Bioaerosol automatic detection platform, detection method and application thereof

Abstract

The present application belongs to the cross field of molecular diagnosis, environmental microorganism detection and nanotechnology, and particularly relates to a biological aerosol automatic detection platform, a detection method and application. The present application realizes specific capture and enrichment of target microorganisms through a silicon nanotip array, and the silicon nanotip array chip is provided with a plurality of regularly arranged silicon nanotips, and the surface of the silicon nanotip is modified with biological capture molecules. The temperature change of the nanotip is monitored in real time by using Raman spectrum, and the feedback optimization of the lysis / amplification temperature curve is realized, the double signal amplification and verification of LAMP amplification and CRISPR detection are combined, the high-resolution melting curve is analyzed through a machine learning algorithm, and the automatic identification and molecular typing of the strain are realized. The present application has the advantages of high detection sensitivity, strong specificity, simple operation, short detection time and the like. The present application is suitable for on-site rapid detection and real-time monitoring of pathogenic bacteria in various environments such as atmosphere, exhaled air and water.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of molecular diagnostics, environmental microbial detection and nanotechnology, and specifically relates to an automated detection platform for bioaerosols, as well as detection methods and applications. Background Technology

[0002] Microbial aerosols are solid or liquid biological particles suspended in the air, typically ranging in size from 0.1 to 100 μm, and can contain various microbial components such as bacteria, fungi, and viruses. These aerosols can penetrate the respiratory tract and spread widely in the environment, causing not only infections, allergies, and respiratory diseases, but also public health emergencies in high-risk locations such as hospitals, farms, and wastewater treatment plants. Therefore, developing technologies and equipment for real-time online monitoring and early warning of airborne microbial pollution is of great significance for public health prevention and control and environmental safety management.

[0003] Nucleic acid amplification detection technology is an important means of microbial identification and characterization, evolving from polymerase chain reaction (PCR) to isothermal amplification technology. While traditional PCR technology offers high sensitivity, it relies on sophisticated thermal cycling equipment, is complex to operate, and is time-consuming, making it difficult to meet the needs of rapid on-site detection. Loop-mediated isothermal amplification (LAMP) technology achieves rapid nucleic acid amplification under isothermal conditions, offering significant advantages in detection speed and equipment simplicity. However, it carries the risk of non-specific amplification and lacks stability in detecting low-abundance targets in complex aerosol matrices. In recent years, the CRISPR-Cas12a system, due to its high specificity and signal amplification capabilities, has been introduced into molecular detection systems. Its combination with LAMP can significantly improve detection accuracy and has been gradually applied to integrated microfluidic detection platforms.

[0004] However, existing LAMP / CRISPR-based microfluidic detection platforms still face several key bottlenecks in practical applications: limited efficiency in capturing and enriching low concentrations of microorganisms in aerosols, making direct and efficient integration with downstream nucleic acid analysis difficult; insufficient temperature uniformity and real-time control during the reaction process, affecting amplification efficiency and result consistency; and the inability to simultaneously acquire morphological (phenotypic) and genotypic information of microorganisms, limiting comprehensive identification and source tracing analysis of microbial species. Currently used aerosol sampling and enrichment methods (such as membrane sampling and centrifugation) are often cumbersome, result in significant sample loss, and easily damage microbial activity, making true integration with microfluidic systems difficult.

[0005] To address the aforementioned issues, silicon nanoparticle-based manipulation techniques, grounded in atomic force microscopy (AFM), show promising applications. Research indicates that the localized electric field enhancement effect ("lightning rod effect") of silicon nanoparticles can capture single bacteria or viruses at relatively low laser power. Combined with their ability to enhance Raman spectroscopy, in-situ, highly sensitive fingerprinting of captured targets is possible. However, existing single-probe capture technologies suffer from low throughput, making it difficult to meet the practical needs of simultaneous and rapid monitoring of multiple airborne microorganisms. Therefore, there is an urgent need to develop novel, integrated, high-throughput real-time aerosol microbial detection systems. Summary of the Invention

[0006] The purpose of this invention is to provide an automated bioaerosol detection platform, detection method, and application, thereby overcoming the shortcomings of existing technologies. It integrates the "probe array method" with the microfluidic-LAMP / CRISPR platform to construct a new generation of bioaerosol detection system that integrates high-throughput physical enrichment, specific biochemical recognition, and precise optical detection.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides an automated bioaerosol detection platform, comprising a microfluidic chip module for carrying and testing samples; a nanocapture and thermal sensing module, wherein the nanocapture is integrated into a dedicated capture chamber at the sample inlet of the microfluidic chip for specific capture and enrichment of target microorganisms; a thermal sensing module employing Raman technology to invert local temperature in real time by monitoring the full width at half maximum (FWHM) of the Raman characteristic peaks at silicon nanotip, and feeding the temperature data back to the temperature control module for dynamic optimization of lysis or amplification temperature curves; a fluid flow drive and control module for sample transfer, nucleic acid extraction, and reagent dispensing; a reagent storage module for storing reagents and primers required for LAMP and CRISPR reactions; a temperature control module including an amplification unit providing a constant temperature environment and a reaction container for nucleic acid amplification; an optical detection module for real-time acquisition of fluorescence signals within the reaction container and analysis of melting curves; a main control and data analysis module with built-in machine learning algorithms capable of normalizing and differentially processing high-resolution melting curve data, and comparing it with standard curves in a database to achieve automatic identification and typing of strains; and a power supply module providing power to the device.

[0009] The nanocapture and thermal sensing module includes a silicon nano-tip array chip and a thermal sensing device. The silicon nano-tip array chip has several regularly arranged silicon nano-tips, and the surface of the silicon nano-tips is modified with biocapture molecules.

[0010] The thermal sensing device comprises a laser, a dichroic mirror, an objective lens, and a Raman spectrometer connected in sequence. The laser beam emitted by the laser is focused onto a silicon nanoparticle array chip by the dichroic mirror and the objective lens. Specifically, the Raman thermal sensing module uses a 530-535 nm laser, which guides the Raman scattered light to the spectrometer through the dichroic mirror, while simultaneously reflecting the Rayleigh scattered light to the camera, thus achieving the dual functions of temperature monitoring and particle tracking.

[0011] In some other embodiments, the biocapture molecule is one or more of antibodies, nucleic acid aptamers, and lectins; the surface of the silicon nanotip also contains a blocking protein layer. The silicon nanotip array chip is used to specifically capture one or a class of target microorganisms. The silicon nanotip is treated with bovine serum albumin (BSA) and ethanolamine solution to block all unoccupied crosslinking agent sites, thereby reducing background noise.

[0012] In some other embodiments, the microfluidic chip includes an upper layer, a middle upper layer, a middle layer, a middle lower layer, and a bottom layer that are stacked and bonded from top to bottom.

[0013] The upper layer is equipped with a sample inlet and a vent.

[0014] The upper and middle layers are equipped with silicon nano-tip trapping regions for fixing silicon nano-tip array chips;

[0015] The middle layer is pre-embedded with magnetic beads for nucleic acid extraction;

[0016] The middle and lower layers contain a LAMP / CRISPR reaction chamber array, which includes multiple detection channels, specifically a positive control channel, a negative control channel, and a negative independent control channel.

[0017] In some other embodiments, the silicon nanoparticle tip array chip is fabricated as follows:

[0018] A regularly arranged array of microwells was fabricated on a substrate using photolithography, with a silicon pillar formed at the center of each microwell.

[0019] An array of silicon nano-tippies was grown at the top of a silicon pillar using a metal-assisted chemical etching method.

[0020] The array of silicon nanotippies was sequentially hydroxylated, aminated, and aldehyde-treated; different biocapture molecules were then immobilized at designated positions on the aldehyde-treated array.

[0021] The solution is used to block the active sites on the array that are not occupied by biocapture molecules.

[0022] In some other embodiments, the silicon pillar has a height of 3-5 μm and a diameter of 150-250 nm;

[0023] The silicon nanotip has a radius of curvature of less than 10 nm and a height of 400-500 nm. The coefficient of variation of the geometry of the silicon nanotip is less than 5%.

[0024] In some other embodiments, the automated bioaerosol detection platform also includes a reagent storage module. The reagent storage module includes lyophilized LAMP reaction premix, LAMP-specific primer set, strand displacement DNA polymerase, dNTPs, a CRISPR / Cas12a buffer system, specific crRNA, and fluorescent dye.

[0025] In some other embodiments, the automated bioaerosol detection platform also includes a temperature control module. The temperature control module includes a cooling chip and a temperature sensor, and is connected to the underlying microfluidic chip. The temperature control module achieves a temperature control error of ±0.1 °C, enabling precise control of nucleic acid lysis (95 °C), LAMP amplification (60-65 °C), and Ct and HRM analysis (65-95 °C).

[0026] In some other embodiments, the automated bioaerosol detection platform also includes an optical detection module and a fluid flow drive and control module. The optical detection module includes a multi-channel excitation light source and a detector; the fluid flow drive module includes an inlet motor and a peristaltic pump; and the control module includes a processor and a display screen.

[0027] Specifically, the optical detection module integrates a multi-channel LED excitation source and a PMT / CCD detector, supporting real-time fluorescence monitoring and data acquisition. The fluid flow drive and control module uses a high-speed motor to drive the chip rotation, relying on centrifugal force and capillary valves to control liquid flow; an integrated micro-pump (peristaltic pump) is used to deliver samples and reagents. The online high-precision detection technology for bioaerosols uses the CFX96 touchscreen real-time PCR detection system for real-time monitoring (1 min at 37 ℃, 30 cycles).

[0028] In some other embodiments, the LAMP pre-primers include two primer pairs, FIP / BIP and F3 / B3, synthesized by Sangon Biotech Ltd. The CRISPR pre-primers include ssDNA-FQ and crRNA, synthesized by Sangon Biotech Ltd.

[0029] The LAMP reaction solution is 2×Lamp PCR Master Mix (Universal). The reaction system includes 5 μL 2×Lamp PCR Master Mix, 0.5-1.5 μL FIP / BIP primer pair (10 µM), 0.5-1 μL F3 / B3 (10 µM) primer pair, 1-5 μL sample, 0.5-1 μL DNA polymerase, and ddH2O added to 10 μL.

[0030] The CRISPR reaction consists of 0.5 μL EnGen® Lba Cas12a nuclease, 0.5–1 μL ssDNA-FQ probe, 0.5–1 μL RNase inhibitor, 1–2 μL NE Buffer 2.1 reaction buffer, 0.5–1.5 μL LAMP reaction product, and enzyme-free water to a final volume of 10 μL. The mixture is incubated at 37 °C for 20 min.

[0031] The CRISPR reaction products were observed under LED blue light to detect the endpoint fluorescence. The excitation wavelength was 450 nm-490 nm, and the receiving wavelength range was 518 nm-525 nm.

[0032] Secondly, the present invention provides the application of the bioaerosol automated detection platform of the first aspect in the identification of pathogen species, gene detection and molecular typing of strains in atmospheric / exhaled / seawater pathogens.

[0033] Thirdly, the present invention provides an automated detection method for bioaerosols, employing the automated bioaerosol detection platform of the first aspect, comprising the following steps:

[0034] The sample to be tested is added to the sample inlet, and the microorganisms are captured and enriched by the silicon nanoparticle array. In-situ Raman spectroscopy scanning and screening are then performed to obtain the target microorganisms.

[0035] The target microorganism is lysed, purified, and washed to obtain a nucleic acid template;

[0036] The nucleic acid template was mixed with loop-mediated isothermal amplification reagent for LAMP isothermal amplification, and the first fluorescence signal was monitored in real time. The LAMP amplification product was mixed with the CRISPR-Cas detection system, and isothermal detection was performed, while the second fluorescence signal generated by the trans-cleavage activity of the Cas protein was monitored in real time.

[0037] Based on the first and second fluorescence signals, and combined with the analysis of the melting curve of the amplification products, data fusion and analysis were performed to obtain the strain identification results.

[0038] In some other embodiments, the sample access method involves automatically or manually injecting the sample liquid (such as sterile aqueous suspension) collected by the electrostatic field graded aerosol sampler into the sample inlet of the microfluidic chip.

[0039] In some other embodiments, the strain identification method involves extracting the strain's nucleic acid, mixing it with CRISPR crRNA and reaction solution using pre-set LAMP primers in the device, reacting at 65 °C for 30 min, and then inactivating it at 90 °C for 2 min. The strain is identified by detecting the fluorescence signal intensity of the reaction system under a 485 nm laser.

[0040] The beneficial effects of this invention are:

[0041] (1) The bioaerosol automated detection platform based on microfluidics and nanoprobe arrays constructed in this invention integrates modules for silicon nanoparticle array capture, nucleic acid extraction, LAMP amplification, CRISPR / Cas12a detection, and real-time fluorescence signal reading, establishing a fully automated integrated system from aerosol sample introduction to strain typing results output. When using Escherichia coli as a model, the system detection limit reaches 2 CFU / mL, requiring no manual intervention throughout the process, greatly reducing operational errors and sample contamination risks, and significantly improving the reliability and efficiency of detection.

[0042] (2) The detection platform of this invention upgrades the traditional "serial" detection to "parallel" detection by introducing a silicon nanoparticle array, enabling simultaneous, high-throughput capture and enrichment of multiple microorganisms, with the detection limit increased to 0.1-2 CFU / mL, an order of magnitude higher than traditional methods. Simultaneously, the platform integrates a dual-signal system of Raman spectroscopy and fluorescence detection, enabling not only genotypic identification of bacterial strains but also the acquisition of biochemical phenotypic information of microorganisms through in-situ Raman spectroscopy, thereby providing a more comprehensive assessment of strain activity, metabolic state, and potential pathogenicity.

[0043] (3) Through verification with actual samples such as air and exhaled air, the platform can complete the entire process from sampling to reporting within 1 hour, meeting the timeliness requirements of rapid on-site detection. The equipment adopts a compact design, supports dual-mode power supply, and takes into account the usage needs of laboratory and outdoor mobile scenarios. It has successfully built a complete real-time online monitoring and early warning system, providing a reliable technical tool for timely early warning and intervention of bioaerosol risks in high-risk environments such as hospitals, farms, and public places. Attached Figure Description

[0044] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0045] Figure 1 This is a three-dimensional schematic diagram of the automatic bioaerosol detection platform based on microfluidics and nanoprobe array in Embodiment 2 of the present invention;

[0046] Figure 2 This is a perspective view of the automated bioaerosol detection platform based on microfluidics and nanoprobe array in Embodiment 2 of the present invention.

[0047] Figure 3 The diagram shows the overall structure and exploded structure of the microfluidic chip in Embodiment 1 of the present invention; wherein, A is the overall structure diagram and B is the exploded structure diagram;

[0048] Figure 4 This is a flowchart illustrating the LAMP / CRISPR reaction principle in Embodiment 3 of the present invention; wherein, A is a flowchart illustrating the LAMP amplification reaction principle and B is a flowchart illustrating the CRISPR amplification reaction principle.

[0049] Figure 5 This is a schematic diagram of the silicon nano-tip array in Embodiment 2 of the present invention;

[0050] Figure 6 This is a diagram showing the optical path flow of the silicon nanotip probe in Embodiment 2 of the present invention;

[0051] Figure 7 This is a flowchart of the automatic detection platform for bioaerosols based on microfluidics and nanoprobe arrays in Embodiment 2 of the present invention.

[0052] The components include: 1. Microfluidic inlet; 2. Touchscreen display; 3. LAMP reagent storage inlet; 4. Sample inlet; 5. CRISPR reagent storage inlet; 6. Microfluidic chip; 7. Excitation source and miniature camera; 8. Thin-film Peltier element and high-precision temperature sensor; 9. Control and power module; 10. Data processing system; 11. LAMP reagent storage area; 12. Sample injection, high-speed motor rotation drive, and port temperature control drive assembly; 13. CRISPR reagent. 14. Storage area; 15. Waste liquid treatment unit; 16. Raman spectrometer; 17. OB / DM system; 18. Raman excitation light; 19. Upper layer of microfluidic chip; 20. Upper middle layer of microfluidic chip; 21. Middle layer of microfluidic chip; 22. Lower middle layer of microfluidic chip; 23. Bottom layer of microfluidic chip; 24. Photodetector; 25. Fluorescence collecting lens; 26. Detection filter; 27. LED light source; 28. Excitation filter; 29. ​​Dichroic mirror; 30. Objective lens; 41. Sample to be tested. Detailed Implementation

[0053] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0054] The test bacteria, Bacillus subtilis and Escherichia coli, were purchased from the China Center for Type Culture Collection. The test primers E.BIP / E.FIP, E.F3 / E.B3, B.BIP / B.FIP, B.F3 / B.B3, ssDNA-FQ, E.crRNA, and B.crRNA were all synthesized by Shanghai Sangon Biotech Co., Ltd. The LAMP and CRISPR primers for other atmospheric and exhaled gas microorganisms were the same.

[0055] Various microbial antibodies are synthesized by General Biotech Ltd.

[0056] The culture media and solutions involved are as follows:

[0057] LB solid medium: 1% peptone, 1% NaCl, 1% yeast extract, 2% agar powder.

[0058] LB liquid medium (5 mL): peptone 0.5 g, NaCl 0.5 g, yeast extract 0.25 g.

[0059] 1× TE Buffer: pH 8.0: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0).

[0060] Example 1

[0061] First, this embodiment provides a detailed construction and preparation method for the nano-capture and thermal sensing module in the automated bioaerosol detection platform, as follows:

[0062] (1) Design and substrate fabrication of silicon nanoprobe array: Low-stress silicon nitride thin film was used as the array substrate. Regularly arranged microwell structures were fabricated on the substrate through photolithography and anisotropic etching processes. At the center of each microwell is a silicon pillar with a height of 4 μm and a diameter of 200 nm, which is used to grow silicon nanowires. The array size can be flexibly designed according to the detection requirements to achieve simultaneous screening of more than ten to dozens of target microorganisms.

[0063] (2) In-situ growth and morphology control of silicon nanotip: Single-crystal silicon nanotips were grown on the top of silicon pillars using metal-assisted chemical etching. By precisely controlling the concentration, temperature, and time of the etching solution, an array of tips with a radius of curvature of less than 10 nm and a uniform height (approximately 500 nm) could be fabricated. The geometry of the tips (such as height and cone angle) directly affects their electric field enhancement factor and capture efficiency. Through process optimization, the coefficient of variation of the enhancement factor (M_loc) of each tip in the array can be made less than 5%, ensuring the consistency of detection.

[0064] (3) Multifunctional modification of probe surfaces: The entire array is treated with oxygen plasma to generate abundant silanol groups (-OH) on the surface of silicon nanotip and remove organic contaminants; the array is then immersed in APTES (3-aminopropyltriethoxysilane) solution to form an aminated surface. Subsequently, a bifunctional crosslinking agent (glutaraldehyde) is used to convert the amino groups into active groups (aldehyde groups) that can covalently bind to biomolecules; microfluidic spotting technology and digital micromirror device (DMD) mediated photolithography are used to precisely and separately spot the captured molecules (specific antibodies, nucleic acid aptamers, and lectins, etc.) onto the designated nanotip or tip clusters in the array. For example: anti-Escherichia coli O antigen antibody is spotted onto tip 1; anti-Staphylococcus aureus protein A antibody is spotted onto tip 2; nucleic acid aptamer targeting influenza virus hemagglutinin is spotted onto tip 3; then bovine serum albumin and casein solutions are used to block the unoccupied active sites on the array to minimize nonspecific adsorption.

[0065] (4) Integration and Packaging: The prepared functionalized probe array chip is bonded to the wall of the dedicated capture chamber of the microfluidic chip using precision alignment technology. The chamber has a sample inlet at the top and transparent sidewalls to facilitate laser focusing and signal collection by the objective lens. The inlet and outlet are connected to the sample loading unit and nucleic acid extraction unit through microchannels.

[0066] More specifically, this embodiment provides a process optimization procedure for nanocapture in an automated bioaerosol detection platform, as follows:

[0067] (1) Material and substrate selection optimization: Low-stress silicon nitride (Si3N4) thin film was selected as the array substrate, whose thermal expansion coefficient matches that of silicon, reducing deformation caused by temperature changes. Single crystal silicon wafers with (100) crystal orientation and moderate resistivity were used to ensure the anisotropy and consistency of etching.

[0068] (2) Optimization of photolithography and microwell array fabrication: Spin coating was used, with the rotation speed controlled at 3000-4000 rpm and the time at 30 s to ensure uniform resist layer thickness (1.5 μm ± 0.05 μm). Exposure parameters were optimized, using 365 nm ultraviolet light and controlling the exposure dose at 80-100 mJ / cm². 2 To avoid overexposure or underexposure leading to microwell size deviation, develop in standard MF-319 developer for 45-60 seconds, monitoring the pattern transfer effect in real time to ensure consistency between microwell diameter (150-250 nm) and depth (3-5 μm).

[0069] (3) Optimization of silicon nanotip morphology control: The tip curvature radius was controlled to be <10 nm by adjusting the concentration of oxidant in the etching solution and the etching time. SEM was used for periodic sampling and inspection to provide feedback for adjusting process parameters. In-situ optical monitoring was used to monitor the height of the silicon pillars during the etching process to achieve high consistency within the range of 400-500 nm. In addition, ≥30 tips were sampled from each batch for SEM imaging and three-dimensional morphology analysis to calculate the coefficient of variation (CV) of height, cone angle, and radius of curvature, ensuring that CV <5%.

[0070] This embodiment also provides a method for preparing a centrifugal microfluidic chip in an automated bioaerosol detection platform, as detailed below:

[0071] The materials used to prepare the microfluidic device are as follows:

[0072] Polymethyl methacrylate (PMMA) sheet (thickness: 1.5 mm); laser cutting machine (Universal LaserSystems); double-sided pressure-sensitive adhesive (thickness: 100 μm); hot press bonding machine (temperature: 120 ℃, pressure: 0.5 MPa, time: 10 min); magnetic beads for nucleic acid purification (diameter: 1 μm, surface modified with silanol groups).

[0073] The fabrication steps of the microfluidic device are as follows:

[0074] (1) Design of chip structure: Solidworks software was used to draw the five-layer structure diagram of the microfluidic chip (including upper layer, upper middle layer, middle layer, lower middle layer and bottom layer). The upper layer is the sample inlet and vent, the upper middle layer is the silicon nanotip capture area, the middle layer is the nucleic acid extraction area (pre-embedded magnetic beads), the lower middle layer is the LAMP / CRISPR reaction area (with 16 reaction chambers), and the bottom layer is the temperature control processing structure.

[0075] (2) Microfluidic chip fabrication: The positive mold is fabricated using soft photolithography, and the microfluidic chip is made by molding. PMMA is poured onto the positive mold and then bonded to the glass sheet after curing.

[0076] (3) Laser cutting: Use a laser cutting machine to cut and modify the five-layer PMMA board to obtain the structure of each layer.

[0077] (4) Magnetic bead pre-embedding: The magnetic bead suspension (magnetic bead concentration: 10 mg / mL) is evenly coated in the nucleic acid extraction area of ​​the middle layer and fixed after drying at room temperature.

[0078] (5) Lamination bonding: The five-layer PMMA board is aligned and bonded together with double-sided adhesive, and then placed in a hot press bonding machine. It is hot-pressed for 10 minutes at 120℃ and 0.5 MPa to ensure that each layer is firmly bonded.

[0079] (6) Quality inspection: Check the chip's sealing and flow channel patency to ensure no leakage or blockage. The resulting microfluidic chip structure has a sample inlet, a silicon nanoparticle tip capture area, a nucleic acid extraction area, a reaction chamber array, and a passive valve system, which can realize automatic sample dispensing and reaction process control.

[0080] The LAMP / CRISPR reaction region of this chip has 16 detection channels, each with a volume of approximately 10 μL and a bottom area of ​​7 mm². 3 The height is 10 mm. A fluid control channel is connected to the top of each detection channel; a positive control channel P, a negative control channel N, and a negative independent control channel N are also provided. This serves as a quality control measure for the reaction system.

[0081] Example 2

[0082] This embodiment provides the composition, assembly, and operation method of an automated bioaerosol detection platform, as detailed below:

[0083] First, this embodiment provides an automated bioaerosol detection platform, including the following modules:

[0084] Microfluidic chip modules are used to hold and test samples. A microfluidic chip module mainly consists of a microfluidic chip (such as...) Figure 3 As shown in Figure A), the microfluidic chip includes, from top to bottom, an upper microfluidic chip layer 18, a middle upper microfluidic chip layer 19, a middle microfluidic chip layer 20, a middle lower microfluidic chip layer 21, and a bottom microfluidic chip layer 22 (as shown in Figure A). Figure 3 (As shown in B in the diagram). The upper layer 18 of the microfluidic chip includes a sample inlet and vent holes; the upper middle layer 19 is the silicon nanoparticle-tip capture region; the middle layer 20 is the nucleic acid extraction region (with embedded magnetic beads); and the lower middle layer 21 is the LAMP / CRISPR reaction region, including 16 detection channels (reaction chambers), each with a volume of approximately 10 μL and a bottom area of ​​7 mm². 3 The height is 10 mm. Each detection channel is connected to a fluid control channel at the top; it also includes a positive control channel P, a negative control channel N, and a negative independent control channel N. This serves as quality control for the reaction system. The bottom layer 22 of the microfluidic chip is a temperature control structure.

[0085] The nanocapture and thermal sensing module integrates a nanocapture device within a dedicated capture chamber at the sample inlet of a microfluidic chip for the specific capture and enrichment of target microorganisms. The nanocapture within the module comprises an array of regularly arranged silicon nanotippies on a substrate and its surface. For example... Figure 5As shown, the silicon nanotip array has a regularly arranged microwell structure, with a silicon pillar at the center of each microwell, 3-5 μm high and 200 nm in diameter, used for growing silicon nanowires. The array size can be flexibly designed according to detection needs to achieve simultaneous screening of dozens of target microorganisms. Single-crystal silicon nanotip is grown at the top of the silicon pillar, forming a tip array with a radius of curvature of less than 10 nm and a uniform height (approximately 500 nm). The geometry of the tip (such as height and cone angle) directly affects its electric field enhancement factor and capture efficiency. Through process optimization, the coefficient of variation of the enhancement factor (M_loc) of each tip in the array can be made less than 5%, ensuring the consistency of detection. Tip arrays with a radius of curvature of less than 10 nm and a uniform height (approximately 500 nm) can be prepared. The surface of the silicon nanotip has abundant silanol groups (-OH) and active groups (aldehyde groups) that can covalently bind to biomolecules; it can accurately and separately spot captured molecules (specific antibodies, nucleic acid aptamers, and lectins, etc.) onto designated nanotip or tip clusters in the array.

[0086] The thermal sensing module in the nanocapture and thermal sensing module uses Raman sensing technology to invert the local temperature in real time by monitoring the changes in the full width at half maximum (FWHM) of the Raman characteristic peaks at the silicon nanotip, and feeds the temperature data back to the temperature control module for dynamic optimization of the lysis or amplification temperature curve; the liquid flow drive and control module is used to complete sample transfer, nucleic acid extraction and reagent dispensing.

[0087] The reagent storage module is used to store the reagents and primers required for LAMP and CRISPR reactions.

[0088] Fluid drive module: high-speed stepper motor (speed: 100-6000 rpm) and miniature peristaltic pump.

[0089] The temperature control module includes an amplification unit that provides a constant temperature environment and a reaction vessel for nucleic acid amplification. The temperature control module includes a thin-film Peltier element (model: TEC1-12706) and a PT100 temperature sensor.

[0090] The optical detection module is used for real-time acquisition of fluorescence signals and melting curve analysis of the samples within the reaction vessel. The optical detection module includes a multi-channel LED excitation source (wavelengths: 450 nm, 485 nm, 532 nm), a PMT detector, a fluorescence collecting lens, a detector filter, a dichroic mirror, and an objective lens. Figure 6As shown, the optical detection module includes, from top to bottom, a photodetector 23, a fluorescence collecting lens 24, a detection filter 25, a dichroic mirror 28, an objective lens 29, and a sample 30 to be tested; it also includes an LED light source 26 and an excitation filter 27. The LED light source 26 is used to project light into the dichroic mirror 28. The LED light source 26 and the photodetector 23 are aligned with the sample 30 to be tested in the LAMP / CRISPR reaction chamber array, and the optical path is adjusted to the optimal focusing state. It is ensured that the fluorescence detection optical path is independent of the Raman optical path of the nanocavity to avoid signal interference. The optical path is adjusted to ensure that the laser beam can be precisely focused on the probe array region, and that the generated Raman scattered light can be efficiently collected by the objective lens 29 and guided to the Raman spectrometer 15.

[0091] The main control and data analysis module has a built-in machine learning algorithm that can normalize and differentially process high-resolution melting curve data and compare it with standard curves in the database to achieve automatic identification and typing of strains. The main control and data analysis module includes an embedded processor (ARM Cortex-A7) and a touch screen.

[0092] The power module provides power to the device; the power module includes a lithium-ion battery (12 V, 10 Ah) and an AC adapter.

[0093] Secondly, this embodiment provides an assembly step for an automated bioaerosol detection platform:

[0094] (1) Mechanical structure construction: An aluminum alloy frame is used as the main structure to fix the installation position of each module.

[0095] (2) Install the temperature control module: attach the Peltier element and temperature sensor to the bottom of the LAMP / CRISPR reaction chamber and connect the temperature control drive circuit. At the same time, integrate another set of micro Peltier elements and sensors on the outer wall of the nanocapture chamber for preliminary temperature control and monitoring of the capture process.

[0096] (3) Integrated nano-capture and thermal sensing module:

[0097] a. Array chip bonding: The prepared functionalized silicon nanoparticle array chip is aligned and bonded to the bottom of the dedicated capture chamber of the microfluidic chip using a precision fixture. Sealant is used to ensure its airtightness.

[0098] b. Optical Path Integration: The low-power 532 nm laser, dichroic mirror, and Raman signal collection optical path are mounted on the side of the capture chamber. The optical path is carefully adjusted to ensure that the laser beam can be precisely focused on the probe array region and that the generated Raman scattered light can be efficiently collected by the objective lens and guided to the Raman spectrometer.

[0099] c. Circuit and signal connection: Connect the signal lines of the laser and Raman spectrometer to the main control board to ensure that the main control system can control the laser's on / off state and power, and receive temperature and spectral data from the spectrometer.

[0100] (4) Install the optical detection module: Align the LED light source and PMT detector with the LAMP / CRISPR reaction chamber array and adjust the optical path to the optimal focusing state. Ensure that the fluorescence detection optical path is independent of the Raman optical path of the nano-chamber to avoid signal interference.

[0101] (5) Integrated fluid drive: The motor is connected to the rotating shaft of the microfluidic chip, and the peristaltic pump is connected to the reagent delivery tube. The inlet of the capture chamber is connected to the sample loading unit through a Teflon microtube to ensure that the flow path is sealed and leak-free.

[0102] (6) Circuit connection: Connect each module to the control board, write the control program (based on Linux system), and realize automated process control through system calculation.

[0103] (7) Overall testing: After powering on, check the functions of each module to ensure that the temperature control accuracy is ±0.1 ℃ and the fluorescence detection signal-to-noise ratio is >200:1.

[0104] The assembled equipment, such as Figure 1 and Figure 2 As shown, specifically, by Figure 1 As can be seen, this automated bioaerosol detection platform is an integrated, desktop cuboid analytical instrument, including a microfluidic inlet 1 on the side and a display touchscreen 2, LAMP reagent storage inlet 3, sample inlet 4, and CRISPR reagent storage inlet 5 on the top. The microfluidic inlet 1 has a slotted structure for carrying sample capture, nucleic acid extraction, and reaction chips. The display touchscreen 2 is a rectangular touch screen, forming the human-machine interface for control and result reading. The sample inlet 4 is a circular sample injection port. On either side of the sample inlet 4 (for sample loading) are the LAMP reagent storage inlet 3 (for nucleic acid amplification) and the CRISPR reagent storage inlet 5 (for reagent input for specific detection). All components are connected by microtubes and equipped with a micro-peristaltic pump. When needed, the micro-peristaltic pump program is activated to inject the sample into the sample inlet 4, forming an integrated function of sample loading, nucleic acid extraction, amplification detection, and data analysis.

[0105] Depend on Figure 2As can be seen, this automated bioaerosol detection platform includes components for sample introduction, reaction-multimodal detection, waste liquid treatment, and data analysis. These components are tightly connected to the internal flow path and circuit system, working together to achieve fully automated analysis. Specifically, the sample introduction component includes a LAMP reagent storage area 11, sample injection, a high-speed motor rotation drive and port temperature control drive assembly 12, and a CRISPR reagent storage area 13. The reaction-multimodal detection component includes a microfluidic chip 6, an excitation light source and a miniature camera 7, a thin-film Peltier element and a high-precision temperature sensor 8, a Raman spectrometer 15, an OB / DM system 16, and a Raman excitation light 17. The microfluidic chip 6 is placed on a temperature control module composed of a thin-film Peltier element and a high-precision temperature sensor 8 to ensure accurate LAMP isothermal amplification. After the reaction, the excitation light source and the miniature camera 7 image the fluorescence signal. The Raman excitation light 17 is focused onto the chip detection area by the OB / DM system 16, and the generated Raman signal is received by the Raman spectrometer 15 for biochemical characterization. A waste liquid treatment unit 14 is also provided at the top to collect reaction waste. The LAMP reagent storage area 11 and the CRISPR reagent storage area 13 are connected by a flow path and pumped to the sample injection by a micro peristaltic pump, driven by a high-speed motor rotation and port temperature. The control drive assembly is located at the bottom outlet position of the sample injection site. When the microfluidic chip 6 is attached to the bottom of the sample inlet 4 by the telescopic moving rod, the reaction system is accurately injected into the microfluidic chip 6 under the control of the high-speed motor and temperature drive.

[0106] The entire system is driven and powered by the control and power module 9. All optical, temperature control and fluid actions are executed synchronously under the instructions of the data processing system 10, forming a closed, intelligent and integrated detection platform from "sample introduction-reaction-multimode detection-waste liquid treatment-data analysis".

[0107] Next, this embodiment provides the fully automated workflow of the above-mentioned automated bioaerosol detection platform, such as... Figure 7 As shown, the specific steps include:

[0108] (1) Sample loading (step S101): The prepared liquid sample (bacterial culture medium and exhaled air eluent) is added to the sample inlet of the chip through a microfluidic pump or manually.

[0109] (2) Chip loading and startup (step S102): Place the chip into the instrument and close the door. The user selects the preset detection program (such as "Identification and Typing") on the touch screen and clicks the "Start" button.

[0110] (3) High-throughput specific capture (step S103):

[0111] The fluid-driven module circulates the sample solution within the array chamber at an optimal flow rate of approximately 10 μL / min for 15 minutes. This allows the target microorganisms in the sample to specifically bind to the corresponding functionalized probes on the array, firmly capturing them on their respective nanotippies and enriching and concentrating the target microorganisms from a large volume of sample solution to 30 μL.

[0112] (4) Washing and in-situ Raman screening (step S104): Rinse the capture chamber with sterile PBS buffer to remove unbound impurities and non-target microorganisms. Switch the optical detection module to Raman spectroscopy mode to perform a rapid scan of the probe array. Analyze the TERS spectrum of each probe site in real time using a machine learning algorithm.

[0113] (5) Controlled release and transfer (step S105): The main control system injects low-pH glycine-hydrochloric acid buffer into the capture chamber according to the screening results or preset program. This mild elution condition breaks the antigen-antibody binding, releasing the captured microorganisms intact and viable from the probe. The released microbial suspension is precisely transported by centrifugation to the downstream nucleic acid extraction area for subsequent LAMP-CRISPR processes.

[0114] (6) Nucleic acid extraction and purification (steps S106-S108):

[0115] Thermal lysis (S106): The instrument's temperature control module heats the lysis chamber to 95 °C for 5 min to fully lyse the cells and release nucleic acids.

[0116] Magnetic bead purification (S107): The instrument control motor rotates the chip according to a specific program, and centrifugal force drives the lysis buffer to flow through the nucleic acid purification zone (magnetic bead area), where nucleic acids are specifically captured.

[0117] Washing and elution (S108): Washing buffer and elution solution flow sequentially through the purification zone under centrifugal force to remove impurities. Finally, a small amount of elution solution (50-100 μL) is used to elute the purified nucleic acid and transfer it to the pre-dispensed channel.

[0118] (7) Parallel amplification and detection (steps S109-S111):

[0119] Distribution and Reconstitution (S109): By increasing the rotation speed, the eluent is precisely distributed under centrifugal force and flows into the circumferentially arranged reaction chambers, storing the pre-placed reaction reagents in the chambers.

[0120] LAMP isothermal amplification real-time fluorescence monitoring (S110): The temperature control module precisely controls the reaction chamber array at 65 ℃. The optical detection module cyclically scans the fluorescence signal of each reaction chamber at set intervals (every 5 s) to monitor the LAMP amplification process in real time.

[0121] CRISPR Real-Time Fluorescence Monitoring (S111): After LAMP amplification, the pre-prepared CRISPR reaction solution and reaction products are mixed by centrifugation, and then kept at 37 °C for 20 min. The optical detection module scans the fluorescence signal of each reaction chamber cyclically at set intervals (every 5 s) to monitor the CRISPR reaction fluorescence signal in real time.

[0122] (8) Analysis of Ct and HRM melting curves (step S112): After the reaction is completed, the temperature control module immediately executes the melting program and slowly heats up to 95 ℃ at a rate of 0.3 ℃ / s. At the same time, the optical detection module continuously collects the fluorescence signal changes of each reaction chamber at high frequency and plots the melting curve.

[0123] (9) High-precision analysis and reporting (steps S113-S115):

[0124] Data reading (S113): The software first determines the positive or negative status of each target gene based on the real-time fluorescence curve.

[0125] Strain identification (S114): The data from positive wells are normalized and differentially processed, and their melting curves or differential curves are compared with the standard curves (corresponding to different strain types) in the instrument's built-in database. Predictions are made using machine learning models.

[0126] Report generation (S115): The final results (including target positivity / negativity, strain identification and typing results, melting curve, etc.) are automatically displayed on the screen and can be connected to a printer to output a report.

[0127] Specifically, the fluorescence detection method is as follows:

[0128] Extracted genomic DNA was used as a template for qPCR. Fluorescence signals were measured using LAMP primers, and the results were compared with those from an automated bioaerosol detection platform based on microfluidics and nanoprobe arrays to optimize experimental details and equipment parameters. qPCR reactions were performed using the SYBR Green Premix Pro Taq HS qPCR Kit on a Light-Cycler 96 real-time PCR system. PCR reaction conditions were consistent with those of the online high-precision bioaerosol detection equipment: denaturation at 95 °C for 5 min, 50 isothermal cycles (5 s at 65 °C under fluorescence illumination), and a melting step (0.3 °C / s, from 60–95 °C).

[0129] Specifically, the algorithm of the main control and data analysis module adopts a dual-signal fusion analysis model, which includes a Raman signal analysis submodule, a fluorescence signal analysis submodule, and signal fusion and decision-making.

[0130] The working process of the Raman signal analysis submodule is as follows:

[0131] (1) Data preprocessing: Baseline correction (using adaptive iterative reweighted penalized least squares method), cosmic ray removal and vector normalization were performed on the raw TERS spectra.

[0132] (2) Feature extraction and classification: A pre-trained one-dimensional convolutional neural network (1D-CNN) was used for microbial identification. The CNN model consists of two convolutional layers (kernel sizes of 5 and 3, and filter numbers of 32 and 64, respectively), one global average pooling layer, and two fully connected layers. The model was trained on our self-built standard microbial TERS spectral database (containing 15 common environmental pathogens, each with more than 500 spectra) and achieved preliminary identification at the genus level with an accuracy of >96%.

[0133] (3) Output: This submodule outputs a preliminary list of microbial species and their relative abundance estimates.

[0134] Specifically, the working process of the fluorescence signal analysis submodule is as follows:

[0135] (1) Real-time fluorescence curve analysis: curve fitting was performed on the real-time fluorescence data of LAMP and CRISPR processes, the cycle threshold (Ct) was calculated, and negative or positive results were determined.

[0136] (2) HRM data analysis: The melting curve data were normalized (fluorescence values ​​were scaled to the 0-100% range), and then differential processing (negative derivative calculation) was performed to obtain the differential melting curve. The peak value (T) of the differential curve was extracted. m The peak value, peak shape, and peak area are used as characteristic values.

[0137] (3) Machine learning typing: The extracted HRM feature vectors are input into a support vector machine (SVM) classifier. This classifier is trained on a standard HRM curve database of known strains and can achieve accurate molecular typing at the strain level.

[0138] Specifically, the signal fusion and decision-making process is as follows:

[0139] The platform doesn't simply juxtapose the two results; instead, it performs a confidence-weighted fusion. For example, if the Raman submodule identifies "Escherichia coli" with high confidence (90%), and the fluorescence HRM submodule also types it as "0157:H7 strain" with high confidence, the system generates a comprehensive report with high confidence. If the Raman result has a lower confidence level (e.g., 40%) due to spectral overlap, but the HRM typing result is clear and has high confidence, the system will prioritize the nucleic acid typing result and note in the report "Raman spectroscopy suggests, confirmed by nucleic acid," effectively avoiding the limitations of a single technology.

[0140] Example 3

[0141] This embodiment is used to verify the detection performance of the equipment, using Bacillus subtilis 168 and Escherichia coli as standard strains.

[0142] Materials: Bacillus subtilis (CCTCC AB 2014293), Escherichia coli (ATCC 25922), LAMP primers (targeting the 16S rDNA genes of Bacillus subtilis and Escherichia coli), CRISPR crRNA (synthesized by Sangon Biotech), 2×LampPCR Master Mix (Universal), and EnGen® Lba Cas12a nuclease (NEB).

[0143] The specific steps are as follows: Bacillus subtilis and Escherichia coli were inoculated into LB liquid medium and cultured at 37 ℃ and 200 rpm until OD reached. 600 =0.6, take 1 mL of bacterial culture, centrifuge, and resuspend in 1×TE buffer to prepare 10 1-5 CFU / mL gradient dilution buffer. Inject 100 μL of bacterial culture into the sample inlet of the microfluidic chip. The microfluidic chip and storage module are pre-loaded with LAMP and CRISPR reaction primers (as shown in Table 1) and the reaction buffer required for the reaction. Select the "Strain Identification" program and start the device. The device automatically completes nucleic acid extraction, LAMP amplification (65 ℃, 30 min), CRISPR detection (37 ℃, 20 min), and C... t And HRM analysis (65-95 ℃).

[0144] Table 1: Primer sequences

[0145]

[0146] The LAMP-CRISPR reaction principle flowchart is as follows: Figure 4As shown, this LAMP-CRISPR reaction is a cascaded amplification system of LAMP amplification and CRISPR detection. Specifically, in the LAMP amplification stage, multiple primers (B3, F3, BIP, and FIP) are used to efficiently and exponentially amplify the target DNA template at a constant temperature of 65°C, generating a large amount of DNA product and completing the first signal amplification of the target sequence (e.g., ...). Figure 4 (As shown in A in the diagram); Subsequently, in the CRISPR detection stage, the Cas12a protein assembles with specific crRNA into a complex, precisely recognizing and activating the target sequence in the LAMP product, thereby exerting its "trans-cleavage" activity, non-specifically cleaving the fluorescent reporter molecule ssDNA in the system, causing the quencher group to separate from the fluorescent group and releasing a detectable fluorescent signal, thus achieving a second signal conversion and amplification (e.g., as shown in A in the diagram). Figure 4 (As shown in B in the diagram). The entire system combines efficient LAMP amplification with highly specific CRISPR recognition, ultimately converting the presence of trace nucleic acid targets into a strong fluorescent signal output.

[0147] Results Analysis: Real-time fluorescence curves showed that both Bacillus subtilis and Escherichia coli exhibited significant amplification signals during the LAMP stage (Ct value <30). After CRISPR detection, the fluorescence signal was further enhanced, with the signal-to-noise ratio increasing by more than 10-fold. HRM analysis revealed significantly different characteristic peaks in the melting curves of the two strains, enabling accurate typing. Sensitivity testing showed that the detection limit for Bacillus subtilis was 6 CFU / mL, and for Escherichia coli, it was 2 CFU / mL.

[0148] To compare the differences in sensitivity, operational complexity, and detection time between the platform of this invention and traditional detection methods, graded dilutions of Bacillus subtilis and Escherichia coli at the same concentration (10... 1-5 (CFU / mL). Samples were collected using a commercial aerosol sampler (Anderson sampler) with a polycarbonate filter membrane. After sampling, the filter membrane was placed in sterile PBS and eluted by shaking. DNA was extracted using a commercial nucleic acid extraction kit (Full Gold). Detection was performed using qPCR (primers as above), with reaction system and conditions following the kit instructions.

[0149] The results showed that the detection limits of traditional methods for Bacillus subtilis and Escherichia coli were 50 CFU / mL and 20 CFU / mL, respectively. The time from sampling to obtaining results was approximately 3-4 hours, requiring multiple manual operations. Compared to the platform of this invention (detection limits of 6 CFU / mL and 2 CFU / mL, respectively, with a total process time ≤1.5 hours), it exhibits lower sensitivity, longer processing time, and more cumbersome operation. The results demonstrate that this device possesses the advantages of high sensitivity, high specificity, and a high degree of automation.

[0150] Example 4

[0151] This embodiment demonstrates the application of the device in real environmental samples, detecting microorganisms in the atmosphere and human exhaled breath.

[0152] Sampling method: Air samples were collected from the hospital outpatient lobby using an electrostatic field graded aerosol sampler (flow rate: 20 L / min, time: 30 min). The sampling solution was sterile PBS buffer. Exhaled breath samples were collected from volunteers, and the sampler collection solution was rinsed with sterile saline.

[0153] Testing steps:

[0154] 1. Inject the collected sample solution into the sample inlet of the microfluidic chip.

[0155] 2. Run the device and select the "Multiple Target Detection" program.

[0156] 3. The equipment automatically performs silicon nanoparticle tip capture and Raman detection, nucleic acid extraction, and LAMP / CRISPR coupled detection, with targets including specific genes of common atmospheric microorganisms.

[0157] Test results: Eight potential pathogens, including Acinetobacter baumannii, Legionella pneumophila, and Streptococcus pneumoniae, were successfully detected. The entire test took less than 1 hour, and the results were 98% consistent with those of conventional methods.

[0158] Comparative Example 1

[0159] The detection method of Embodiment 4 of the present invention is compared with the efficiency and coverage of conventional methods in actual environmental sample detection. The specific method is as follows:

[0160] 1. For the same batch of air and exhaled air samples, the following methods were used respectively:

[0161] Culture method: The sampling solution is spread on agar plates, MacConkey plates, etc., and incubated at 37 ℃ for 24-48 h. Single colonies are picked for biochemical identification.

[0162] Conventional PCR method: After extracting total DNA, PCR amplification is performed using multiple pairs of specific primers, followed by electrophoresis detection.

[0163] 2. Record the detected bacterial species, detection time, and operating procedures.

[0164] The results showed that the culture method detected only 3 bacteria (Acinetobacter baumannii, Streptococcus pneumoniae, and Staphylococcus aureus), taking more than 48 hours. The conventional PCR method detected 5 bacteria, taking about 3 hours, but could not perform strain typing. The platform of this invention detected 8 potential pathogens, supported strain typing, and took ≤1 hour in the entire process, with a high degree of automation.

[0165] This embodiment 4 demonstrates that the device is suitable for rapid on-site monitoring and can provide technical support for infection control in public places.

[0166] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An automated bioaerosol detection platform, characterized in that, It includes a microfluidic chip module for carrying and testing samples; the nanocapture and thermal sensing module integrates nanocapture into a dedicated capture chamber at the sample inlet of the microfluidic chip for the specific capture and enrichment of target microorganisms; The thermal sensing module employs Raman spectroscopy to invert local temperature in real time by monitoring the changes in the full width at half maximum (FWHM) of the Raman characteristic peaks at silicon nanoparticle tips. This temperature data is then fed back to the temperature control module for dynamic optimization of lysis or amplification temperature curves. The fluid flow drive and control module handles sample transfer, nucleic acid extraction, and reagent dispensing. The reagent storage module stores reagents and primers required for LAMP and CRISPR reactions. The temperature control module includes an amplification unit providing a constant temperature environment and a reaction vessel for nucleic acid amplification. The optical detection module acquires fluorescence signals within the reaction vessel in real time and analyzes melting curves. The main control and data analysis module incorporates machine learning algorithms to normalize and differentially process high-resolution melting curve data, comparing it with standard curves in a database to achieve automatic identification and typing of bacterial strains. The power module provides the power source for the equipment; The nanocapture and thermal sensing module includes a silicon nanoparticle array chip and a thermal sensing device. The silicon nanoparticle array chip is provided with a number of regularly arranged silicon nanoparticles, and the surface of the silicon nanoparticles is modified with biocapture molecules. The thermal sensing device includes a laser, a dichroic mirror, an objective lens, and a Raman spectrometer connected in sequence; the laser beam emitted by the laser is focused onto a silicon nanoparticle array chip by the dichroic mirror and the objective lens. The microfluidic chip comprises, from top to bottom, an upper layer, a middle upper layer, a middle layer, a middle lower layer, and a bottom layer, which are stacked and bonded together sequentially. The upper layer is provided with a sample inlet and a ventilation hole; The upper and middle layers are provided with silicon nano-tip trapping regions for fixing silicon nano-tip array chips; The middle layer is pre-embedded with magnetic beads for nucleic acid extraction; The middle and lower layers are provided with a LAMP / CRISPR reaction chamber array, which includes multiple detection channels, and also includes a positive control channel, a negative control channel, and a negative independent control channel. Silicon nanotip is grown at the top of a silicon pillar, which has a height of 3-5 μm and a diameter of 150-250 nm. The silicon nanotip has a radius of curvature of less than 10 nm and a height of 400-500 nm. The coefficient of variation of the geometry of the silicon nanotip is less than 5%.

2. The automated bioaerosol detection platform according to claim 1, characterized in that, The biocapture molecule is one or more of antibodies, nucleic acid aptamers, and lectins; the surface of the silicon nanotip also contains a blocking protein layer.

3. The automated bioaerosol detection platform according to claim 1, characterized in that, The method for fabricating the silicon nanoparticle array chip is as follows: A regularly arranged array of microwells was fabricated on a substrate using photolithography, with a silicon pillar formed at the center of each microwell. An array of silicon nano-tippies was grown at the top of a silicon pillar using a metal-assisted chemical etching method. The array of silicon nanotippies was sequentially hydroxylated, aminated, and aldehyde-treated; different biocapture molecules were then immobilized at designated positions on the aldehyde-treated array. The solution is used to block the active sites on the array that are not occupied by biocapture molecules.

4. The automated bioaerosol detection platform according to claim 1, characterized in that, The aforementioned automated bioaerosol detection platform also includes a reagent storage module, which comprises lyophilized LAMP reaction premix, LAMP-specific primer set, strand displacement DNA polymerase, dNTPs, CRISPR / Cas12a buffer system, specific crRNA, and fluorescent dye.

5. The automated bioaerosol detection platform according to claim 1, characterized in that, The aforementioned bioaerosol automatic detection platform also includes a temperature control module, which includes a cooling chip and a temperature sensor, and the temperature control module is connected to the underlying layer of the microfluidic chip.

6. The automated bioaerosol detection platform according to claim 1, characterized in that, The aforementioned automated bioaerosol detection platform further includes an optical detection module, a fluid flow drive module, and a control module. The optical detection module includes a multi-channel excitation light source and a detector; the fluid flow drive module includes an inlet motor and a peristaltic pump; and the control module includes a processor and a display screen.

7. The application of the bioaerosol automated detection platform according to any one of claims 1-6 in the identification of atmospheric / seawater pathogen species, gene detection, and molecular typing of strains.

8. An automated method for detecting bioaerosols, characterized in that, The automated bioaerosol detection platform according to any one of claims 1-6 includes the following steps: The sample to be tested is added to the sample inlet, and the microorganisms are captured and enriched by the silicon nanoparticle array. In-situ Raman spectroscopy scanning and screening are then performed to obtain the target microorganisms. The target microorganism is lysed, purified, and washed to obtain a nucleic acid template; The nucleic acid template was mixed with loop-mediated isothermal amplification reagent for LAMP isothermal amplification, and the first fluorescence signal was monitored in real time. The LAMP amplification product was mixed with the CRISPR-Cas detection system, and isothermal detection was performed, while the second fluorescence signal generated by the trans-cleavage activity of the Cas protein was monitored in real time. Based on the first fluorescence signal and the second fluorescence signal, and combined with the analysis of the melting curve of the amplification product, data fusion and analysis were performed to obtain the strain identification results; The detection method described is not intended for disease diagnosis or treatment.

Images