Up-conversion light-transmitting self-driven micro-fluidic chip for respiratory tract pathogen detection and preparation method of up-conversion light-transmitting self-driven micro-fluidic chip

Abstract

The invention discloses an up-converting light self-driven micro-fluidic chip for respiratory pathogen detection and a preparation method of the up-converting light self-driven micro-fluidic chip. The core is to provide a self-driven micro-fluidic chip for combined quantitative detection of influenza A virus (IAV), influenza B virus (IBV) and respiratory syncytial virus (RSV) antigens. The chip integrates microfluidic and up-conversion luminescence (UCP) technologies, and comprises a sample adding area, a labeling reaction area, a space-time valve and a micro-channel detection area, the labeling reaction area contains UCP nano-particles to label an anti-IAV / IBV / RSV specific monoclonal antibody 1, and the micro-channel detection area labels a corresponding anti-IAV / IBV / RSV specific monoclonal antibody 2 so as to synchronously capture three virus antigens and read signals. The target antigen concentration is quantified by detecting the fluorescence peak area ratio of each detection point to a quality control point, and the method has the advantages of short detection period, high sensitivity and low background interference, can provide a precise tool for rapid typing diagnosis, illness state evaluation and curative effect monitoring of respiratory virus infection, and can also be applied to preparation of related detection test paper.

Description

Technical Field

[0001] This invention relates to an upconversion luminescence self-driven microfluidic chip for the detection of respiratory pathogens and its preparation method, particularly to the preparation of a microfluidic chip for the quantitative detection of influenza A / B virus and respiratory syncytial virus using this method, belonging to the field of medical product technology, and used for the diagnosis of acute respiratory infections. Background Technology

[0002] Respiratory viral infections are a major cause of illness, hospitalization, and even death worldwide. Common viruses such as respiratory syncytial virus (RSV), influenza A virus (IAV), and influenza B virus (IBV) share highly similar early clinical symptoms (such as fever, cough, and sore throat), yet their treatment regimens differ significantly. Therefore, rapid and accurate detection of respiratory viral antigens is a crucial prerequisite for achieving clinical subtyping and treatment, avoiding antibiotic overuse, and blocking viral transmission.

[0003] Current respiratory virus antigen detection technologies have significant limitations: Immunochromatographic detection of respiratory virus antigens has advantages such as low operational threshold, rapid and efficient operation, strong adaptability to various scenarios, and controllable cost. It does not require complex instruments or professional technical expertise, and sample processing is simple. The detection cycle is only 5-15 minutes, and qualitative results can be obtained immediately. Moreover, the test strips are portable, easy to store, and have low transportation costs, making them suitable for various scenarios such as primary healthcare, large-scale population screening, emergency epidemic investigation, and home self-testing. However, its detection sensitivity is lower than that of nucleic acid detection (RT-PCR) and chemiluminescence technology. False negatives are prone to occur when the viral load is low or in the early stages of infection. Its anti-interference ability is weak, and it is easily affected by sample collection quality, viral mutation, matrix effect, environmental temperature and humidity, and batch differences of reagents. There is also a risk of false positives. At the same time, it lacks quantitative capabilities, only providing "positive / negative" results, and cannot accurately quantify viral load to assist in disease assessment and efficacy monitoring. The repeatability of the results is also inferior to that of instrumental detection methods.

[0004] Microfluidics, as an emerging detection technology, boasts core advantages such as low sample volume, high reaction efficiency, and strong functional integration, making it an ideal platform for portable and rapid antigen detection. Through the precise microchannel structure of microfluidic chips, automated sample introduction, transfer, and reaction can be achieved, eliminating the need for complex manual operations and significantly shortening the detection cycle. Simultaneously, chip miniaturization reduces the size and weight of the detection system, meeting the "sampling and testing" requirements of point-of-care testing (POCT). However, microfluidics alone cannot provide high-sensitivity detection signals for viral antigen detection. When combined with a chemiluminescence signal system, substrate storage and release units must be integrated within the chip, increasing design complexity and failing to avoid substrate instability issues. Furthermore, when combined with traditional fluorescence, background interference and photobleaching problems persist.

[0005] Upconversion luminescence technology is based on rare earth ions (such as Yb). 3+ Er 3+ Tm 3+ Ho 3+ A novel optical technology for "photon avalanche" energy level transitions in matrix materials, the core principle of which is: using Yb 3+ As a sensitizer, Yb is excited by near-infrared light (980 nm). 3+ After absorbing a photon, it transitions from the ground state to an excited state, and then transfers energy to an activator (such as Er) through nonradiative energy transfer. 3+ Tm 3+ The activator ions absorb energy and successively transition to higher energy levels, eventually releasing photons from these higher energy levels via radiative transitions, emitting visible light with wavelengths shorter than the excitation light. Currently, the core carrier of UCP technology is rare-earth-doped fluoride nanoparticles (such as NaYF4:Yb). 3+ Er 3+ NaGdF4:Yb 3+ ,Tm 3+ These particles possess the following key characteristics suitable for respiratory viral antigen detection:

[0006] (1) Excellent optical stability: The physical shielding effect of the outer electrons of rare earth ions makes them less affected by the external environment (such as temperature, pH value, solvent polarity), which is significantly better than traditional organic fluorescent dyes and can ensure the long-term stability of antigen detection signals.

[0007] (2) Extremely low background interference: UCP technology can avoid background fluorescence interference from the sample matrix through near-infrared light excitation, and the signal-to-noise ratio can be much higher than that of traditional fluorescence technology, providing a high-specific signal basis for the detection of low-concentration antigens;

[0008] (3) Controllable detection sensitivity: By adjusting the size of nanoparticles, the concentration of rare earth ion doping, and surface modification strategies, the quantum yield of UCP can be optimized. Combined with the enrichment effect of immune capture, the detection limit can be as low as femtogram (fg / mL), which can capture early infection (viral load <10). 3 Trace amounts of antigen (PFU / mL);

[0009] (4) Multi-target encoding capability: By selecting different activator ions or adjusting the doping ratio, the emission wavelength of UCP can be precisely controlled, and parallel detection of RSV, IAV and IBV antigens can be achieved without the need for multiple independent detections.

[0010] The combination of microfluidic technology and upconversion luminescence technology for respiratory viral antigen detection can create a synergistic effect of "complementary functions and superimposed advantages": On the one hand, the microchannel structure of the microfluidic chip can construct an integrated process of "sample introduction-filtration-antigen capture-UCP detection", achieving autonomous fluid flow driven by capillary force or osmotic pressure; at the same time, the laminar flow effect within the microchannel can accelerate the immunobinding of antigens and UCP probes, further improving detection sensitivity; on the other hand, the low background and high stability characteristics of UCP technology can directly achieve signal reading within the microfluidic chip, and when paired with a portable near-infrared excitation-visible light detection module, it can meet the needs of POCT scenarios; in addition, by constructing three independent detection channels within the chip, modifying them with RSV, IAV, and IBV capture antibodies respectively, and pairing them with UCP probes of different emission wavelengths, simultaneous quantitative detection of three viral antigens can be completed at one time, significantly improving detection efficiency compared to immunochromatography technology.

[0011] Based on the aforementioned technical advantages, this invention develops a self-driven, highly stable, and low-cost microfluidic-upconversion luminescence integrated chip for rapid and accurate detection of respiratory viral antigens. This breakthrough overcomes the limitations and operational pain points of chemiluminescence immunoassay technology, providing a valuable tool for studying the pathogenesis of respiratory infectious diseases and for intelligent detection of pathogens in different scenarios. Summary of the Invention

[0012] The purpose of this invention is to develop a self-driven microfluidic chip for the diagnosis of respiratory pathogens based on upconversion nanomaterials, which is sensitive and accurate.

[0013] In order to achieve the above-mentioned objectives of the present invention, the present invention proposes to adopt the following technical solution:

[0014] The self-driven microfluidic chip of this invention detects upconversion luminescent material UCP nanoparticles by modifying and activating a series of surface chemical groups, and covalently binding them with functional bioactive molecules. This allows for precise scanning and analysis of UCP (upconversion luminescent) nanoparticles that bind to the detection and control bands through specific immune reactions. Furthermore, relying on a two-photon fluorescence rapid quantitative detection system, it enables accurate quantitative detection or highly specific qualitative determination of respiratory viral antigens.

[0015] Specifically, the present invention provides a self-driven microfluidic chip based on upconversion luminescence technology for detecting proteins A, B, and C, characterized in that it contains upconversion luminescent material (UCP) nanoparticles that bind specific antibodies against influenza A virus, influenza B virus, and respiratory syncytial virus.

[0016] The self-driven microfluidic chip described above includes a sample loading area, a labeled reaction area, a time-space valve, and a microfluidic detection area. During detection, the sample is added to the loading area and passes through a filtered blood membrane into the reaction area. In the reaction area, the antigen specifically binds to the target fluorescent antibody, forming a "fluorescent antibody-antigen" complex. Then, under capillary force, the "fluorescent antibody-antigen" complex flows with the sample to the microfluidic detection area, where it is specifically recognized and captured by the capture antibody immobilized there, forming a "fluorescent antibody-antigen-antibody" double-antibody sandwich immune system. Once the sample has completely passed through the microfluidic detection area, the fluorescence signal at the capture site can be collected, and the target antigen in the sample can be quantified based on the fluorescence signal intensity.

[0017] The labeled reaction region described above may contain UCP (upconversion luminescence) nanoparticle-labeled anti-A antibody 1, anti-B antibody 1, anti-C antibody 1, and quality control material.

[0018] The microfluidic detection zone described above may contain detection points A, B, C, and a quality control detection point. Anti-A antibody 2 may be immobilized at detection point A, anti-B antibody 2 at detection point B, and anti-C antibody 2 at detection point C. An antibody against the quality control material may be immobilized at the quality control detection point. Anti-A antibody 1 and anti-A antibody 2 may be different or the same anti-A antibody. Anti-B antibody 1 and anti-B antibody 2 may be different or the same anti-B antibody. Anti-C antibody 1 and anti-C antibody 2 may be different or the same anti-C antibody. The line connecting the centers of the four detection points (A, B, C, and quality control detection point) is a straight line, which may be perpendicular to the direction of sample movement (chromatographic direction).

[0019] In the self-driven microfluidic chip described above, the quality control material can be a known protein. The detection points A, B, C, and quality control detection points on the microchannel detection area can be arranged longitudinally. The longitudinal direction is parallel to the direction of sample movement.

[0020] In the self-driven microfluidic chip described above, detection points A, B, C, and quality control can all be circular detection points.

[0021] The A protein mentioned above can be an influenza A antigen, the B protein can be an influenza B antigen, and the C protein can be a respiratory syncytial virus antigen.

[0022] In the self-driven microfluidic chip described above, the antibody is a monoclonal antibody.

[0023] The preparation process of the detection point A, the detection point B, the detection point C and the quality control detection point in the microfluidic detection area described above may include the process of spotting the anti-A antibody 2 at the detection point A, the anti-B antibody 2 at the detection point B, the anti-C antibody 2 at the detection point C and the antibody against the quality control material at the quality control detection point.

[0024] The liquid flow direction of the self-driven microfluidic chip described above can be from the sample loading area to the labeling reaction area, from the labeling reaction area to the time-space valve, and from the time-space valve to the microchannel detection area. The self-driven microfluidic chip comprises a substrate and a top cover, both made of polymethyl methacrylate (PMMA). The sample loading area is the location for adding liquid samples. A blood filtration membrane can be placed in the sample loading area for separating plasma from whole blood samples. The time-space valve is the flow rate control area. The time-space valve can be composed of densely packed horizontal grooves, which can reduce the sample flow rate and increase the reaction time between antigens and antibodies.

[0025] In the self-driven microfluidic chip described above, the concentrations of A and / or B and / or C can be determined by detecting the fluorescence signal values ​​of the upconversion nanomaterials on the microchannel detection region. This includes detecting the upconversion emission signal values ​​at the A detection point, B detection point, C detection point, and quality control detection point on the microchannel detection region to obtain detection peaks; determining the concentration of A by calculating the ratio of the detection peak area of ​​the A detection point to that of the quality control detection point; determining the concentration of B by calculating the ratio of the detection peak area of ​​the B detection point to that of the quality control detection point; and determining the concentration of C by calculating the ratio of the detection peak area of ​​the C detection point to that of the quality control detection point.

[0026] This invention also provides the application of the self-driven microfluidic chip described above in the preparation of test strips for detecting influenza A antigen and / or influenza B antigen and / or respiratory syncytial virus (RSV) influenza antigen. Test strips for detecting influenza A antigen and / or influenza B antigen and / or RSV influenza antigen prepared using the self-driven microfluidic chip described above are also within the scope of protection of this invention.

[0027] This invention provides a self-driven microfluidic chip for the combined quantitative detection of influenza A antigen, influenza B antigen, and respiratory syncytial virus (RSV) influenza antigen based on upconversion luminescence. This chip has advantages such as small size, rapid and accurate quantification, low background interference, and minimal interference between different detection items. It provides a quantitative detection reagent for influenza A antigen, influenza B antigen, and RSV influenza antigen based on upconversion luminescence, enabling rapid, accurate, and quantitative detection of these antigens.

[0028] The beneficial effects of this invention are as follows:

[0029] By constructing three independent detection channels within the chip, modifying them with RSV, IAV, and IBV capture antibodies respectively, and pairing them with UCP probes of different emission wavelengths, simultaneous quantitative detection of three viral antigens can be achieved in one operation, significantly improving detection efficiency compared to chemiluminescence technology. Combining microfluidic technology with upconversion luminescence technology for respiratory viral antigen detection can create a synergistic effect of "functional complementarity and synergistic advantages." Attached Figure Description

[0030] Figure 1 This is a schematic diagram of a microfluidic chip structure.

[0031] Figure 2 These are the results of microfluidic chip detection of three viral antigens. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] A combined quantitative detection method using upconversion luminescent immunochromatographic chips for detecting RSV, IAV, and IBV

[0035] 1. A method for fabricating an upconversion luminescence self-driven microfluidic chip for detecting RSV, IAV, and IBV.

[0036] 1.1 Reagents

[0037] (1) UCP particles used in the fabrication of self-driven microfluidic chips.

[0038] (2) Anti-IAV antibodies (including anti-IAV monoclonal antibody 1 (Hangzhou Boyue Biotechnology Co., Ltd., catalog number IAV107) and anti-IAV monoclonal antibody 2 (Suzhou Nearshore Protein Technology Co., Ltd., catalog number DA074)), anti-IBV antibodies (anti-IBV monoclonal antibody 1 (Nanjing Fuxiao Biotechnology Co., Ltd., catalog number M011308M) and anti-IBV monoclonal antibody 2 (Xiamen Tongrenxin Biotechnology Co., Ltd., catalog number Flu24)), and anti-RSV antibodies (anti-RSV monoclonal antibody 1 (Hangzhou Boyue Biotechnology Co., Ltd., catalog number RSV101) and anti-RSV monoclonal antibody 2 (Hangzhou Boyue Biotechnology Co., Ltd., catalog number RSV102)).

[0039] (3) IAV antigen (Guangdong Feipeng Biotechnology Co., Ltd., catalog number FLUA-SC-G1-001), IBV antigen (Guangdong Feipeng Biotechnology Co., Ltd., catalog number FLUB-SC-G2-001), RSV antigen (Chongqing Tansheng Technology Co., Ltd., catalog number FAP-IE0182023110310)

[0040] (4) Other Tris-HC1, phosphate buffer (PBS), BSA (blocking), sample dilution (BSA+Tween20+TritonX100).

[0041] 1.2 Chip Structure

[0042] 1.2.1 Overall Chip Structure: The microfluidic chip mainly consists of two parts: a substrate and a top cover. The microchip substrate mainly consists of five parts: a sample loading area, a labeling reaction area, a time-space valve (flow rate control area), and a microchannel detection area.

[0043] 1.2.2 Reaction Zone: The reaction zone contains UCP-labeled anti-IAV antibody 1 (UCP-anti-IAV monoclonal antibody 1), UCP-labeled anti-IBV antibody 1 (UCP-anti-IBV monoclonal antibody 1), UCP-labeled anti-RSV antibody 1 (UCP-anti-RSV monoclonal antibody 1), and UCP-labeled goat IgG (UCP-goat IgG) as a quality control.

[0044] 1.2.3 Microfluidic detection area: The microfluidic detection area contains anti-IAV monoclonal antibody 2, anti-IBV monoclonal antibody 2, anti-RSV monoclonal antibody 2 and anti-quality control antibody (rabbit anti-goat IgG) arranged longitudinally.

[0045] 1.3 Method for preparing upconversion luminescent immunochromatographic chips for detecting RSV, IAV, and IBV

[0046] An upconversion luminescence-microfluidic immunoassay method was established using a double antibody sandwich approach to enable rapid quantitative detection of IAV and / or IAV and / or RSV.

[0047] The detection principle is as follows:

[0048] (1) Positive standard sample: After the sample is added to the sample loading area of ​​the microfluidic chip, it flows forward (from the sample pad to the absorbent pad) under the siphon effect, passing through the labeling reaction area and the microfluidic detection area respectively. The IAV and / or IAV and / or RSV in the positive sample first combine with the UCP-anti-IAV monoclonal antibody 1 and / or UCP-anti-IBV monoclonal antibody 1 and / or UCP-anti-RSV monoclonal antibody 1 in the labeling reaction area to form the UCP-anti-IAV monoclonal antibody 1-IAV complex and / or UCP-anti-IBV monoclonal antibody 1-IBV complex and / or UCP-anti-RSV monoclonal antibody 1-RSV complex. The formed complex and free labeled antibody continue to flow forward to the microfluidic detection zone: anti-IAV monoclonal antibody 2, anti-IBV monoclonal antibody 2, and anti-RSV monoclonal antibody 2, immobilized in the microfluidic detection zone, respectively capture the UCP-anti-IAV / IBV / RSV monoclonal antibody 1-IAV / IBV / RSV complex to form a solid-phase-anti-IAV / IBV / RSV monoclonal antibody 2-IAV / IBV / RSV-anti-IAV / IBV / RSV monoclonal antibody 1-UCP new complex; at the quality control location, QC captures the free quality control material UCP-sheep IgG to form a solid-phase rabbit anti-sheep IgG-sheep IgG-UCP.

[0049] (2) Negative samples: The sample, along with UCP-anti-IAV monoclonal antibody 1, UCP-anti-IBV monoclonal antibody 1, UCP-anti-RSV monoclonal antibody 1, and UCP-goat IgG, enters the microfluidic detection zone, forming a solid-phase rabbit anti-goat IgG-goat IgG-UCP only on the control band. Therefore, in this microfluidic chip detection mode, positive samples exhibit specific signal peaks at both the detection and control points, while negative samples only show specific signal peaks at the control point.

[0050] 1.3.1 Preparation of the reaction zone

[0051] Surface-modified UCP particles (Beijing Kaijing Gene Technology Co., Ltd.) were prepared into a 10 mg / mL UCNPs solution. 100 μL of the solution was centrifuged and the supernatant was discarded. 100 μL of M ES buffer (10 mM, pH 5.4), 0.02 mg EDC, and 0.02 mg NHS were added and sonicated. The mixture was stirred at room temperature for 0.5 h. The supernatant was discarded by centrifugation (13000 rpm, 15 min). The mixture was washed once with 100 μL HEPES buffer (10 mM, pH 7.4) and resuspended. 50 μg of antibody (0.5 mg / mL, diluted with HEPES buffer) was added, and the mixture was stirred at room temperature for 2 h. The mixture was then centrifuged (13000 rpm, 15 min, 4℃) and the supernatant was discarded. 100 μL of blocking buffer was added, and the mixture was stirred at room temperature for 2 h for blocking. The mixture was centrifuged (13000 rpm, 15 min, 4℃), washed twice with 100 μL HEPES buffer (10 mM, pH 7.4), and resuspended. Four UCP monoclonal antibody complexes were obtained: UCP-anti-IAV monoclonal antibody 1, UCP-anti-IBV monoclonal antibody 1, UCP-anti-RSV monoclonal antibody 1, and UCP-goat IgG. These four UCP monoclonal antibody complexes were mixed and poured onto the reaction area of ​​the microfluidic chip. They were pre-frozen at -80℃ for 2 hours, then freeze-dried for 3 hours and stored in a desiccator. 1.3.2 Spotting in the Microfluidic Detection Area: The upconversion luminescent self-driven microfluidic chip samples used for detecting IAV, IBV, and RSV were anti-IAV monoclonal antibody 2, anti-IBV monoclonal antibody 2, anti-RSV monoclonal antibody 2, and anti-control antibody (rabbit anti-goat IgG). Longitudinal (parallel to the flow direction) spotting was performed on the microfluidic chip's microfluidic detection area. The spotting method involved spraying four separate dots longitudinally (parallel to the flow direction) (corresponding to positions 100-200, 400-500, 600-700, and 800-900 on the chip). Anti-IAV monoclonal antibody 2, anti-IBV monoclonal antibody 2, anti-RSV monoclonal antibody 2, and anti-control antibody (rabbit anti-goat IgG) were each sampled in 600 nL sequentially to obtain detection point 1 (T1), detection point 2 (T2), detection point 3 (T3), and quality control point (QC). Specifically, T1 (sample is anti-RSV monoclonal antibody 2) is the RSV detection point, T2 (sample is anti-IAV monoclonal antibody 2) is the IAV detection point, T3 (sample is anti-IBV monoclonal antibody 2) is the IBV detection point, and C (sample is rabbit anti-goat IgG) is the quality control point. The line connecting the centers of these four points (T1, T2, T3, and QC) is a straight line parallel to the direction of sample flow. After spotting, the microfluidic chip is placed in an electric heating constant temperature drying oven at 37°C for 1 hour to obtain an upconversion light-emitting self-driven microfluidic chip for detecting IAV, IBV and RSV.

[0052] 2. Detection of IAV, IBV, and RSV using a chip and analysis of the results.

[0053] IAV, IBV, and RSV standards were diluted to create six concentration gradients of IAV / IBV / RSV: 1, 5, 10, 15, 20, and 25 mg / L. These diluted samples were then mixed with a sample diluent (BSA + Tween 20 + Triton X100) at a volume ratio of 1:99 to obtain the test samples. 300 μL of the test sample was added to the up-transfer self-driven microfluidic chip used for the combined detection of IAV, IBV, and RSV, and allowed to stand for 15 min. A two-photon fluorescence detection system was used to scan the microfluidic channel detection area. Detection peaks were observed at the detection points (T1, T2, and T3) and quality control points (QC). The ratio of the peak area at each detection point to the peak area at the quality control point (T / C value) was used as the detection result. T1 / C represents the RSV detection result, T2 / C represents the IAV detection result, and T3 / C represents the IBV detection result.

Claims

1. An upconversion luminescence self-driven microfluidic chip for respiratory pathogen detection and its fabrication method, characterized in that, This invention includes a method for preparing an upconversion luminescent self-driven microfluidic chip and the application of this method for quantitative detection of influenza A / B virus and respiratory syncytial virus. The self-driven microfluidic chip structure comprises a sample loading area, a labeling reaction area, a space-time valve, and a microfluidic detection area. The labeling reaction area contains upconversion nanoparticle-labeled anti-A monoclonal antibody 1, anti-B monoclonal antibody 1, anti-C monoclonal antibody 1, and a quality control substance. The microfluidic detection area contains detection point 1 (T1), detection point 2 (T2), detection point 3 (T3), and a quality control point (QC), which are anti-A monoclonal antibody 2, anti-B monoclonal antibody 2, anti-C monoclonal antibody 2, and anti-quality control antibody, respectively. The detection points 1 (T1), 2 (T2), 3 (T3), and QC are arranged vertically, and the line connecting the centers of the detection points is parallel to the direction of fluid flow.

2. The upconversion light-emitting self-driven microfluidic chip as described in claim 1, characterized in that, The labeled reaction area contains UCP-labeled anti-influenza A antigen antibody 1 (UCP-anti-IAV monoclonal antibody 1), UCP-labeled anti-influenza B antigen antibody 1 (UCP-anti-IBV monoclonal antibody 1), UCP-labeled anti-respiratory syncytial virus antigen antibody 1 (UCP-anti-RSV monoclonal antibody 1), and UCP-labeled goat IgG (UCP-goat IgG) as a quality control.

3. The upconversion light-emitting self-driven microfluidic chip as described in claim 1, characterized in that, The marked reaction zone is designed with three types of surface topography. First, there is a high capillary action zone with a relatively dense array structure (the array structure is arranged obliquely), followed by a short section of low capillary action zone without an array structure, and then a medium capillary action zone with a relatively sparse array structure (the array structure is arranged laterally).

4. The upconversion light-emitting self-driven microfluidic chip as described in claim 1, characterized in that, The microfluidic detection area contains, in a longitudinal sequence, anti-RSV monoclonal antibody 2, anti-IAV monoclonal antibody 2, anti-IBV monoclonal antibody 2, and anti-quality control antibody (rabbit anti-sheep IgG).

5. The upconversion light-emitting self-driven microfluidic chip as described in claim 1, characterized in that, The antibody spotting volume in the labeled reaction area is 1.6 μl, and the antibody spotting volume in the microfluidic detection area is 0.6 μl.

6. The upconversion light-emitting self-driven microfluidic chip as described in claim 1, characterized in that, The quantitative detection of influenza A / B virus and respiratory syncytial virus is specifically performed as follows: the sample is added to the sample application area, and the sample enters the reaction area through the filtered blood membrane. The antigen specifically binds to the target fluorescent antibody in the reaction area to form a "fluorescent antibody-antigen" complex. Then, under capillary force, the "fluorescent antibody-antigen" complex flows with the sample to the microfluidic detection area, where it is specifically recognized and captured by the capture antibody fixed in the microfluidic detection area, forming a "fluorescent antibody-antigen-antibody" double-antibody sandwich immune system. Once the sample has completely passed through the microfluidic detection zone, the fluorescence signal at the capture site can be collected, and the target antigen in the sample can be quantified based on the fluorescence signal intensity.

7. The upconversion light-emitting self-driven microfluidic chip as described in claim 1, characterized in that, The concentrations of A and / or B and / or C are determined by detecting the fluorescence signal values ​​of the upconversion nanomaterials on the microfluidic detection region. This includes detecting the upconversion emission signal values ​​at the A, B, C, and quality control detection points on the analytical membrane to obtain detection peaks; determining the concentration of A by calculating the ratio of the detection peak area at the A detection point to that at the quality control detection point; determining the concentration of B by calculating the ratio of the detection peak area at the B detection point to that at the quality control detection point; and determining the concentration of C by calculating the ratio of the detection peak area at the C detection point to that at the quality control detection point.

8. The application of the upconversion luminescence self-driven microfluidic chip prepared by the preparation method according to any one of claims 1 to 7, characterized in that, The application involves using the upconversion luminescence self-driven microfluidic chip and its related antibody reagents for the combined immunoassay of RSV, IAV, and IBV.

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