrode chip, thereby completing the detection of the presence and concentration of microorganisms in the target gas.

2. The integrated method for sampling and capturing microorganisms in aerosols using an integrated sampling and enrichment device and immunomagnetic bead incubation technology according to claim 1, characterized in that, In step 1), the sample collection solution contains functional substances that specifically recognize target microorganisms. These functional substances include, but are not limited to, magnetic beads that modify antibodies, aptamers, and nucleic acids. The magnetic beads had a diameter of 20 nm, the BSA concentration for preparing the magnetic beads was 1%, and the sealing time with BSA during the preparation of the magnetic beads was 30 min.

3. The integrated method for sampling and capturing microorganisms in aerosols using an integrated sampling and enrichment device and immunomagnetic bead incubation technology according to claim 1, characterized in that, In step 1): The target gas sources include aerosols resulting from the vaporization of space gases or liquids, and the liquids include allantoic fluid, blood, saliva, and swab diluent.

4. The integrated method for sampling and capturing microorganisms in aerosols using an integrated sampling and enrichment device and immunomagnetic bead incubation technology according to claim 1, characterized in that, In step 2): The sampling enrichment time using an impact-type aerosol sampling pump is 10-30 minutes. The sampling flow rate of the impact-type aerosol sampling pump is 5-15 lpm.

5. The integrated method for sampling and capturing microorganisms in aerosols using an integrated sampling and enrichment device and immunomagnetic bead incubation technology according to claim 1, characterized in that, The nanochannel electrode chip in step 4): The membrane pore size of nanochannels ranges from 0.01 to 10 μm, including single nanopores or nanochannel arrays; Nanochannel materials include porous anodic alumina membranes, block copolymer self-assembled membranes, silicon nitride porous membranes, and carbon nanotube membranes; The conductive layer materials on the surface of nanochannels include, but are not limited to, silver, gold, copper, and carbon. The nanochannel electrode uses a dual-electrode connection method.

6. The integrated method for sampling and capturing microorganisms in aerosols using an integrated sampling and enrichment device and immunomagnetic bead incubation technology according to claim 1, characterized in that, In step 4), The EIS impedance changes of the nanochannel electrode chip were characterized before and after filtration. If the impedance changes before and after filtration, it indicates that there are microorganisms in the target gas. The measured impedance change is compared with the standard curve model established by fitting the impedance change and the target microorganism concentration obtained by pre-calibration to obtain the corresponding microorganism concentration result. If the impedance does not change before and after filtration, it indicates that there are no microorganisms in the target gas.

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