Paper-based sensor chip for multiplex respiratory virus detection and preparation method and application thereof

Abstract

The application discloses a paper-based sensor chip for multiple respiratory virus detection and a preparation method and application thereof. The paper-based sensor chip takes filter paper as a base material and is provided with a sample adding area (100), a working electrode area (200), a reference electrode area (300) and a counter electrode area (400). The working electrode area (200) is provided with a plurality of working electrodes which are arranged in a row matrix in a direction away from the sample adding area (100). The working electrode takes graphene as a conductive channel, and the surface of the working electrode is fixed with a nano-gold-streptavidin composite material and a capture probe covalently combined with biotin. The upper side of the working electrode is the reference electrode area (300), the lower side of the working electrode is the counter electrode area (400), and the working electrode, the reference electrode area (300) and the counter electrode area (400) form a three-electrode system. The paper-based sensor chip can realize efficient and synchronous detection of multiple viruses and has good application value.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and in particular to a paper-based sensor chip for the detection of multiple respiratory viruses, its preparation method, and its application. Background Technology

[0002] Acute respiratory infections are a leading cause of morbidity and mortality worldwide, with viruses being the primary pathogens. Novel coronavirus pneumonia (SARS-CoV-2), influenza virus (IV), and respiratory syncytial virus (RSV) can all cause highly similar clinical symptoms, such as fever, cough, fatigue, and pneumonia. However, they differ significantly in treatment regimens, prognostic assessments, and public health management strategies. Therefore, establishing a rapid, accurate, and simultaneous diagnostic method to identify multiple pathogens is crucial for guiding precise clinical medication, controlling nosocomial infections, and effectively responding to epidemics.

[0003] Currently, common respiratory virus detection methods mainly include viral culture, immunological detection, and molecular biological detection. Viral culture, as the traditional "gold standard," is accurate, but its cumbersome operation and long processing time (several days to weeks) cannot meet the needs of rapid clinical diagnosis. Immunological detection methods (such as colloidal gold immunochromatographic strips) are rapid and simple, but their sensitivity and specificity are relatively low, prone to false negatives, and often difficult to achieve multiplexing. Molecular biological detection methods, such as real-time quantitative PCR, multiplex PCR, and isothermal amplification, have become mainstream detection methods. Real-time quantitative PCR, as the current "gold standard" for molecular diagnostics, has high sensitivity and specificity. However, this method requires expensive and bulky thermal cyclers, has a complex operation procedure, must be performed by technicians in a specialized laboratory, and has a long testing time (usually 1-3 hours), making low-cost, high-throughput multiplexing difficult. Multiplex PCR is often limited by the number of fluorescence channels, resulting in limited detection throughput. While isothermal amplification techniques (such as RPA and LAMP) eliminate the need for thermal cycling and simplify the amplification process, their detection relies heavily on fluorescence signal reading, and they also face limitations in multiple detection capabilities and high equipment costs.

[0004] In recent years, electrochemical biosensors have shown great potential in the field of in vitro diagnostics due to their advantages such as simple equipment, low cost, high sensitivity, ease of miniaturization, and real-time detection capabilities. However, applying electrochemical sensing technology to the multiplex detection of respiratory viruses still faces significant challenges: ① Crosstalk in multiplex detection: It is difficult to achieve parallel detection of multiple targets on a single chip, and there is mutual interference between signals. ② Balancing sensitivity and specificity: In complex clinical samples (such as saliva and nasal swabs), it is difficult to achieve high sensitivity and high specificity for low viral loads. ③ Integration and automation: Sample pretreatment, nucleic acid amplification (if applicable), and electrochemical detection are difficult to integrate seamlessly, making them unsuitable for "sample-in-result-out" devices for rapid on-site detection.

[0005] Therefore, there is an urgent need in this field to develop a novel respiratory virus detection device and method that combines high sensitivity, high specificity, multiplex detection capability, rapid response, and portability to meet the pressing needs of current clinical and public health prevention and control. Summary of the Invention

[0006] The first objective of this invention is to provide a paper-based sensor chip.

[0007] The second objective of this invention is to provide a method for fabricating a paper-based sensor chip.

[0008] The third objective of this invention is to provide the application of the above-mentioned paper-based sensor chip in the preparation of a detection kit.

[0009] The fourth aspect of the present invention is to provide a detection system.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a paper-based sensor chip, which uses filter paper as a substrate and is provided with a sample application area, a working electrode area, a reference electrode area, and a counter electrode area, wherein: The working electrode area is provided with a number of working electrodes, which are arranged in a row matrix along the direction away from the sample application area. The working electrodes use graphene as a conductive channel, and nano-gold-streptavidin composite material and capture probe covalently bound to biotin are fixed on the surface. The upper side of the working electrode is the reference electrode region, and the lower side of the working electrode is the counter electrode region. The working electrode, the reference electrode region, and the counter electrode region together form a three-electrode system.

[0011] The paper-based sensor chip according to embodiments of the present invention has at least the following beneficial effects: (1) This invention constructs a complete three-electrode system (graphene working electrode, graphene counter electrode, and Ag / AgCl reference electrode) by directly printing on paper-based materials, which integrates the detection area and the electrode in terms of physical and functional aspects. Furthermore, the chromatography, immunoreaction, and electrochemical detection of the sample to be tested are completed on the same plane and continuously without external assembly or disassembly, thus achieving true integrated detection. It is particularly suitable for automated and portable on-site detection and has great application value in fields such as virus detection.

[0012] (2) In the detection process of the paper-based sensor chip of the present invention, the sample to be tested is first mixed with pre-labeled SiO2 antibodies (such as SARS-CoV-2 N protein antibody, IV NP protein antibody and RSV-F protein antibody) for pretreatment; then it is dropped into the sample application area and migrated to the working electrode area. When the sample to be tested passes through the working electrode area, the viral antigens therein are specifically captured by the capture antibodies (such as anti-SARS-CoV-2 N protein antibody, anti-IV NP protein antibody and anti-RSV-F protein antibody) fixed in the working electrode area of ​​the paper-based sensor, thereby forming a stable "antibody-antigen-antibody" sandwich structure in the working electrode area. This structure not only ensures the specificity of detection, but also achieves effective amplification of the impedance signal (SiO2 can significantly reduce electron transfer on the electrode surface) through the synergistic effect of the two antibodies. Finally, the reading is obtained by a portable electrochemical detector, completing the rapid detection of the three viral antigens with high sensitivity and high specificity.

[0013] In some embodiments of the present invention, the working electrode region is provided with not less than two working electrodes. Preferably, there are 3 to 10 electrodes, for example, 3, 4, 5, 6, 7, 8, 9 or 10 electrodes.

[0014] In some embodiments of the present invention, when the working electrode region is provided with four working electrodes, the capture probes covalently bound to biotin on the first, second, and third working electrodes arranged in sequence are independently biotin-conjugated antiviral protein antibodies; the capture probe covalently bound to biotin on the fourth working electrode is a biotin-conjugated quality control antibody.

[0015] In some embodiments of the present invention, the spacing between the working electrodes is 1 to 5 mm, preferably 2 to 4 mm.

[0016] In some embodiments of the present invention, the biotin-conjugated antiviral protein antibody is independently selected from any one of anti-SARS-CoV-2 N protein antibody-biotin, anti-IV NP protein antibody-biotin, and anti-RSV-F protein antibody-biotin.

[0017] In some embodiments of the present invention, the capture probes covalently bound to biotin and immobilized on the surfaces of the first working electrode, the second working electrode, the third working electrode, and the fourth working electrode are respectively anti-SARS-CoV-2 N protein antibody-biotin, anti-IV NP protein antibody-biotin, anti-RSV-F protein antibody-biotin, and goat anti-mouse secondary antibody-biotin.

[0018] This paper-based sensor chip enables rapid and simultaneous detection of three pathogens: the novel coronavirus (SARS-CoV-2), influenza virus (IV), and respiratory syncytial virus (RSV).

[0019] In some embodiments of the present invention, the biotin-conjugated quality control antibody is goat anti-mouse secondary antibody-biotin.

[0020] In some embodiments of the present invention, the working electrode is printed with a graphene oil-based conductive coating. And / or, the reference electrode region is sprayed with Ag / AgCl slurry; And / or, the counter electrode area is coated with a graphene oil-based conductive coating.

[0021] In some embodiments of the present invention, the working electrode region is isolated from the reference electrode region and the counter electrode region by a wax seal layer.

[0022] On the paper base of the printed electrode, a microfluidic boundary (i.e., wax seal) is drawn using solid paraffin. This helps to ensure that the sample to be tested is in full contact with the working electrode during detection. Furthermore, after the reaction is complete, the paraffin can be melted by heating. By actively controlling the reaction time and exposing the electrode surface for detection, the detection process can be effectively controlled.

[0023] A second aspect of the present invention provides a method for fabricating a paper-based sensor chip as described in the first aspect, comprising the following steps: S1. Using filter paper as a substrate, a graphene oil-based conductive coating is coated in the working electrode area to serve as the working electrode, and Ag / AgCl slurry and graphene oil-based conductive coating are coated in the reference electrode area and the counter electrode area respectively, and then cured. S2. Spray the nano-gold-streptavidin composite material and the corresponding biotin-covalently bound capture probe onto the surface of the working electrode, and then dry it after sealing treatment.

[0024] In some embodiments of the present invention, the concentration of the nano-gold-streptavidin composite material is 0.01~0.5 mg / mL.

[0025] In some embodiments of the present invention, the concentration of the capture probe covalently bound to biotin is 0.2~10 mg / mL.

[0026] In some embodiments of the present invention, the sealing treatment includes treatment with a solution containing 0.05-0.2% bovine serum albumin (BSA).

[0027] A third aspect of the present invention provides the application of the paper-based sensor chip as described in the first aspect in the preparation of a detection kit.

[0028] In some embodiments of the present invention, the detection kit includes a virus detection kit, such as a respiratory virus detection kit.

[0029] In some embodiments of the present invention, the detection kit further comprises a silica nanoparticle-antibody conjugate.

[0030] In some embodiments of the present invention, the antibody in the silica nanoparticle-antibody conjugate is a viral protein antibody.

[0031] In some embodiments of the present invention, the antibody in the silica nanoparticle-antibody conjugate includes at least one of SARS-CoV-2 N protein antibody, IV NP protein antibody, and RSV-F protein antibody.

[0032] A fourth aspect of the present invention provides a detection system comprising: The pretreatment module is used to mix the sample to be tested with the silica nanoparticle-antibody conjugate to obtain a mixture; The detection module includes the paper-based sensor chip described in the first aspect, the paper-based sensor chip being used to detect the mixture; The information acquisition module is used to detect and analyze the signals on the paper-based sensor chip; A storage module is used to store the results of the detection and analysis.

[0033] In some embodiments of the present invention, the antibody in the silica nanoparticle-antibody conjugate is a viral protein antibody.

[0034] In some embodiments of the present invention, the antibody in the silica nanoparticle-antibody conjugate includes at least one of SARS-CoV-2 N protein antibody, IV NP protein antibody, and RSV-F protein antibody.

[0035] In some embodiments of the present invention, the silica nanoparticle-antibody conjugate includes at least one of Ab-N-2-SiO2, Ab-NP-2-SiO2, and Ab-RSV-F-2-SiO2.

[0036] In some embodiments of the present invention, the mass ratio of the silica nanoparticles to the antibody in the silica nanoparticle-antibody conjugate is 1:35~50.

[0037] This invention couples SiO2 nanoparticles, acting as signal tags, to a viral protein antibody for detection. Upon forming an immune sandwich complex, the high insulating properties of SiO2 significantly increase the electron transfer impedance (Ret) at the electrode interface, thereby converting the biorecognition process into a significantly amplified electrochemical impedance signal. This is a built-in, enzyme-free, and fluorescence-free signal amplification strategy that contributes to improved detection accuracy and sensitivity.

[0038] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram of the overall structure of the paper-based sensor chip for multiple respiratory virus detection according to the present invention.

[0040] Figure 2 The images show fluorescence intensity diagrams and fluorescence intensity-concentration standard curves of different concentrations of FITC-Ab-N-2 according to the present invention, where A is the fluorescence intensity-wavelength spectrum of different concentrations of FITC-Ab-N-2; and B is the fluorescence intensity-concentration standard curve.

[0041] Figure 3 This diagram illustrates the optimized mass ratio of silica nanoparticles to antibodies in this invention. In the diagram, A represents the optimized coupling ratio of NH2-SiO2 to Ab-N-2; B represents the optimized coupling ratio of NH2-SiO2 to Ab-NP-2; and C represents the optimized coupling ratio of NH2-SiO2 to Ab-RSV-F-2.

[0042] Figure 4 A comparison of EIS results for the detection of the novel coronavirus (SARS-CoV-2) with and without SiO2-antibody linkage.

[0043] Figure 5 EIS comparison of influenza virus (IV) detection with and without SiO2-antibody linkage.

[0044] Figure 6 EIS comparison of respiratory syncytial virus (RSV) detection with and without SiO2-antibody.

[0045] Figure 7This invention uses differential pulse voltammetry (DPV) to detect three viral proteins, and the resulting graphs show the sensitivity and calibration curves. A represents the DPV response curves for different concentrations of SARS-CoV-2 N protein; B represents the calibration curves of the corresponding peak current versus N protein concentration; C represents the DPV response curves for different concentrations of influenza virus (IV) NP protein; D represents the calibration curves of the corresponding peak current versus NP protein concentration; E represents the DPV response curves for different concentrations of respiratory syncytial virus (RSV) F protein; and F represents the calibration curves of the corresponding peak current versus RSV-F protein concentration.

[0046] Figure 8 The images show the specific detection results of the paper-based sensor chip of this invention for three target viral proteins. A is a comparison of the DPV signals of SARS-CoV-2 N protein and its interfering protein; B is a comparison of the DPV signals of influenza virus (IV) NP protein and its interfering protein; and C is a comparison of the DPV signals of respiratory syncytial virus (RSV) F protein and its interfering protein.

[0047] Figure label: Sample loading area 100; working electrode area 200; first working electrode 210; second working electrode 220; third working electrode 230; fourth working electrode 240; reference electrode 300; counter electrode 400; wax seal layer 500. Detailed Implementation

[0048] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0049] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0050] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0051] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0052] In the description of this invention, the Ab-N-2, Ab-NP-2, and Ab-RSV-F-2 antibodies were purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0053] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0054] Example 1: A paper-based sensor chip for multiple respiratory virus detection like Figure 1 As shown, this embodiment provides a paper-based sensor chip for multiple respiratory virus detection. The paper-based sensor chip uses filter paper as a substrate and is provided with a sample application area 100, a working electrode area 200, a reference electrode area 300 and a counter electrode area 400. The working electrode area is provided with a first working electrode 210, a second working electrode 220, a third working electrode 230 and a fourth working electrode 240.

[0055] Specifically, the filter paper substrate of this invention can be chromatographic filter paper. Chromatographic filter paper has a smoother surface than other ordinary filter papers, which is beneficial to the uniformity of electrode printing and signal stability. The thickness of the chromatographic filter paper can be 0.15~0.25mm, and the flow rate can be 110~150 mm / 30min. For example, Whatman No. 1 chromatographic filter paper can be used. The sample loading area 100 is used to absorb the pretreated sample to be tested, so that the sample to be tested enters the working electrode area 200 for reaction.

[0056] In the working electrode area 200 of the paper-based sensor chip, four graphene lines with a size of 10 mm × 2 mm are printed in parallel to form a graphene line array. Each graphene line corresponds to the first working electrode 210, the second working electrode 220, the third working electrode 230, and the fourth working electrode 240, respectively, with a line spacing of 3 mm. In addition, a nano-gold-streptavidin composite material and a capture probe are sprayed onto the surface of the graphene lines. The capture probe is prepared by conjugating a capture antibody with NHS-biotin and is used to capture the corresponding viral antigen in the sample. When the detection targets are novel coronavirus (SARS-CoV-2), influenza virus (IV), and respiratory syncytial virus (RSV), the capture probes sprayed on the surfaces of the first working electrode 210, the second working electrode 220, the third working electrode 230, and the fourth working electrode 240 are respectively anti-SARS-CoV-2 N protein antibody-biotin (such as Ab-N-1-biotin), anti-IV NP protein antibody-biotin (such as Ab-NP-1-biotin), anti-RSV-F protein antibody-biotin (such as Ab-RSV-F-1-biotin), and goat anti-mouse secondary antibody-biotin (such as Sa-biotin).

[0057] The reference electrode region 300 and the counter electrode region 400 are located on the upper and lower sides of the graphene line array, respectively, and Ag / AgCl paste and graphene oil-based conductive coating are printed in these regions. The working electrode region 200, the reference electrode region 300, and the counter electrode region 400 constitute a complete three-electrode system.

[0058] In some embodiments, the working electrode region 200 is isolated from the reference electrode region 300 and the counter electrode region 400 by a wax seal layer 500 to ensure that the sample to be tested is in full contact with and reacts with the detection line of the working electrode region 200 during the detection process.

[0059] When using this paper-based sensor chip to detect pretreated samples, the sample is first mixed with pre-labeled SiO2 antibodies (such as SARS-CoV-2 N protein antibody, IV NP protein antibody, and RSV-F protein antibody) for pretreatment. Then, the sample is added to the sample application area, allowing it to migrate towards the working electrode area along the absorption direction. As the sample passes through the working electrode area, the viral antigens are specifically captured by the capture antibodies (such as anti-SARS-CoV-2 N protein antibody, anti-IV NP protein antibody, and anti-RSV-F protein antibody) immobilized in the working electrode area of ​​the paper-based sensor, forming a stable "antibody-antigen-antibody" sandwich structure in the working electrode area. This structure not only ensures the specificity of the detection but also effectively amplifies the impedance signal (SiO2 can significantly reduce electron transfer on the electrode surface) through the synergistic effect of the two antibodies. Finally, the results are read using a portable electrochemical detector, achieving highly sensitive and specific rapid detection of the three viral antigens.

[0060] The paper-based sensor chip for multiple respiratory virus detection in this embodiment can achieve rapid and simultaneous detection of three pathogens: novel coronavirus (SARS-CoV-2), influenza virus (IV), and respiratory syncytial virus (RSV).

[0061] Example 2: A method for fabricating a paper-based sensor chip for multiplex respiratory virus detection This embodiment provides a method for fabricating a paper-based sensor chip for multiple respiratory virus detection, specifically including the following:

[0062] 1. Electrode Printing Using precision spraying equipment, four 10 mm × 2 mm graphene oxide lines were printed in parallel on Whatman No. 1 chromatography filter paper (3001-861, Whatman) using graphene oil-based conductive coating (HQNANO-GR-017-2, Suzhou Carbon Graphene Technology Co., Ltd.) as raw materials. These lines served as working electrodes, with a line spacing of 3 mm.

[0063] Above and below the aforementioned graphene oxide line array, a reference electrode was printed using Ag / AgCl paste (Elec-H230), and a counter electrode was printed using graphene oil-based conductive coating, forming a complete three-electrode system. The printed material was then cured in an 80°C oven for 30 minutes for later use.

[0064] 2. Preparation of capture probes Four capture antibodies—Ab-N-1, Ab-NP-1, Ab-RSV-F-1, and goat anti-mouse secondary antibody (Sa)—were diluted to 1 mg / mL with PBS. NHS-biotin was added to each capture antibody solution at a molar ratio of 10:1, and the reaction was carried out at room temperature in the dark for 1 hour. The reaction solutions were then loaded onto pre-equilibrated desalting columns to remove unreacted biotin. The purified capture probes were collected, their concentrations determined, and diluted to 2 mg / mL for later use.

[0065] The resulting products were named Ab-N-1-biotin, Ab-NP-1-biotin, Ab-RSV-F-1-biotin, and Sa-biotin, respectively.

[0066] 3. Functional treatment of the working electrode (1) Streptavidin layer fixation: The nano gold-streptavidin composite material (0.05 mg / mL, Nanjing Shennuoqing Biotechnology Co., Ltd.) was sprayed evenly onto the above 4 graphene lines (i.e. working electrodes) in sequence using a spray gun and dried at room temperature.

[0067] (2) Antibody fixation: Using a micro-spotting instrument, Ab-N-1-biotin (2 mg / mL), Ab-NP-1-biotin (2 mg / mL), Ab-RSV-F-1-biotin (2 mg / mL), or Sa-biotin (2 mg / mL) were precisely spotted onto the first, second, third, and fourth working electrodes, respectively, to obtain the first, second, third, and fourth working electrodes. The fourth working electrode was used for quality control.

[0068] (3) Blocking: Immerse the material prepared above in 1% BSA solution and block at room temperature for 1 hour to block non-specific binding sites. Then rinse gently with PBS and dry to obtain the paper-based sensor chip. Store the dried paper-based sensor chip in a sealed container under cold storage for later use.

[0069] Example 3: A detection method based on a paper-based sensor chip for multiple respiratory virus detection This embodiment optimizes silica nanoparticle-antibody conjugates and screens out a virus detection method for a paper-based sensor chip used for multiplex respiratory virus detection, as detailed below: 1. Screening for the optimal silica nanoparticle-antibody conjugate In this experiment, aminated silica (NH2-SiO2, purchased from Nanjing JK Biotechnology Co., Ltd.) was used as a conjugate, and Ab-N-2, Ab-NP-2, or Ab-RSV-F-2 were used as antibodies. The optimal fit ratio between the antibody and NH2-SiO2 was screened and optimized. The specific experimental steps are as follows: S1. Activation: Wash 1 mL of NH2-SiO2 (100 nm, 1 mg / mL) twice with MES buffer (12,000 rpm, 10 min), and resuspend in 1 mL of MES. Add 50 μL of glutaraldehyde solution and incubate at room temperature in the dark with shaking for 2 hours.

[0070] S2. Washing: Centrifuge (12,000 rpm, 10 min) to remove excess glutaraldehyde, wash 3 times with PBS, and then resuspend with PBS.

[0071] S3. Antibody Conjugation: Fluorescently labeled antibodies (Fitc-Ab-N-2, FITC-Ab-NP-2, FITC-Ab-RSV-F-2) were conjugated to NH2-SiO2 at different mass ratios (NH2-SiO2:antibody = mg:µg). Ten gradient ratios were set for each antibody group, labeled 1-10, representing (1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50), respectively. One unconjugated NH2-SiO2 sample was added to each group as a control for calibration. The conjugates were then placed on a constant-temperature shaker and slowly shaken overnight at 4°C.

[0072] S4. Detection: Centrifuge (12,000 rpm, 10 min), collect the supernatant, wash three times with PBS, and detect using a fluorescence spectrophotometer. Fluorescent antibodies not bound to the SiO2 surface will remain free in the supernatant and will not be centrifuged.

[0073] S5. Data Processing: The amount of antibody bound to SiO2 (μg) = [Total amount of antibody added (μg) - Total amount of antibody in the supernatant (μg)]. The total amount of antibody in the supernatant is calculated using an established standard curve.

[0074] Test results as follows Figure 2 and Figure 3 As shown, where Figure 2 In the figure, A represents the fluorescence intensity-wavelength spectra of different concentrations of Fitc-Ab-N-2; B represents the fluorescence intensity-concentration standard curve. The results show that the fluorescence intensity of Fitc-Ab-N-2 exhibits a good regularity with concentration, and its fluorescence intensity-concentration standard curve shows a significant linear relationship (R0). 2=0.9792), indicating that the fluorescent label has stable optical response characteristics within the experimental concentration range and can be used to evaluate the coupling between SiO2 and antibody.

[0075] Figure 3 The optimal ratios for preparing silica nanoparticle-antibody conjugates by coupling NH2-SiO2 with different antibodies are shown in the diagram, where A represents Ab-N-2, B represents Ab-NP-2, and C represents Ab-RSV-F-2. The results show that when NH2-SiO2 is coupled with Ab-N-2, Ab-NP-2, or Ab-RSV-F-2, the fluorescence intensity inflection point is reached at the mass ratios of (1:40, 1:35, and 1:35), indicating that the binding of NH2-SiO2 to the antibody is saturated at these ratios. Therefore, this ratio was subsequently used for the preparation of silica nanoparticle-antibody conjugates.

[0076] The above results indicate that effective coupling between antibodies and silica carriers can be achieved by optimizing the ratio, laying a material foundation for subsequent applications such as biosensing and targeted detection based on these coupling materials. It also provides experimental basis for optimizing the coupling process of different antibodies with NH2-SiO2.

[0077] 2. Electrochemical Impedance Spectroscopy (EIS) Experiment Antibodies conjugated with three types of silicon nanoparticles—Ab-N-2-SiO2, Ab-NP-2-SiO2, and Ab-RSV-F-2-SiO2—can be electrochemically captured by binding to antigens. In the experimental system, the SiO2-antibody and the capture probe operate independently, targeting different epitopes of the same antigen, forming a typical antibody-sandwich recognition structure. The core function of the capture antibody is to specifically anchor the target antigen in solution, while the SiO2-antibody acts as a signal enhancement element to improve the sensitivity of the detection system. The following experiments, using electrochemical impedance spectroscopy data, tested whether the introduction of SiO2-antibody could significantly increase the electrode surface impedance, thereby effectively amplifying the detection signal. The specific experimental methods are as follows: In the simulated sample, the three antigens—novel coronavirus, influenza virus, and respiratory syncytial virus—were each divided into two portions at a final concentration of 10 ng / mL. One portion was supplemented with the corresponding SiO2- antibody at a final concentration of 1 mg / mL, while the other portion was left unsupplied. The measurement buffer contained 5 mM [Fe(CN)6]. 3- / 4- A solution of 0.1 M KCl was used, and the EIS test frequency was 0.1-10. 5 Hz.

[0078] Figures 4-6 This is a graph of electrochemical impedance spectroscopy data measured by an electrochemical workstation, in which... Figure 4A comparison of novel coronavirus detection using silica (SiO2)-antibody and unlinked SiO2-antibody; Figure 5 Comparison of influenza virus detection with and without SiO2-linked antibodies; Figure 6 To compare the detection of respiratory syncytial virus with and without SiO2-antibody-linked antibodies.

[0079] Test results showed that the impedance values ​​of all three antigen systems increased after the addition of SiO2-antibody. This is presumably because the antibody forms a "barrier layer" on the electrode surface, hindering charge transfer and increasing charge transfer resistance. This strongly demonstrates that SiO2 antibody modification can alter the electrochemical behavior of the electrode-solution interface. In short, SiO2 antibody can act as a "signal amplification / regulation element," altering interfacial impedance by binding to the virus, enhancing signal differences during virus detection, and thus achieving more accurate detection and identification.

[0080] 3. Virus detection methods (1) Preparation of test samples: Select about 500 μL of test sample (such as saliva sample), mix with 500 μL of PBS treatment solution containing 0.5% Tween-20, and vortex; then add the prepared suspensions containing Ab-N-2-SiO2 (final concentration 1 mg / mL), Ab-NP-2-SiO2 (final concentration 1 mg / mL) and Ab-RSV-F-2-SiO2 (final concentration 1 mg / mL) respectively to the above treatment solution, incubate at room temperature for 15 minutes to allow the virus to bind with the corresponding antibody-SiO2, and obtain the pretreated mixed sample to be tested.

[0081] (2) Chip reaction: The above-mentioned mixed sample to be detected is dropped into the sample application area of ​​the prepared multiple respiratory virus paper-based sensor chip. The mixed sample flows through the detection lines of the four working electrodes through capillary action.

[0082] (3) Wax sealing and control: Use solid paraffin to draw microfluidic boundaries upstream and downstream of the detection line, control the liquid flow rate and ensure sufficient reaction with the antibody, and react at room temperature for 20 minutes.

[0083] (4) Signal detection and readout: Gently rinse the chip surface with PBST buffer to remove unbound SiO2 labeling; then place the chip on a 40°C hot plate until the paraffin melts and is absorbed by the filter paper, exposing the complete electrode surface. Next, connect the paper-based sensor chip to an electrochemical workstation and place it in a solution containing 5 mM [Fe(CN)6]. 3- / 4-Mix 0.1 M KCl solution with the following parameters: DPV potential -0.2-0.5 V, pulse amplitude 50 mV, pulse period 0.5 s, and pulse width 0.05 s. Record the peak current value of each detection line.

[0084] (5) Results analysis: Qualitative or quantitative analysis is performed based on the electrochemical detection results (DPV data).

[0085] Example 4: Sensitivity Detection In this embodiment, the differential pulse voltammetry (DPV) method is used to characterize the sensitivity of the paper-based sensor chip for multiple respiratory virus detection prepared above, as detailed below: SARS-CoV-2 N protein solution, NP protein solution, and RSV-F protein solution with different concentration gradients of 0, 0.1, 1, 10, 100, and 1000 ng / mL were prepared as samples to be tested; then, the above protein solutions of different concentrations (all containing 5 mM [Fe(CN)6]) were respectively used as samples to be tested. 3- / 4- The sample was incubated with 0.1 M KCl on the surface of a paper-based sensor chip. After washing three times with distilled water to remove non-specific adsorption, the peak current change was measured under DPV parameters (potential range -0.2~0.5 V, pulse amplitude 50 mV, pulse period 0.5 s, pulse width 0.05 s). Finally, a standard curve was plotted by comparing the current response value with the logarithm of the protein concentration to evaluate the detection sensitivity and linear range.

[0086] The DPV detection results and calibration curves for three respiratory viral proteins are as follows: Figure 7 As shown, A is the DPV response curve of SARS-CoV-2 N protein at different concentrations; B is the calibration curve of the corresponding peak current versus N protein concentration; C is the DPV response curve of influenza virus (IV) NP protein at different concentrations; D is the calibration curve of the corresponding peak current versus NP protein concentration; E is the DPV response curve of respiratory syncytial virus (RSV) F protein at different concentrations; F is the calibration curve of the corresponding peak current versus RSV-F protein concentration.

[0087] The test results showed that for the SARS-CoV-2 N protein, the DPV response current exhibited a regular change with increasing concentration. The peak current and the logarithm of the protein concentration showed a linear relationship within the test range, and the fitting equation was Y = 3.05 × 10⁻⁶. -4 -2.49×10 -5 Lg(X) (R²=0.97), according to LOD = 3×σ blank / S, where σ blankThe standard deviation of the blank response and the absolute value of the slope of the S-calibration curve yielded a detection limit (LOD) of 0.0625 ng / mL for N protein, indicating that the sensor possesses high sensitivity and laying the foundation for accurate quantification. In the detection of influenza virus (IV) NP protein, the DPV response curve exhibited a characteristic shift with increasing concentration, and the peak current was linearly correlated with the logarithm of the NP protein concentration, with the fitting equation being: Y = 2.83 × 10⁻⁶. -4 -2.43×10 -5 Lg(X)(R 2 =0.97), with a corresponding detection limit of 0.0421 ng / mL. This result confirms the effective quantification capability of this method for NP protein. For respiratory syncytial virus (RSV) F protein, the DPV response also showed a significant concentration dependence, with the linear equation of the calibration curve being Y = 2.69 × 10⁻⁴ - 2.07 × 10⁻⁵. Lg(X)(R 2 =0.98), and the detection limit was 0.0725 ng / mL, indicating that this method can also achieve sensitive and accurate quantitative analysis of F protein.

[0088] The above results demonstrate that the paper-based sensor chip for multiplex respiratory virus detection of this invention exhibits excellent detection sensitivity and quantitative analysis performance. It demonstrates good applicability and quantitative performance in the detection of three respiratory virus proteins, showing a stable linear dependence between peak current and the logarithm of protein concentration. This provides a reliable method for highly sensitive quantitative analysis of respiratory viruses. This invention provides an effective means for early diagnosis and epidemiological monitoring of viral infections and has significant clinical application value.

[0089] Example 5: Specificity Detection In this embodiment, the differential pulse voltammetry (DPV) method is used to specifically characterize the paper-based sensor chip for multiple respiratory virus detection prepared above, as follows: At a uniform concentration of 10 μg / mL, the target protein and its related interfering substances were independently measured, as follows: For the SARS-CoV-2 N protein, its DPV response with H1N1 N protein, RSV-G protein, RSV-F protein, and influenza virus (IV) NP protein was measured; for the influenza virus (IV) NP protein, its DPV response with RSV-F protein, SARS-CoV-2 N protein, human metapneumovirus (HMPV), and varicella-zoster virus glycoprotein E (VZV-gE) was measured; for the RSV-F protein, its DPV response with SARS-CoV-2 N protein, IV NP protein, porcine epidemic diarrhea virus (PEDV), and Marek's disease virus (MDV) was measured. The parameter settings of the DPV program were consistent with those of the above sensitivity tests.

[0090] DPV-specific detection results of three respiratory viral proteins are as follows: Figure 8 As shown, A represents the SARS-CoV-2 N protein, B represents the IV NP protein, and C represents the RSV-F protein. By comparing the signal responses of each target protein and its corresponding interfering protein in their respective detection regions, the results indicate that the sensor only generates significant current changes in response to the target protein, and no obvious cross-reaction was observed, confirming its high selectivity and specificity in recognition.

[0091] The above results demonstrate that the paper-based sensor chip for multiple respiratory virus detection of the present invention has excellent detection specificity and anti-interference effect.

[0092] Example 6: Accuracy Detection This embodiment uses the detection method of Embodiment 3 above, employing 10 randomly obtained real saliva samples as test samples to characterize the detection accuracy of the paper-based sensor chip for multiplex respiratory virus detection of the present invention. The control group is detected using the gold standard molecular biology diagnostic method PCR. The specific experimental procedure is as follows: (1) Preparation of test samples: saliva samples from 10 individuals (including healthy individuals and patients) were randomly selected and mixed with 500 μL of PBS containing 0.5% Tween-20 and vortexed. Then, suspensions containing Ab-N-2-SiO2 (final concentration 1 mg / mL), Ab-NP-2-SiO2 (final concentration 1 mg / mL), and Ab-RSV-F-2-SiO2 (final concentration 1 mg / mL) were added to the mixture in proportion and incubated at room temperature for 15 minutes to allow the virus to bind with the corresponding antibody-SiO2, thus obtaining the pretreated mixed sample to be tested.

[0093] (2) Chip reaction: The above-mentioned mixed sample to be detected is dropped into the sample application area of ​​the prepared multiple respiratory virus paper-based sensor chip. The mixed sample flows through the detection lines of the four working electrodes through capillary action.

[0094] (3) Wax sealing and control: Use solid paraffin to draw microfluidic boundaries upstream and downstream of the detection line, control the liquid flow rate and ensure sufficient reaction with the antibody, and react at room temperature for 20 minutes.

[0095] (4) Signal detection and readout: Gently rinse the chip surface with PBST buffer to remove unbound SiO2 labeling; then place the chip on a 40°C hot plate until the paraffin melts and is absorbed by the filter paper, exposing the complete electrode surface. Next, connect the paper-based sensor chip to an electrochemical workstation and place it in a solution containing 5 mM [Fe(CN)6]. 3- / 4- Mix 0.1 M KCl solution with the following parameters: DPV potential -0.2-0.5 V, pulse amplitude 50 mV, pulse period 0.5 s, and pulse width 0.05 s. Record the peak current value of each detection line.

[0096] (5) Results analysis: The viral infection status was determined based on the electrochemical detection results (DPV data). Positive results were recorded as (+) and negative results as (-), and the results were compared with the PCR gold standard.

[0097] The test results are shown in Table 1.

[0098] Table 1:

[0099] Note: Samples 1-3 are normal human samples, and Samples 4-10 are patient samples; "+" indicates that the corresponding virus was detected, and "-" indicates that it was not detected.

[0100] By comparing the detection method of this invention with PCR diagnosis (the gold standard) for the detection of SARS-CoV-2, IV, and RSV, the results showed that both methods were negative in normal human samples (Samples 1-3). In patient samples, the two methods were highly consistent in determining viral infection (e.g., Samples 4, 7, 9, and 10 detected IV positive, Sample 6 detected SARS-CoV-2 positive, and Sample 8 detected RSV positive). This indicates that the detection method of this invention has a high degree of consistency with PCR diagnosis results, with an accuracy rate of 100%, and possesses high accuracy and potential application value in clinical viral detection.

[0101] In summary, this invention provides a paper-based sensor chip for multiplex respiratory virus detection, its fabrication method, and its applications. The chip uses chromatographic filter paper as a substrate and employs screen printing and spraying processes to construct a three-electrode system comprising multiple graphene working electrodes, a counter electrode containing Ag / AgCl, and a reference electrode. Specific capture antibodies against SARS-CoV-2, influenza virus, and respiratory syncytial virus are immobilized in their respective detection areas (i.e., on the working electrodes) using streptavidin-biotin interaction. Simultaneously, silica nanoparticle-labeled signal antibodies are introduced as impedance amplification probes, achieving efficient detection of three different novel respiratory viruses. The paper-based sensor chip of this invention enables highly sensitive and specific simultaneous detection of multiple viral antigens, offering advantages such as low cost, ease of operation, and suitability for rapid on-site screening. It has significant application value in the point-of-care diagnosis of respiratory pathogens.

[0102] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A paper-based sensor chip, characterized in that, The paper-based sensor chip uses filter paper as its substrate and is provided with a sample application area (100), a working electrode area (200), a reference electrode area (300), and a counter electrode area (400), wherein: The working electrode region (200) is provided with a plurality of working electrodes, which are arranged in a row matrix in a direction away from the sample application region (100). The working electrodes use graphene as a conductive channel and have nano-gold-streptavidin composite material and a capture probe covalently bound to biotin fixed on their surface. The upper side of the working electrode is the reference electrode region (300), and the lower side of the working electrode is the counter electrode region (400). The working electrode, the reference electrode region (300), and the counter electrode region (400) together form a three-electrode system.

2. The paper-based sensor chip according to claim 1, characterized in that, The working electrode area (200) is provided with not less than two working electrodes; Preferably, when the working electrode area (200) is provided with four working electrodes, the capture probes covalently bound to biotin on the first working electrode (210), the second working electrode (220) and the third working electrode (230) arranged in sequence are independently biotin-conjugated antiviral protein antibodies; the capture probe covalently bound to biotin on the fourth working electrode (240) is a biotin-conjugated quality control antibody.

3. The paper-based sensor chip according to claim 2, characterized in that, The biotin-conjugated antiviral protein antibody is independently selected from any one of the following: anti-SARS-CoV-2 N protein antibody-biotin, anti-IV NP protein antibody-biotin, and anti-RSV-F protein antibody-biotin; And / or, the biotin-conjugated quality control antibody is goat anti-mouse secondary antibody-biotin.

4. The paper-based sensor chip according to any one of claims 1 to 3, characterized in that, The working electrode is printed with a graphene oil-based conductive coating. And / or, the reference electrode region (300) is coated with Ag / AgCl slurry; And / or, the counter electrode region (400) is coated with a graphene oil-based conductive coating.

5. The paper-based sensor chip according to claim 4, characterized in that, The working electrode area (200) is isolated from the reference electrode area (300) and the counter electrode area (400) by a wax seal layer (500).

6. The method for fabricating a paper-based sensor chip as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Using the filter paper as a substrate, a graphene oil-based conductive coating is sprayed onto the working electrode area (200) as the working electrode, and Ag / AgCl slurry and graphene oil-based conductive coating are respectively coated onto the reference electrode area (300) and the counter electrode area (400), and then cured. S2. Spray the nano-gold-streptavidin composite material and the corresponding biotin-covalently bound capture probe onto the surface of the working electrode, and then dry it after sealing treatment.

7. The preparation method according to claim 6, characterized in that, The concentration of the nano-gold-streptavidin composite material is 0.01~0.5 mg / mL; And / or, the concentration of the capture probe covalently bound to biotin is 0.2~10 mg / mL; And / or, the sealing treatment includes treatment with a solution containing 0.05-0.2% bovine serum albumin.

8. The use of the paper-based sensor chip as described in any one of claims 1 to 5 in the preparation of a detection kit; Preferably, the detection kit includes a virus detection kit; Preferably, the test kit further comprises a silica nanoparticle-antibody conjugate.

9. A detection system, characterized in that, include: The pretreatment module is used to mix the sample to be tested with the silica nanoparticle-antibody conjugate to obtain a mixture; The detection module includes a paper-based sensor chip according to any one of claims 1 to 5, wherein the paper-based sensor chip is used to detect the mixture; The information acquisition module is used to detect and analyze the signals on the paper-based sensor chip; A storage module is used to store the results of the detection and analysis.

10. The detection system according to claim 9, characterized in that, The antibody in the silica nanoparticle-antibody conjugate includes at least one of SARS-CoV-2 N protein antibody, IV NP protein antibody, and RSV-F protein antibody; And / or, in the silica nanoparticle-antibody conjugate, the conjugation mass ratio of the silica nanoparticles to the antibody is 1:35~50.

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