Application of plasma-modified amino magnetic beads in aerosol pathogen enrichment

Abstract

This invention discloses the application of plasma-modified amino magnetic beads in the enrichment of aerosol pathogens. Belonging to the field of pathogen enrichment technology, and addressing the problems of cumbersome procedures, long processing times, and organic reagent residues in existing chemical modification methods, this invention employs low-temperature plasma technology to generate high-energy active particles (such as NH2 free radicals and NH3 free radicals) through dielectric barrier discharge in an ammonia or ammonia / argon mixed atmosphere. + (e.g., amino groups) react directly with the surface of silanol magnetic beads to graft amino groups in a one-step process, achieving rapid, solvent-free dry modification. The modified magnetic beads were used for the enrichment of pathogenic microorganisms. Results showed that the adsorption rates of both modified and original magnetic beads were greater than 80%, but the enrichment efficiency of the modified magnetic beads was higher than that of the original magnetic beads. This invention achieves rapid amino functionalization of magnetic beads, significantly improving the enrichment efficiency of pathogenic aerosols, and is applicable to fields such as respiratory infectious disease prevention and control, biosafety monitoring, and environmental public health assessment.

Description

Technical Field

[0001] This invention belongs to the field of pathogenic microorganism enrichment technology, specifically relating to the application of plasma-modified amino magnetic beads in aerosol pathogen enrichment. Background Technology

[0002] Aerosols contain pathogenic microorganisms such as bacteria, viruses, and fungi, and are important vectors for the transmission of respiratory infectious diseases (such as influenza, COVID-19, and tuberculosis). According to the World Health Organization, airborne transmission is a major route of transmission for many highly infectious pathogens. Therefore, achieving efficient collection and enrichment of pathogenic microorganisms in aerosols is of great significance for the prevention and control of respiratory infectious diseases, biosafety monitoring, and environmental public health assessment.

[0003] Magnetite beads, due to their superparamagnetic properties and surface functionalizability, are widely used in the biomedical field, including nucleic acid extraction, immunoassay, cell sorting, and pathogen enrichment. In aerosol pathogen detection, magnetic beads are often used to capture and enrich target pathogens from sampling solutions to improve detection sensitivity. However, unmodified magnetic beads lack active functional groups on their surface and cannot effectively bind biomolecules. Therefore, it is necessary to modify the surface of magnetic beads to introduce active groups such as amino and carboxyl groups, and then couple them with antibodies or nucleic acid probes to achieve specific capture of pathogens.

[0004] Currently, the mainstream modification method is chemical modification. Patent CN118022690A discloses a silane coupling agent method, which uses nano-iron oxide magnetic beads to react with a silane coupling agent to introduce amino groups and then couple antibodies for immunoassay. Patent CN116525234A discloses a novel oleic acid-modified superparamagnetic iron oxide silanol magnetic beads, which are modified with oleic acid for 1-3 hours and then coated with silicon for 8-16 hours to enhance hydrophobicity. Patent CN116732505A discloses a modification method using a sol-gel method, which requires stirring for 18 hours to complete the coating. Patent CN104538168A discloses a multi-step chemical coupling method, which uses a crosslinking agent to connect multiple surface-functionalized magnetic nanoparticles to prepare high-magnetic-content modified magnetic beads. The aforementioned chemical modification methods generally suffer from common problems such as multiple reaction steps (2-5 steps), long processing time (several hours to tens of hours), reagent residue caused by the use of large amounts of organic solvents and crosslinking agents, and easy aggregation of magnetic beads. Moreover, most methods focus on the application of magnetic beads in specific scenarios rather than the innovation of the modification method itself. Summary of the Invention

[0005] To address the shortcomings of chemical modification methods in the prior art, such as cumbersome steps, long processing time, residual organic reagents inhibiting detection, and easy aggregation of magnetic beads, this invention proposes a one-step amino modification of magnetic beads using low-temperature plasma technology. Although plasma surface modification technology has been extensively studied in the fields of surface modification of metallic and polymer materials, its application to the surface amino modification of magnetic beads for the enrichment of aerosol pathogens is currently unreported in the literature or patents. Existing magnetic bead enrichment technologies all employ chemical modification methods for functionalization, which share common problems such as numerous reaction steps and long processing times. Therefore, applying rapid, efficient, and solvent-free plasma surface modification technology to the amino modification of magnetic beads and achieving efficient enrichment of aerosol pathogens has significant innovative and practical value.

[0006] The technical solution of the present invention is as follows:

[0007] An application of plasma-modified amino magnetic beads in the enrichment of aerosol pathogens is disclosed. The plasma modification method employs low-temperature plasma technology, in which high-energy active particles generated by dielectric barrier discharge in a mixed atmosphere of ammonia, argon, or ammonia gas react directly with the surface of silanol magnetic beads. After grafting amino groups, the beads are used to enrich pathogenic microorganisms in aerosols.

[0008] In the above technical solution, the particle size of the siloxane magnetic beads is 200 nm.

[0009] In the above technical solution, the plasma modification device includes a high-voltage electrode, a quartz dielectric tube, a copper mesh ground electrode, a gas inlet, a gas outlet, and a flange connection structure. The high-voltage electrode is located at the center of the quartz dielectric tube, and the copper mesh ground electrode is wrapped around the quartz dielectric tube and connected to the grounding wire to seal the gap between the magnetic bead and the gas.

[0010] In the above technical solution, the ratio of ammonia to argon is 1:1, and the gas flow rate is 0.04–0.1 slm.

[0011] In the above technical solution, the parameters of the dielectric barrier discharge include: discharge voltage of 5–8kV, discharge frequency of 500–2000Hz, rise time of 50–400ns, pulse width of 100–400ns, fall time of 50–400ns, and processing time of 5–30min.

[0012] In the above technical solution, the enrichment of pathogenic microorganisms includes the following steps:

[0013] The plasma-modified magnetic bead suspension was injected into the cyclone cup of the wet-wall cyclone aerosol sampler;

[0014] High-speed cyclone airflow captures pathogenic microorganisms and brings them into contact with magnetic beads;

[0015] The functional groups on the surface of magnetic beads bind to pathogenic microorganisms through electrostatic interactions;

[0016] After sampling, an external magnetic field is used to separate the magnetic beads from the bound pathogenic microorganisms, thereby obtaining enriched pathogenic microorganisms.

[0017] In the above technical solution, after plasma modification, amino groups are introduced into the surface of the magnetic beads, making their enrichment efficiency significantly higher than that of unmodified magnetic beads.

[0018] In the above technical solution, the mass concentration of the magnetic bead suspension is 0.02 mg / mL, and the concentration of the pathogenic microorganism suspension is 1 × 10⁻⁶. 5 –1×10 7 CFU / mL.

[0019] In the above technical solution, after enrichment, the complex of magnetic beads and pathogens is transferred to sterile PBS for washing and resuspending. The enriched portion, the original sample solution and the unbound portion are counted by the coating method to calculate the enrichment efficiency.

[0020] In the above technical solution, the pathogenic microorganisms include, but are not limited to, bacteria, viruses or fungi, and the application fields are respiratory infectious disease prevention and control, pathogen monitoring and biosafety detection.

[0021] Beneficial effects

[0022] This invention marks the first application of low-temperature plasma surface modification technology to the amino modification of silicon hydroxyl iron oxide magnetic beads, and its use for the efficient enrichment of aerosol pathogenic microorganisms. Compared with existing chemical modification methods, the technological innovation of this invention lies in: utilizing active particles (NH2 free radicals, NH3...) in high-energy plasma. + The method involves directly grafting amino groups onto the surface of magnetic beads via a one-step, dry, solvent-free process, rather than the traditional multi-step chemical coupling reaction. This eliminates the problem of residual chemical reagents. Furthermore, the plasma-modified magnetic beads are used for the enrichment of aerosol pathogens, solving the problem of low enrichment efficiency of existing magnetic beads. Attached Figure Description

[0023] Figure 1 Flowchart of plasma-modified amino-beaded magnetic beads for pathogen enrichment;

[0024] Figure 2 Diagram of plasma modification device;

[0025] Figure 3 Infrared spectra of magnetic beads before and after modification;

[0026] Figure 4 Image of Escherichia coli enriched by magnetic beads;

[0027] In the diagram: 1 is the ammonia valve, 2 is the argon valve, 3 is the flow controller, 4 is the mixing tank, 5 is the inlet, 6 is the plasma source, 7 is the high-voltage wire, 8 is the stainless steel high-voltage electrode, 9 is the PTFE flange, 10 is the quartz dielectric tube, 11 is the plasma zone, 12 is the copper mesh ground electrode, 13 is the grounding wire, 14 is the ferric oxide bead, 15 is the quartz wool, and 16 is the outlet. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the scope of protection of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0029] Example:

[0030] This invention proposes a method for generating high-energy active particles (NH2 free radicals, NH3 free radicals) using dielectric barrier discharge in an ammonia or ammonia / argon mixed atmosphere. + A method involving the direct reaction of amino groups with the surface of silanol magnetic beads, followed by the application of the modified magnetic beads to aerosol pathogen enrichment, is described in the following process: Figure 1 As shown, this includes magnetic beads containing silanol, plasma-modified amino groups, and magnetic beads enriched with pathogens.

[0031] The technical solution includes two steps: (1) modification of dielectric barrier discharge beads, and (2) pathogen enrichment of modified magnetic beads.

[0032] (1) Dielectric barrier discharge device such as Figure 2 As shown: A stainless steel high-voltage electrode 8, a quartz dielectric tube 10, and a copper mesh ground electrode 12 are coaxially arranged. The stainless steel high-voltage electrode 8, which serves as the high-voltage end, is fixed at the center of the quartz dielectric tube 10 and is fixedly connected to the quartz dielectric tube 10 through a polytetrafluoroethylene flange 9. A copper mesh ground electrode 12 is wrapped around the outside of the quartz dielectric tube 10.

[0033] The operation of this device is as follows: Ferrite beads 14 are filled between the stainless steel high-voltage electrode 8 and the quartz dielectric tube 10, and the bottom is sealed with quartz wool 15 to prevent the ferrite beads 14 from falling out, but gas can pass through. Ammonia valve 1 and argon valve 2 are opened, and the mixed gas enters the reaction chamber through the quartz tube inlet 5 via the flow controller 3 and mixing tank 4, and then exits through the outlet 16. The stainless steel high-voltage electrode 8 is connected to the plasma source 6 via a high-voltage wire 7. A high voltage is applied between the stainless steel high-voltage electrode 8 and the copper mesh ground electrode 12, causing the gas between the two electrodes to ionize and form a plasma region 11. The discharged plasma directly modifies the silanol magnetic beads by grafting amino groups.

[0034] (2) Plasma-modified magnetite beads were used to enrich pathogenic microorganisms. The method was as follows: Before sampling, the magnetic bead solution was injected into the cyclone cup of the wet-wall cyclone aerosol sampler. After the cyclone sampling device was started, the aerosol containing pathogens was captured by the high-speed cyclone and incorporated into the liquid phase, and fully mixed and contacted with the suspended magnetic beads. The functional groups on the surface of the magnetic beads efficiently bound the pathogens through electrostatic and affinity interactions, forming a magnetic bead-pathogen complex. After sampling, the complex was rapidly separated and enriched using an external magnetic field, and then it could be used with a detection device for subsequent analysis.

[0035] The steps of this method are as follows:

[0036] (1) Sample preparation: Select siloxane magnetic beads with a particle size of 0~200nm, clean them with ultrasonic waves and dry them for later use.

[0037] (2) Constructing a dielectric barrier discharge device: Connect the argon cylinder, ammonia cylinder, gas flow meter, and plasma device in sequence according to the gas path. After the connection is completed, check the airtightness and directionality of the entire gas path to ensure that there is no leakage. Place the quartz wool 15 into the stainless steel high-voltage electrode 8 and the quartz dielectric tube 10, and then place the silicon hydroxyl magnetic bead and fix the upper flange. Connect the plasma excitation circuit, connect the high voltage output terminal of the high voltage power supply to the upper end of the stainless steel high-voltage electrode 8 of the plasma, connect the copper mesh ground electrode 12 to the copper mesh, set up a high voltage probe, current coil and oscilloscope to monitor the voltage and current waveforms in real time, and check whether the entire circuit has good contact to ensure that each part is safely grounded.

[0038] (3) Magnetic bead modification: Adjust the ratio of argon to ammonia to 0-1 and the flow rate to 0.04-0.1 slm. Introduce argon and ammonia to purge the device and ensure that there is no air in the device. Set the frequency (500-2000Hz), voltage (5-8kV), rise time (50-400ns), pulse width (100-400ns), fall time (50-400ns), and processing time (5-30min) of the pulse repetition high voltage power supply and conduct magnetic bead modification preparation experiments.

[0039] (4) Magnetic bead pathogen enrichment experiment: The enrichment efficiency of magnetic beads was evaluated in the bioaerosol detection and evaluation chamber: the convection fan was run at a wind speed of 15-20 m / min to purify the chamber for 15 min. A magnetic bead-physiological saline suspension with a mass concentration of 0.01-0.05 mg / mL and a magnetic bead-physiological saline suspension with a concentration of 1×10 5 -1×10 7CFU / mL bacterial suspension. Inject 5-10 mL of the magnetic bead suspension into the cyclone cup of the wet-wall cyclone sampler and place it at the center of the wind tunnel. Set the liquid injection rate of the aerosol generator to 0.1-0.2 mL / min and the generation time to 5-10 min to ensure uniform distribution of pathogen aerosols. Start the cyclone sampling device, setting the sampling flow rate to the equipment calibration value (100-300 L / min) and the sampling time to 5-10 min. After sampling, purify the chamber for 15 min. Remove the mixture from the cyclone cup, separate the magnetic bead-bacterial complex using an external magnet, and remove the supernatant. Wash the magnetic beads 2-3 times with sterile PBS and resuspend them in PBS. Take 200 μL of the resuspension (enriched portion), 200 μL of the original sampling solution (total bacteria), and 200 μL of the supernatant (unbound portion), respectively, and spread them on solid culture medium. Incubate at 37°C for 24 h. Count the number of colonies and calculate the enrichment efficiency (%) = (number of colonies in the enriched portion / total number of colonies) × 100%.

[0040] Example:

[0041] 1g of magnetic beads was weighed and placed in a dielectric barrier discharge apparatus. Discharge was performed using a 1:1 mixture of argon and ammonia gas at a flow rate of 0.06 slm. A pulsed high-voltage power supply was used, with a discharge voltage of 8kV, a discharge frequency of 1kHz, a rise and fall time of 200ns, a pulse width of 400ns, and a processing time of 10min. The infrared spectra of the magnetic beads before and after treatment are as follows: Figure 3 As shown, after plasma modification, an amino peak was added to the surface of the magnetic beads, indicating successful modification. Subsequently, the modified magnetic beads and the original magnetic beads were mixed to prepare a magnetic bead-physiological saline suspension with a concentration of 0.02 mg / mL, and a solution with a concentration of 1 × 10⁻⁶ was prepared. 5 E. coli suspension was prepared. 10 mL of the magnetic bead suspension was injected into the cyclone cup of the wet-wall cyclone sampler and placed at the center of the wind tunnel. The aerosol generator was set to a liquid injection rate of 0.2 mL / min and a generation time of 10 min. The cyclone sampling device was started, with a sampling flow rate set to 300 L / min according to the device calibration, and a sampling time of 10 min. The mixture in the cyclone cup was removed, and the magnetic bead-bacterial complex was separated using an external magnet. The supernatant was removed. The magnetic beads were washed three times with sterile PBS and resuspended in PBS. 200 μL of the resuspension (enriched fraction), 200 μL of the original sampling solution (total bacteria), and 200 μL of the supernatant (unbound fraction) were respectively spread onto solid culture medium and incubated at 37°C for 24 h. The results of magnetic bead enrichment of E. coli are shown below. Figure 4 As shown, the adsorption rates of both the original magnetic beads and the modified magnetic beads exceeded 80%, but the enrichment efficiency of the modified magnetic beads was greater than that of the original magnetic beads, indicating that the modified beads were more effective.

[0042] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Use of plasma-modified amino magnetic beads for the enrichment of aerosol pathogens, characterized in that, The plasma modification method employs low-temperature plasma technology. In an atmosphere of ammonia, ammonia, or argon, high-energy active particles generated by dielectric barrier discharge react directly with the surface of silanol magnetic beads. After grafting amino groups, these particles are used to enrich pathogenic microorganisms in aerosols.

2. Use of a plasma-modified amino magnetic bead according to claim 1 for the enrichment of aerosol pathogens, characterized in that, The particle size of the silanol magnetic beads is 200 nm.

3. Use of a plasma-modified amino magnetic bead according to claim 1 for aerosolized pathogen enrichment, characterized in that, The device used for plasma modification includes a high-voltage electrode, a quartz dielectric tube, a copper mesh ground electrode, a gas inlet, a gas outlet, and a flange connection structure. The high-voltage electrode is located at the center of the quartz dielectric tube, and the copper mesh ground electrode is wrapped around the quartz dielectric tube and connected to the grounding wire to seal the gap between the magnetic bead and the gas.

4. Use of a plasma-modified amino magnetic bead according to claim 1 for aerosolized pathogen enrichment, characterized in that, The ratio of ammonia to argon in the mixed atmosphere is 1:1, and the gas flow rate is 0.04–0.1 slm.

5. Use of a plasma-modified amino magnetic bead according to claim 1 for aerosolized pathogen enrichment, characterized in that, The parameters of the dielectric barrier discharge include: discharge voltage of 5–8kV, discharge frequency of 500–2000Hz, rise time of 50–400ns, pulse width of 100–400ns, fall time of 50–400ns, and processing time of 5–30min.

6. Use of a plasma-modified amino magnetic bead according to claim 1 for aerosolized pathogen enrichment, characterized in that, The enrichment of the pathogenic microorganisms includes the following steps: The plasma-modified magnetic bead suspension was injected into the cyclone cup of the wet-wall cyclone aerosol sampler; The cyclone airflow captures pathogenic microorganisms and brings them into contact with the magnetic beads; The functional groups on the surface of magnetic beads bind to pathogenic microorganisms through electrostatic interactions; After sampling, an external magnetic field is used to separate the magnetic beads from the bound pathogenic microorganisms, thereby obtaining enriched pathogenic microorganisms.

7. Use of a plasma-modified amino magnetic bead according to claim 1 for aerosolized pathogen enrichment, characterized in that, After plasma modification, amino groups are introduced onto the surface of the magnetic beads, making their enrichment efficiency higher than that of unmodified magnetic beads.

8. The application of plasma-modified amino magnetic beads according to claim 6 in aerosol pathogen enrichment, characterized in that, The concentration of the pathogenic microorganism suspension is 1 x 10 5 CFU / mL. The concentration of the pathogenic microorganism suspension is 1 x 10 7 CFU / mL.

9. The application of plasma-modified amino magnetic beads according to claim 6 in aerosol pathogen enrichment, characterized in that, After enrichment, the complex of magnetic beads and pathogenic microorganisms was transferred to sterile PBS for washing and resuspending. The enriched portion, the original sample solution, and the unbound portion were counted by the spread method to calculate the enrichment efficiency.

10. The application of plasma-modified amino magnetic beads according to claim 1 in aerosol pathogen enrichment, characterized in that, The pathogenic microorganisms include bacteria, viruses or fungi, and the application areas are respiratory infectious disease prevention and control, pathogen monitoring and biosafety testing.

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