System for quantitatively detecting and analyzing aerosol virus by using AuNRs (at) ZIF-8 microfluidic SERS (Surface Enhanced Raman Scattering) chip

Abstract

The invention discloses a quantitative detection and analysis system for aerosol viruses by using an AuNRs (at) ZIF-8 microfluidic SERS chip. The quantitative detection and analysis system comprises an aerosol sampling instrument, biological enrichment magnetic beads, an AuNRs (at) ZIF-8-SERS substrate, a microfluidic chip, a Raman spectrum detection system, a PLC control module, a Hall coil, a quantitative analysis model and the like. Wherein the aerosol sampling system adopts a low-shear-force air inlet structure, and biological enrichment magnetic beads are preloaded in sampling liquid; the biological enrichment magnetic beads are constructed in a way that ferroferric oxide nanospheres are coated with silicon dioxide, and the surfaces of the ferroferric oxide nanospheres are modified with an antibody pDNA2; an inner core of the AuNRs (at) ZIF-8-SERS substrate is formed by wrapping a gold nanorod with a metal organic framework, and pDNA1 is coupled to the surface of the gold nanorod; the micro-fluidic chip is provided with a three-channel inlet, a cell culture layer is enhanced, the biological activity is kept, and mixing enhancement is realized; the PLC main control module and the Hall coil mainly control the magnetic beads to operate; according to the quantitative analysis model, a concentration-toxicology quantitative structure-activity model is packaged in a PC, and direct output of the toxicology of microorganisms is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of novel biological detection instruments, and mainly relates to a quantitative detection and analysis system for aerosol viruses using an AuNRs@ZIF-8 microfluidic SERS chip. Background Art

[0002] Compared to non-biological aerosols, the core challenges of aerosol microbial detection lie in: 1) maintaining the activity of biological samples; 2) efficient enrichment and capture; and 3) analyzing dynamic processes. These key scientific issues currently remain technical bottlenecks hindering the accurate identification of aerosol microorganisms. Therefore, the development of new methods and technologies that enable in situ, non-destructive detection of aerosol microorganisms is of great practical significance.

[0003] Although traditional detection methods (such as real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay (ELISA)) have high sensitivity and specificity, their complicated operation procedures, long detection cycles, high equipment dependence and high costs have limited their application in large-scale screening and rapid on-site detection. Surface-enhanced Raman scattering (SERS) technology has shown great potential in the field of aerosol virus detection due to its unique molecular fingerprint recognition ability (detection limit can reach the single virus level), second-level response speed and non-destructive detection advantages. In particular, its compatibility with portable spectrometers provides an innovative solution for the development of point-of-care testing (POCT), which is expected to break through the technical barriers of traditional methods in real-time monitoring. However, the existing surface-enhanced Raman scattering (SERS) detection technology system still has the following key technical bottlenecks: (1) Damage to biological activity: The existing aerosol sampling device relies on the strong shear force generated by the mechanical pump group (such as the impact sampler), which will cause >60% of the enveloped virus structure to be inactivated; (2) Capture efficiency limitation: The concentration of virus particles in the aerosol is usually at a trace level (10 2 -10 3 particles / mL), the traditional SERS substrate has a limited specific surface area and insufficient surface chemical modification, resulting in low virus adsorption efficiency; (3) System integration defects: There is a lack of a microfluidic platform that integrates aerosol collection, virus enrichment and SERS detection, making it difficult to achieve on-site real-time detection and precise quantitative analysis, which seriously restricts the practical application of this technology in epidemic prevention and control.

[0004] To address the above issues, the present invention proposes a quantitative detection and analysis system for aerosol viruses using an AuNRs@ZIF-8 microfluidic SERS chip. The invention first proposes the use of biospecific enrichment technology to optimize the aerosol collection module: a liquid impact sampler with a low-shear air intake structure is selected, and a buffer solution is used to wet the wall surface to reduce mechanical stress; the sample liquid is pre-loaded with antibody-modified magnetic beads (such as Fe3O4 magnetic beads coupled to respiratory syncytial virus pDNA), and biospecific enrichment is higher than physical retention. Ensure microbial survival rate and high enrichment efficiency;

[0005] Secondly, the AuNRs@ZIF-8 composite material was constructed. The porous structure of ZIF-8 can be used as a carrier to highly disperse the AuNRs and increase their specific surface area. ZIF-8 has a certain adsorption capacity and biocompatibility for biological molecules such as viruses, and synergizes with AuNRs with good SERS activity to further improve the adsorption and capture performance of viruses.

[0006] Finally, a microfluidic-SERS optofluidic detection and analysis platform was deployed, combining the SERS composite substrate with the sample to be tested in a microfluidic microreactor chamber. A cell culture layer was then modified on the microfluidic substrate to maintain viral activity and enable in situ detection. An established "concentration-toxicity" quantitative structure-activity model was encapsulated in a PC, enabling direct conversion of spectral data collected by the Raman detection system into a toxicological analysis of the microorganisms. Through collaborative innovation in materials, devices, and algorithms, highly sensitive aerosol virus detection, dynamic culture, and toxicological analysis were integrated. This approach could be expanded to include the combined detection of multiple pathogens in the future, providing a new tool for public health monitoring. Summary of the Invention

[0007] The present invention aims to overcome the problems of low virus collection efficiency and easy nucleic acid degradation in traditional aerosol sampling, and to break through the technical bottlenecks of uneven mixing, low detection sensitivity, and inability to conduct real-time quantitative analysis in existing microfluidics-SERS combined technology.

[0008] According to some embodiments of the present invention, a system for quantitative detection and analysis of aerosol viruses using an AuNRs@ZIF-8 microfluidic SERS chip has at least the following beneficial effects: a portable detection system with full-chain integration of "sampling-enrichment-detection-analysis", short detection time, high sensitivity, and simultaneous analysis of aerosol virus load and toxicity.

[0009] According to some embodiments of the present invention, a system for quantitative detection and analysis of aerosol viruses using an AuNRs@ZIF-8 microfluidic SERS chip includes an aerosol sampler, bio-enrichment magnetic beads, an AuNRs@ZIF-8-SERS substrate, a microfluidic chip, a Raman spectroscopy detection system, a PLC control module, a Hall coil, and a quantitative analysis model.

[0010] According to some embodiments of the invention, aerosol virus collection is achieved by an aerosol sampler;

[0011] According to some embodiments of the present invention, Raman signal enhancement is achieved, and the SERS detection substrates are bio-enriched magnetic beads and AuNRs@ZIF-8;

[0012] According to some embodiments of the present invention, a microfluidic chip can highly simulate bacterial culture conditions and living environments;

[0013] According to some embodiments of the present invention, the SERS detection of viral RNA is performed using a Raman spectroscopy detection system;

[0014] According to some embodiments of the present invention, the PLC main control module controls the power supply module to generate currents of different magnitudes;

[0015] According to some embodiments of the present invention, a Hall coil is used to generate magnetic force and control the movement of the magnetic beads;

[0016] According to some embodiments of the present invention, a quantitative analysis model for converting Raman spectroscopy data into viral load is implemented;

[0017] According to some embodiments of the present invention, a system for quantitative detection and analysis of aerosol viruses using AuNRs@ZIF-8 microfluidic SERS chip is provided. The aerosol sampler uses a liquid impact sampler with a low shear force air intake structure, and a high-speed air sampling pump to collect aerosols. The wall is wetted with a buffer solution to reduce mechanical stress. Bio-enrichment magnetic beads are preloaded in the sampling liquid for the first step of enrichment of the analyte. The substrates are bio-enrichment magnetic beads and metal AuNRs@ZIF-8, which have spherical structures and prismatic dodecahedral structures, respectively, and have strong Raman scattering enhancement capabilities. The prepared ferroferric oxide nanospheres and gold nanorods are wrapped with silica and a metal organic framework. pDNA1 is coupled to the metal organic framework as a mesoscopic material outside the gold nanorod as a SERS substrate a, and the silica outside the ferroferric oxide nanosphere as a mesoscopic material is coupled to the metal organic framework. pDNA2 is used as the SERS substrate b; the SERS substrate b and the target detection object, that is, tDNA, form a multifunctional microsphere with a satellite structure; the microfluidic chip is prepared using the reverse lithography method and the reverse molding method, which includes an inlet, a channel, a SERS detection chamber and an outlet. The microfluidic chip channel width is 400um and the depth is 100um. The microfluidic chip can be used to complete the cultivation and detection of viral RNA; the main control system composed of the Hall coil, the power module, the PLC main control module and the PC is turned on to generate a magnetic force to control the movement of the magnetic beads, and then the bio-enrichment magnetic beads of the virus-coupled tDNA, the buffer solution and the SERS substrate a are introduced into the microfluidic chip, and the light source of the Raman spectroscopy inspection system is turned on. The Raman signal after the target enhancement is collected and captured by the microscope objective of the Raman spectroscopy detection system. The specific implementation process of the device is as follows:

[0018] The microfluidic chip has three inlets and one outlet. Virus-linked tDNA bio-enrichment magnetic beads, buffer, and SERS substrate a are introduced sequentially through the inlets. The master control system, consisting of a Hall coil, power module, PLC main control module, and PC, is then activated. The PC controls the Hall coil to generate varying magnetic forces, controlling the movement of the magnetic beads and driving the analyte through the specially designed structure within the microfluidic chip, causing the three injection solutions to mix. The Raman spectroscopy detection system's light source is then activated and aligned with the SERS detection chamber on the microfluidic chip. Raman detection of the analyte is performed, generating SERS signals of viral RNA. The measured signals are analyzed and transferred to the PC via USB for processing using custom-developed data analysis software. The software first performs preprocessing operations such as baseline correction and smoothing on the raw spectra to remove noise and background interference. The concentration of the virus in the unknown sample is then calculated based on the calibration curve. The spectral morphology enables toxicological analysis of the virus. Finally, the waste liquid is discharged through the outlet to prevent environmental contamination.

[0019] Compared with the prior art, the present invention is characterized by:

[0020] (1) The present invention uses a liquid impact sampler to reduce mechanical stress and preloads bio-enrichment magnetic beads in the sampling liquid to ensure the survival rate of microorganisms and achieve aerosol sampling with high enrichment efficiency.

[0021] (2) The present invention constructs an AuNRs@ZIF-8 microfluidic SERS chip to meet the needs of dynamic detection and analysis of microorganisms and achieve specific capture and good SERS performance.

[0022] (3) The real-time quantitative analysis system designed by the present invention mainly encapsulates the established "concentration-toxicity" quantitative structure-activity model in a PC, directly converts spectral data into toxicological quantitative output, and provides a simple and convenient new approach for microbial detection and analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic structural diagram of the present invention as a whole.

[0024] Item numbers in the figure: 1—aerosol sampler, 2—bioaccumulation magnetic beads, 2a—iron tetroxide nanospheres, 2b—silicon dioxide, 2c—pDNA1, 3—AuNRs@ZIF-8—SERS substrate, 3a—gold nanorods, 3b—metal organic framework, 3c—pDNA1, 4—microfluidic chip, 5—Raman detection system, 6—PLC control module, 7—Hall coil, 8—quantitative analysis model, 9—PC, 10—target detection object, 11—power module, 12—high-speed flow air sampling pump, 13—main control module.

[0025] Figure 2 This is a schematic diagram of the composition of the main control module in the present invention.

[0026] Part numbers in the figure: 13a—external 25V DC power supply, 13b—power module, 13c—DAC conversion module, 13d—communication module, 13e—PLC control module.

[0027] Figure 3 Schematic diagram of the structure of the microfluidic chip in the implementation of the present invention.

[0028] Item numbers in the figure: 4a—bioenrichment magnetic beads for virus-conjugated tDNA, 4b—buffer, 4c—pDNA2-modified AuNRs@ZIF-8 microspheres, 4d—serpentine mixing channel, 4e—detection chamber, 4f—exit.

[0029] Figure 4 Structure diagram of bioenrichment magnetic beads and AuNRs@ZIF-8.

[0030] Item numbers in the figure: a—Electron microscopy image of ferroferric oxide nanospheres, b—Electron microscopy image of bio-enrichment magnetic beads, c—Schematic diagram of gold nanorods, d—Schematic diagram of AuNRs@ZIF-8 microspheres.

[0031] Figure 5 The model establishment and data analysis of the relationship between Raman spectrum and concentration are mainly based on BP neural network and support vector machine for modeling and analysis.

[0032] Item numbers in the figure: a—artificial neural network BP, b—accuracy, c—training status, d—prediction results, e—support vector machine algorithm SVR, f—CARS feature extraction, g—SVR parameter tuning, h—prediction result confusion matrix. DETAILED DESCRIPTION

[0033] The following describes embodiments of the present invention in detail. Examples of such embodiments are illustrated in the accompanying drawings, where identical or similar reference numerals throughout denote identical or similar elements or elements having identical or similar functions. The embodiments described below with reference to the accompanying drawings are illustrative and intended solely to explain the present invention and are not to be construed as limiting the present invention. In the description of the present invention, it should be understood that references to orientation, such as "upper," "lower," and other references to orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are intended solely to facilitate description and simplify the present invention. They do not indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and are therefore not to be construed as limiting the present invention. In the description of the present invention, "a plurality" refers to more than two. The use of "first" or "second" is solely for the purpose of distinguishing technical features and is not to be construed as indicating or implying relative importance, or as implicitly indicating the number of such features, or as implicitly indicating the order of such features. In the description of the present invention, unless otherwise expressly defined, terms such as "dispose," "install," and "connect" are to be interpreted broadly. Persons skilled in the art can reasonably determine their specific meanings within the present invention based on the specific content of the technical solution.

[0034] The present invention will be further described in detail below with reference to the accompanying drawings:

[0035] According to some embodiments of the present invention, a system for quantitative detection and analysis of aerosol viruses using AuNRs@ZIF-8 microfluidic SERS chip is shown in FIG. Figure 1As shown, a high-speed air sampling pump is turned on for sampling, with a sampling volume of 7 to 10 L / min and a sampling time of 5 to 10 min; silicon dioxide is wrapped around the ferroferric oxide nanospheres, and pDNA2 is coupled to the surface to produce a SERS substrate b as a biological enrichment magnetic bead preloaded in the sample solution to enrich the target detection object; a metal organic framework and gold nanorods are prepared, the metal organic framework is coated on the gold nanorods, and pDNA1 is coupled to the outer layer of the metal organic framework to prepare a SERS substrate a; the mixed solution, buffer solution and substrate a are respectively introduced into the three inlets of the microfluidic chip, the Hall coil, power module and PLC control module are turned on to enrich the fully mixed mixture in the detection chamber, and the mixture is detected by a Raman spectroscopy detection system, and the detection system transmits the detected Raman spectral data to a PC, which encapsulates the established "concentration-toxicity" quantitative structure-activity model, so that the spectral data collected by the Raman detection system can be directly converted into a toxicological analysis of microorganisms, and the collected data can be displayed in real time and data analysis can be performed.

Claims

1. A quantitative detection and analysis system for aerosol viruses using AuNRs@ZIF-8 microfluidic SERS chip, characterized in that: include: Aerosol sampler (1), bio-enrichment magnetic beads (2), AuNRs@ZIF-8—SERS substrate (3), microfluidic chip (4), Raman spectroscopy detection system (5), PLC control module (6), Hall coil (7), quantitative analysis model (8), etc. Among them, the aerosol sampler (1) is used for collecting aerosol viruses; the bio-enrichment magnetic beads (2) are used for the first step of enrichment of the analyte; The AuNRs@ZIF-8-SERS substrate (3) has a structure of gold nanorods (3a) wrapped with a metal organic framework (3b) and pDNA1 (3c) coupled to its surface; the microfluidic chip (4) has polydimethylsiloxane (PDMS) and glass as main materials; the Raman spectroscopy detection system (5) is placed above the detection chamber; the PLC control module (6) can control the operation of the high-speed air sampling pump (12); the Hall coil (7) is used to control the movement speed of the bio-enrichment magnetic beads (2); the quantitative analysis model (8) is to encapsulate the "concentration-toxicity" quantitative structure-activity model in the PC (9) to achieve direct output of the toxicity of microorganisms.

2. The aerosol virus quantitative detection and analysis system using the AuNRs@ZIF-8 microfluidic SERS chip according to claim 1, characterized in that: The aerosol sampling system (1) uses a liquid impact sampler with a low shear force air intake structure and a high-speed air sampling pump (12) to collect aerosols, and a buffer solution is used to wet the wall surface to reduce mechanical stress. Bio-enrichment magnetic beads (2) are pre-loaded in the sampling liquid for the first step of enrichment of the analyte.

3. The aerosol virus quantitative detection and analysis system using the AuNRs@ZIF-8 microfluidic SERS chip according to claim 1, characterized in that: The structure of the bio-enrichment magnetic beads (2) is that ferroferric oxide nanospheres (2a) with a particle size of 200±10 nm are wrapped with a silica shell (2b) with a thickness of 50±5 nm and pDNA2 (2c) is coupled on the surface thereof, and is used for capturing the target detection object (10) and enhancing the Raman signal.

4. The aerosol virus quantitative detection and analysis system using the AuNRs@ZIF-8 microfluidic SERS chip according to claim 1, characterized in that: The structure of the AuNRs@ZIF-8-SERS substrate (3) is that gold nanorods (3a) with a length of 67.02 nm and a width of 15.65 nm are wrapped with a metal organic framework (3b) with a particle size of 222.08 nm and pDNA1 (3c) is coupled on its surface to form a SERS substrate, and multifunctional microspheres with a satellite structure formed with biological enrichment magnetic beads (2) further enhance the Raman effect.

5. The aerosol virus quantitative detection and analysis system using the AuNRs@ZIF-8 microfluidic SERS chip according to claim 1, characterized in that: The main materials of the microfluidic chip (4) are polydimethylsiloxane (PDMS) and glass, which are bonded by oxygen plasma treatment to form a closed detection chamber.

6. The aerosol virus quantitative detection and analysis system using the AuNRs@ZIF-8 microfluidic SERS chip according to claim 1, characterized in that: The PLC control module (6) and the Hall coil (7) can control the operating speed of the high-speed air sampling pump (12) and the biological enrichment magnetic beads (2).

7. The aerosol virus quantitative detection and analysis system using the AuNRs@ZIF-8 microfluidic SERS chip according to claim 1, characterized in that: The Hall coil (7) is used to control the movement speed of the bio-enrichment magnetic beads (2) and is placed below the microfluidic chip (4).

8. The aerosol virus quantitative detection and analysis system using the AuNRs@ZIF-8 microfluidic SERS chip according to claim 1, characterized in that: The quantitative analysis model (8) encapsulates the "concentration-toxicity" quantitative structure-activity model in a PC (9), and mainly uses BP neural network and support vector machine algorithms to establish a model to analyze the impact of changes in aerosol microbial concentration on spectral intensity, and ultimately achieve direct output of the toxicity of microorganisms.

9. The aerosol virus quantitative detection and analysis system using the AuNRs@ZIF-8 microfluidic SERS chip according to claim 1, characterized in that: The power module (11) is connected to the Hall coil (7), and the module outputs current to the Hall coil (7). The current generated by the power module (11) is adjustable.

10. The aerosol virus quantitative detection and analysis system using the AuNRs@ZIF-8 microfluidic SERS chip according to claim 1, characterized in that: The main control module (13) can control the power supply module (11) to generate currents of different magnitudes and the PLC control module (6) to issue control instructions.

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