Light-excited lateral flow immunosensor, detection method and application of light-excited lateral flow immunosensor

Abstract

The invention provides a light-excited lateral flow immunosensor reagent strip, a detection analysis method and application of the light-excited lateral flow immunosensor reagent strip. The reagent strip comprises an electrode substrate, a combination pad, a sample pad layer, a chromatography membrane and a water absorption pad, and the sample pad layer, the combination pad, the chromatography membrane and the water absorption pad of the reagent strip are connected end to end and are sequentially overlapped and pasted on the electrode substrate. The light-excited lateral flow immunosensor reagent strip is applied to quantitative analysis of various disease markers in biological fluids such as blood, saliva, cerebrospinal fluid or urine.

Description

Technical Field

[0001] The present invention belongs to the field of medical diagnosis, and particularly relates to a light-excited lateral flow immunosensor, a detection method and their applications. Background Art

[0002] Immunoassay technology has been widely used in the field of medical diagnosis. Commonly used immunoassay methods include magnetic particle chemiluminescence, electrochemiluminescence, homogeneous chemiluminescence, etc. The principle of magnetic particle chemiluminescence is a new analytical method that combines magnetic separation technology, chemiluminescence technology and immunoassay technology. It utilizes the special properties of magnetic particles and the high sensitivity of chemiluminescence to achieve rapid, sensitive and high-throughput detection of biomolecules. The principle of electrochemiluminescence involves an electrochemical reaction process, and the whole reaction process can be continuously cycled, and the detection signal is continuously amplified, thereby improving the detection sensitivity. Magnetic particle chemiluminescence and electrochemiluminescence instruments need to be equipped with a liquid path system, a reagent refrigeration area, cleaning liquid, etc., and the appearance is relatively large. Homogeneous chemiluminescence does not involve cleaning. In terms of the detection principle, as long as the distance between the photosensitive sphere and the luminescent sphere in the reagent components is <200 nm, light can be emitted. Photons are received by a photomultiplier tube (PMT). The strict design requirements of the optical module make it impossible to miniaturize the instrument appearance, the instrument cost is relatively high, and this technology has background signal problems. For example, (1) there will be non-specific binding between the two spheres; (2) the Brownian motion between the spheres causes them to approach each other. Due to the detection method, whole blood separation is required before testing to ensure accuracy.

[0003] In addition, the visit of patients with mild symptoms or those who need long-term monitoring of detection items to the hospital increases the disease cross-infection rate. The above-mentioned methods are only applicable to hospital scenarios and are difficult to promote for home use. To solve the problems of home use, whole blood testing, and reduced background signals, it is urgent to develop a small instrument with a simple system that can be monitored by mobile phone for disease levels and supporting detection reagents.

[0004] Electrochemical immunoassay is based on immune recognition and realizes specific detection of specific substances through the immune recognition reaction of antigen and antibody. The backend uses electrical signals for output, which has the high specificity of immune technology and the advantages of electrochemical detection, such as low cost, high sensitivity, high cost performance, and small detection instrument. Compared with traditional immunoassay that uses photons as signal output, electrochemistry collects signals such as current and potential, and does not require a complex optical path system and light receiving module. Therefore, the cost of the instrument is relatively low. The sensor can use screen-printed electrodes, with low batch printing cost, good repeatability and no need for maintenance. And because the electrochemical detection instrument does not require a complex structure and optical path components, it can achieve miniaturization and portability of the instrument. At the same time, since electrical signals are detected, whole blood samples can be used for detection, which is more suitable for point-of-care testing (POCT) scenarios.

[0005] Electrochemical detection can use a variety of techniques to collect and analyze the electrical signals of samples. The commonly used test techniques mainly include cyclic voltammetry (CV), linear sweep voltammetry (LSV), differential pulse voltammetry (DPV), square wave voltammetry (SWV), amperometry (IT), and electrochemical impedance spectroscopy (EIS). Among them, DPV is a commonly used analytical technique in electrochemistry. Its detection sensitivity is higher than that of conventional detection techniques and can detect trace substances. The detection principle of DPV is to superimpose a series of pulse signals on the basis of linear scanning, and take the difference between the signals before and after the pulse to eliminate the capacitive current. During the scanning potential change process, the difference is continuously measured, and a differential pulse curve can be obtained by plotting the potential-difference. The electrochemical oxidation-reduction potentials of different substances are different, so a specific current peak curve (including peak potential and peak current information) can be obtained at a specific position. The peak potential can be used for qualitative analysis of the analyte, and the height of the peak current is related to the concentration of the analyte, which can be used for quantitative analysis. At the same time, during the signal acquisition process of DPV, signal acquisition is only performed at the end of each pulse period. The current generated by the oxidation-reduction of substances and the background current are affected differently by the pulse. The decay rate of the background current is much greater than the current generated by the oxidation-reduction of substances. Therefore, collecting signals at the end of the pulse period in DPV can effectively reduce the background signal, so it has better detection sensitivity.

[0006] Lateral flow immunoassay (LFIA) is a rapid, portable, and simple technique that requires almost no infrastructure and is usually used for rapid on-site screening. This method realizes detection by the lateral flow of the sample on a membrane containing antibodies, and the signal is revealed by a label.

[0007] Photoactivated chemiluminescence is to irradiate the acceptor microspheres with excitation light of a certain wavelength to generate singlet oxygen ( 1 O2), 1 O2 can transfer energy to a reducing chemical substance to generate an electrochemical signal only when an immune reaction (specific binding of antigen and antibody) occurs. This invention can effectively exclude non-specific binding without immune reaction and improve the sensitivity and detection specificity of the reagent.

[0008] The combination of electrochemistry, lateral flow immunoassay, and photoactivated chemiluminescence integrates the advantages of high sensitivity and good signal stability of electrochemistry analysis, the convenience and low cost of lateral flow immunoassay, and the high sensitivity and detection specificity of photoactivated chemiluminescence to meet higher application scenarios. Summary of the Invention

[0009] The purpose of the present invention is to provide a photoexcited lateral flow electrochemical immunosensor that uses electrochemical signals for output to solve the problems of high cost and inconvenience caused by complex sensors in the above-mentioned prior art.

[0010] Compared with currently available lateral flow electrochemical assays, the present invention proposes a new method for determining a target substance. Instead, a substance that can react with reactive oxygen species to generate a substance whose electrical signal can be detected by an electrode is introduced, effectively distinguishing immune binding from other background interferences and improving sensitivity. Users can collect fingertip blood at home and drop it into the sample application hole, and it will chromatograph to the detection line of the chromatographic membrane through capillary action.

[0011] To achieve the above object, the main technical solutions of the present invention are as follows:

[0012] In a first aspect, the present invention provides a light-excited lateral flow immunosensor reagent strip, comprising an electrode backing, a conjugate pad, a sample pad layer, a chromatographic membrane, and an absorbent pad.

[0013] Among them, the electrode backing is disposed at the bottommost end of the reagent strip, providing a solid support for the reagent strip;

[0014] One end of the conjugate pad is in contact with the electrode backing, and the conjugate pad contains a first structural substance that can specifically bind to the target analyte. The first structural substance is conjugated with a photosensitive compound that can be activated under preset conditions, and the activated photosensitive compound can generate reactive oxygen species;

[0015] Further, the conjugate pad is made of cross-linked silica, glass fiber, polyester or rayon;

[0016] The sample pad layer is located at the forefront of the reagent strip, with one end placed on the conjugate pad and the other end placed on the electrode backing. The sample pad layer contains a second structural substance, and the second structural substance is conjugated with an oxidized compound that can accept reactive oxygen species to generate a preset electrical signal;

[0017] The chromatographic membrane provides a solid support for the specific binding of the target analyte and guides the capillary flow of the target analyte along the direction of the reagent strip;

[0018] Further, the chromatographic membrane is selected from a nitrocellulose or cellulose acetate membrane;

[0019] The absorbent pad is located at the rearmost end of the reagent strip, with one end placed on the chromatographic membrane and the other end in contact with the electrode backing, and adsorbs excess reagent to maintain lateral flow along the reagent strip.

[0020] Further, the sample pad layer includes at least one of a sample pad and a blood filtering membrane placed on the electrode backing;

[0021] (1) When the target analyte is from a whole blood sample, the sample cushion layer of the test strip comprises a blood filtration membrane and a sample pad. The blood filtration membrane and the sample pad are sequentially pasted on the electrode substrate from left to right, and one end of the blood filtration membrane is placed above the sample pad, and the whole blood sample is filtered and separated; the second structural substance is contained on the sample pad;

[0022] The blood filtration membrane, the sample pad, the conjugate pad, the chromatographic membrane and the absorbent pad of the test strip are connected end to end and sequentially lapped and pasted on the electrode substrate; or

[0023] (2) When the target analyte is from a whole blood sample, the sample cushion layer of the test strip only comprises a blood filtration membrane. The blood filtration membrane and the conjugate pad are sequentially pasted on the electrode substrate from left to right, and one end of the blood filtration membrane is placed above the conjugate pad, and the whole blood sample is filtered and separated; the second structural substance is contained on the blood filtration membrane;

[0024] The blood filtration membrane, the conjugate pad, the chromatographic membrane and the absorbent pad of the test strip are connected end to end and sequentially lapped and pasted on the electrode substrate;

[0025] (3) When the target analyte is not a whole blood sample, the sample cushion layer of the test strip only comprises a sample pad. The sample pad and the conjugate pad are sequentially pasted on the electrode substrate from left to right, and one end of the sample pad is placed above the conjugate pad; the second structural substance is contained on the sample pad;

[0026] The sample pad, the conjugate pad, the chromatographic membrane and the absorbent pad of the test strip are connected end to end and sequentially lapped and pasted on the electrode substrate.

[0027] When the target analyte in the sample is an antigen or a hapten, it is detected in the form of binding to a double antibody or competitively binding to a single antibody. The surface of the electrode is not treated. The detection reagent contains a conjugate pad with photosensitive spheres, a sample cushion layer with an oxidizable compound, and a chromatographic membrane coated with an antibody or an antigen. The photosensitive spheres contain photosensitive substances that can be excited by excitation light of a certain wavelength, such as rose bengal, methylene blue, phthalocyanine complexes, naphthalocyanine complexes and their combinations; the oxidizable compounds do not produce electrochemically signal substances and can undergo redox reactions with reactive oxygen species to produce products that can cause electrochemical signals, such as: hydroquinone, resorcinol, dopamine, paracetamol, p-aminophenol, ferrocene and its derivatives or combinations. Both the photosensitive spheres and the oxidizable compounds contain reactive groups such as amino groups, carboxyl groups, aldehyde groups that can react with proteins. The surface of the photosensitive spheres can be coated with antibodies, antigen carriers, avidin.

[0028] (1) The target analyte antigen forms a sandwich complex with the antibody labeled on the surface of the treated photosensitive beads on the conjugate pad and the antibody coated on the test line of the chromatographic membrane. After excitation by light of a certain wavelength, the photosensitive beads will generate reactive oxygen species upon excitation. The distance of transmission of the reactive oxygen species is limited and can only react with the oxidizable compounds within 200 nm. The reaction product of the reactive oxygen species and the oxidizable compounds generates an electrochemical signal, which can generate an electrical signal under the action of a specific potential. As the concentration of the analyte increases, the electrical signal becomes stronger. The specific potential here can be controlled by different electrochemical techniques, such as CV, IT, DPV, SWV, and combinations of these techniques. The measured signals include but are not limited to current signals, current peak signals, potential signals, potential peak signals, etc.

[0029] (2) The target analyte antigen competes with the antigen coated on the test line of the chromatographic membrane for the antibody labeled on the surface of the treated photosensitive beads on the conjugate pad. After excitation by light of a certain wavelength, the photosensitive beads will generate reactive oxygen species upon excitation. The distance of transmission of the reactive oxygen species is limited and can only react with the oxidizable compounds within 200 nm. The reaction product of the reactive oxygen species and the oxidizable compounds generates an electrochemical signal, which can generate an electrical signal under the action of a specific potential. As the concentration of the target analyte increases, the generated electrical signal becomes weaker.

[0030] (3) The target analyte antigen competes with the antibody coated on the test line of the chromatographic membrane for the antigen labeled on the surface of the treated photosensitive beads on the conjugate pad. After excitation by light of a certain wavelength, the photosensitive beads will generate reactive oxygen species upon excitation. The distance of transmission of the reactive oxygen species is limited and can only react with the oxidizable compounds within 200 nm. The reaction product of the reactive oxygen species and the oxidizable compounds generates an electrochemical signal, which can generate an electrical signal under the action of a specific potential. As the concentration of the target analyte increases, the generated electrical signal becomes weaker.

[0031] (4) The target analyte hapten forms a sandwich complex with the antibody labeled on the surface of the treated photosensitive beads on the conjugate pad and the antibody coated on the test line of the chromatographic membrane. After excitation by light of a certain wavelength, the photosensitive beads will generate reactive oxygen species upon excitation. The distance of transmission of the reactive oxygen species is limited and can only react with the oxidizable compounds within 200 nm. The reaction product of the reactive oxygen species and the oxidizable compounds generates an electrochemical signal, which can generate an electrical signal under the action of a specific potential. As the concentration of the analyte increases, the electrical signal becomes stronger.

[0032] (5) The target analyte hapten competes with the antigen coated on the test line of the chromatographic membrane for binding to the antibody carrier labeled on the surface of the photosensitive beads treated on the conjugate pad. After excitation by light of a certain wavelength, the photosensitive beads will generate reactive oxygen species after being excited. The distance that the reactive oxygen species can transfer is limited and can only react with the compounds susceptible to oxidation within 200 nm. The reaction product of the reactive oxygen species and the compound susceptible to oxidation generates an electrochemical signal, which can generate an electrical signal under the action of a specific potential. As the concentration of the analyte increases, the electrical signal becomes weaker.

[0033] (6) The target analyte hapten competes with the photosensitive bead antigen carrier on the conjugate pad for binding to the antibody coated on the test line of the chromatographic membrane. After excitation by light of a certain wavelength, the photosensitive beads will generate reactive oxygen species after being excited. The distance that the reactive oxygen species can transfer is limited and can only react with the compounds susceptible to oxidation within 200 nm. The reaction product of the reactive oxygen species and the compound susceptible to oxidation generates an electrochemical signal, which can generate an electrical signal under the action of a specific potential. As the concentration of the target analyte increases, the generated electrical signal becomes weaker.

[0034] (7) The target analyte can also form a sandwich with the antibody on the conjugate pad, the photosensitive bead antigen, and the antigen coated on the test line of the chromatographic membrane; after excitation by light of a certain wavelength, the photosensitive beads will generate reactive oxygen species after being excited. The distance that the reactive oxygen species can transfer is limited and can only react with the compounds susceptible to oxidation within 200 nm. The reaction product of the reactive oxygen species and the compound susceptible to oxidation generates an electrochemical signal, which can generate an electrical signal under the action of a specific potential. As the concentration of the analyte increases, the electrical signal becomes stronger.

[0035] In a second aspect, the present invention provides a photoexcited lateral flow immunosensor system, comprising:

[0036] A cassette, including an upper cassette and a lower cassette, which are connected in a plug-in manner; a bayonet for fixing the reagent strip is provided on the lower shell, and after the reagent strip is fixed on the lower shell, it is assembled with the upper shell by pressure;

[0037] A sample addition hole, the upper shell is provided with a sample addition hole, the sample addition hole corresponds to the sample cushion layer of the reagent strip, and the test line on the chromatographic membrane is close to the sample addition hole;

[0038] An observation window, the upper shell is also provided with an observation window, and the observation window corresponds to the test line on the chromatographic membrane;

[0039] A quality control line is also provided on the chromatographic membrane of the reagent strip, the interval between the quality control line and the test line is 6 - 8 mm, and the quality control line is far from the sample addition hole; and

[0040] An electrochemical signal analysis unit, and the reagent strip is connected to the electrochemical signal analysis unit through a cable or wirelessly.

[0041] Third aspect, the present invention provides a method for preparing the reagent strip, comprising the following steps:

[0042] STEP 1: Prepare photosensitive microspheres, which contain a photosensitive compound and are conjugated with an antibody or an antigen;

[0043] STEP 2: Prepare a conjugate pad. After diluting the photosensitive microspheres conjugated with an antibody or an antigen in STEP 1 and the photosensitive microspheres conjugated with DNP-BSA, uniformly spray them on the conjugate pad;

[0044] STEP 3: Prepare a chromatographic membrane, on which a test line and a quality control line are provided; an antibody or an antigen is coated on the test line, and an antibody is coated on the quality control line. Use a coating buffer to adjust the concentrations of the antibody or antigen on the test line and the quality control line respectively, and then dry them;

[0045] STEP 4: Prepare a sample pad layer, including:

[0046] STEP 4-1: Prepare a sample pad. After diluting the oxidized compound with a treatment solution, uniformly spray it on the glass fiber membrane, and then dry it; and / or

[0047] STEP 4-2: Prepare a blood filtering membrane. Uniformly spray the treatment solution on the blood filtering membrane in parallel, and then dry it; STEP5: Prepare an absorbent pad. Cut the absorbent paper into a size of 25mm * 300mm;

[0048] STEP 6: Prepare the reagent strip, including:

[0049] STEP 6-1: Connect the sample pad, conjugate pad, chromatographic membrane, and absorbent pad prepared in STEP 2, STEP 3, STEP 4-1, and STEP 5 end to end, and sequentially lap and paste them on the electrode backing;

[0050] STEP 6-2: Connect the blood filtering membrane, conjugate pad, chromatographic membrane, and absorbent pad prepared in STEP 2, STEP 3, STEP 4-2, and STEP 5 end to end, and sequentially lap and paste them on the electrode backing; or

[0051] STEP 6-3: Connect the blood filtering membrane, sample pad, conjugate pad, chromatographic membrane, and absorbent pad prepared in STEP 2, STEP 3, STEP 4-1, STEP 4-2, and STEP 5 end to end, and sequentially lap and paste them on the electrode backing;

[0052] Fourthly, the present invention provides a method for assembling a photoexcited lateral flow immunosensor system. Place the reagent strip prepared above into the lower shell buckle of the sensor system. Align the sample pad of the reagent strip with the sample adding hole of the system, align the detection line on the chromatography membrane with the observation window, and insert the upper shell into the lower shell by pressing.

[0053] Fifthly, the present invention provides a method for detecting a target analyte, comprising the following steps:

[0054] Step 1, using the immunosensor system, add a sample to the sample adding hole of the system, and chromatograph it to the observation window under the action of capillary suction;

[0055] Step 2, insert the system after the reaction in Step 1 into the electrochemical analysis unit, run the program, and test the background electrical signal of the sample;

[0056] Step 3, use light of a specific wavelength to excite the photosensitive compound, and detect the electrical signal after excitation; and

[0057] Step 4, compare the background electrical signal and the electrical signal after excitation, obtain the sample electrical signal information, and determine the presence and / or quantity of the target analyte.

[0058] Sixthly, the present invention provides a use of a photoexcited lateral flow immunosensor system (non-diagnostic). This system is used for quantitatively analyzing the electrochemical signals generated by various disease markers in biological fluids such as blood, saliva, cerebrospinal fluid, or urine.

[0059] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0060] 1) The photoinduced chemical reaction solves the problem of poor specificity in detection by traditional electrochemical methods;

[0061] 2) The use of electrical signals for the backend signal output has great advantages over optical signals in terms of sample types and some specific interferences, and at the same time, the cost of signal acquisition is relatively low.

[0062] 3) The instrument required for electrochemical detection is relatively simple, and product miniaturization can be achieved.

[0063] 4) The lateral flow immunoassay (LFIA) is a fast, portable, and simple technology, usually used for rapid on-site screening, and the reagent components can be stored at room temperature.

[0064] 5) The instrument system for detecting the target analyte of the present invention uses screen-printed electrodes, and the electrode materials include but are not limited to carbon, silver, gold, copper and their combinations, which solves the problems of high operation difficulty and poor reproducibility of traditional three-electrode systems, resulting in difficulty in commercialization. At the same time, the instrument system of the present invention has low cost, and there is no cross-interference in single-use, making it suitable for POCT usage scenarios such as home use. Users can import the detection results into the mobile phone via Bluetooth, USB, TPC, etc., and monitor the significance and fluctuations of the detection results in real time.

[0065] 6) The detection samples of the present invention are not limited to fingertip blood, and urine, saliva, etc. can also be included in the method of the present invention for detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with the drawings and specific embodiments.

[0067] Figure 1A - 1C It is a schematic diagram of the structural composition of the electrode reagent strip, where Figure 1A is the electrode reagent strip with both a blood filtration membrane and a sample pad; Figure 1B is the electrode reagent strip with a sample pad but without a blood filtration membrane; Figure 1C is the electrode reagent strip with a blood filtration membrane but without a sample pad;

[0068] Figure 2 is the schematic diagram of the reagent strip assembly;

[0069] Figure 3 is the reaction principle diagram;

[0070] Figure 4 is the signal generation principle diagram;

[0071] Figure 5 is the concentration correlation diagram, where 20 clinical samples are detected and compared with Roche turbidimetry by this method;

[0072] Among them, 1-1 is the blood filtration membrane, 1-2 is the sample pad, 2 is the conjugate pad, 3 is the chromatographic membrane, 4 is the absorbent pad, 5 is the electrode backing, and 6 is the test line. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0073] The present invention discloses a light-excited lateral flow immunosensor, a detection method and its application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The method and application of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0074] Glossary of Terms

[0075] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. To better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0076] The term "photosensitive compound" refers to a photosensitive substance that can be excited by excitation light of a certain wavelength, such as rose bengal, methylene blue, phthalocyanine complexes, naphthalocyanine complexes, and combinations thereof.

[0077] The term "oxidizable compound" refers to a compound that can undergo a redox reaction with reactive oxygen species to produce a product that can cause an electrochemical signal. The oxidizable compound itself does not produce an electrochemical signal substance, and it can be any one or combination of hydroquinone, resorcinol, dopamine, paracetamol, p-aminophenol, ferrocene, and its derivatives. The forms of oxidizable compounds can be divided into chemical substances, silica microspheres containing oxygenated chemical substances, and polystyrene microspheres containing oxygenated chemical substances.

[0078] The term "antibody" refers to immunoglobulins and immunoglobulin fragments, whether produced naturally or partially or fully synthetically (e.g., recombinantly), including any fragment that retains the binding specificity of the full-length immunoglobulin and at least contains a portion of the variable region of the immunoglobulin molecule. The term antibody includes polyclonal antibodies, monoclonal antibodies, ScFv, or antibody fragments.

[0079] The term "monoclonal antibody" refers to a population of identical antibodies, indicating that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. "Polyclonal antibody" refers to a population of antibodies containing antibodies with multiple different sequences.

[0080] The term "scFv fragment" refers to an antibody formed by connecting the variable region of the heavy chain of an antibody and the variable region of the light chain through a short peptide (linker) linker of 5-20 amino acids. The linker length allows the two variable domains to bridge without substantial interference.

[0081] The "antibody fragment" of an antibody refers to any part of the full-length antibody that is less than the full length but at least contains a portion of the variable region of the antibody that binds to the antigen (e.g., one or more CDRs and / or one or more antibody binding sites), and thus retains the binding specificity and at least part of the specific binding ability of the full-length antibody.

[0082] The term "antigen" refers to a substance that can be an isolated antigen or present in a biological sample, usually a high molecular weight protein, polysaccharide, lipid, or polypeptide, which can be detected in different immunoassay configurations.

[0083] The term "hapten" refers to a compound that can specifically bind to a corresponding antibody but does not itself serve as an immunogen (or antigen) for the preparation of antibodies. Haptens are usually linked to an antigen carrier used to generate antibodies. Haptens can be drugs, monosaccharides, amino acids, small peptides, phospholipids, or triglycerides, etc.

[0084] The terms "antigen carrier" or "immunogenic carrier" can be used interchangeably and refer to such a group or moiety that, when conjugated to a hapten and injected into a mammal or otherwise used as an immunogen, induces an immune response and elicits the production of antibodies that bind the hapten. The molecular weight range (in daltons) of poly(amino acid) as an antigen carrier is, for example, from about 5,000 to about 10,000,000, or from about 20,000 to about 600,000, or from about 25,000 to about 250,000 molecular weight. Poly(amino acid) antigen carriers include proteins such as, for example, albumin, serum proteins such as globulins, crystallins, and lipoproteins. Exemplary proteins include, but are not limited to, for example, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), ovalbumin, and bovine gamma globulin (BGG). Non-poly(amino acid) antigen carriers include polysaccharides, nucleic acids, and particles (biological and synthetic materials).

[0085] The "specific binding" of an antibody or its antigen-binding fragment refers to the ability of the antibody or antigen-binding fragment to form one or more non-covalent bonds with the homologous antigen through non-covalent interactions between the antibody-binding site of the antibody and the antigen. "Competitive binding" refers to the competition between two or more antigens or haptens for binding to an antibody, antigen carrier, or antigen-binding fragment when the amount of the antibody, antigen carrier, or antigen-binding fragment is limited.

[0086] Figures 1 to 5 schematically show embodiments of the present invention, wherein Figure 1A is a schematic diagram of the structural composition of an electrode reagent strip with both a blood filtration membrane and a sample pad present; Figure 1B is a schematic diagram of the structural composition of an electrode reagent strip with a sample pad present and a blood filtration membrane absent; Figure 1C is a schematic diagram of the structural composition of an electrode reagent strip with a blood filtration membrane present and a sample pad absent.

[0087] See Figure 1A , which schematically shows a reagent strip. In one embodiment, the reagent strip includes: a blood filtration membrane 1-1, a sample pad 1-2, a conjugate pad 2, a chromatographic membrane 3, and an absorbent pad 4 connected end to end and sequentially lapped and pasted on the electrode backing 5.

[0088] See Figure 1B , which schematically shows a reagent strip. In one embodiment, the reagent strip includes: a sample pad 1-2, a conjugate pad 2, a chromatographic membrane 3, and an absorbent pad 4 connected end to end and sequentially lapped and pasted on the electrode backing 5.

[0089] See Figure 1C , which schematically shows a reagent strip. In one embodiment, the reagent strip includes: a blood filtration membrane 1-1, a conjugate pad 2, a chromatographic membrane 3, and a water absorption pad 4 connected end to end and sequentially lapped and pasted on the electrode backing 5. Among them, a detection line 6 is also included on the chromatographic membrane 3.

[0090] Figure 3 Schematically shows the reaction principle diagram. Among them, the target analyte contacts the photosensitive spheres on the conjugate pad of the reagent strip and the antibodies or antigens coated on the chromatographic membrane. The photosensitive spheres contain photosensitive substances that can be excited by excitation light of a certain wavelength. The activated photosensitive compounds can generate reactive oxygen species; the sample cushion layer contains compounds susceptible to oxidation, and the compounds susceptible to oxidation react with the reactive oxygen species on the conjugate pad to produce products that can cause electrochemical signals, which can generate electrical signals under the action of a specific potential.

[0091] Figure 4 Shows the signal generation principle diagram, 其中 ， The test target analyte is the reduction peak signal of the product (BQ) after the oxidation of the compound susceptible to oxidation (HQ). The reduction peak of BQ at -0.15V belongs to the reduction peak of benzoquinone (BQ), and the reaction that occurs is BQ + e- → HQ.

[0092] Figure 5 Shows the concentration correlation of detecting 20 clinical samples (this method is compared with Roche turbidimetry).

[0093] In the examples, unless otherwise specified, the experimental methods used are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be purchased from commercial channels.

[0094] Table 1: Key material procurement information

[0095] Material Name Manufacturer Article Number Photosensitive Microspheres Weidu Biologics 67500100 EDC sigma E7750 - 5G NHS sigma H1759 - 100MG BSA sigma SRE0096 CRP Labeled Antibody Boyue Biologics CRP101 CRP Coated Antibody Boyue Biologics CRP101 DNP - BSA Dongkang Biologics A04501 Rabbit Anti - DNP Antibody Shanghai Qiming Biotechnology Co., Ltd. MAB2059 AN3 Absorbent Pad Shanghai Gold Label SX42

[0096] Example 1. Synthesis of GG-Ab1 microspheres

[0097] Add 1 mL of microspheres to a centrifuge tube, add 100 μL of 50 mM MES buffer (pH 5.5), and quickly mix well by shaking with 3.2 μL of EDC.

[0098] Add 14 μL of NHS and quickly mix well by shaking, then discard the supernatant.

[0099] Add 100 μL of buffer, resuspend by ultrasound, and add 500 μg of CRP-specific monoclonal antibody.

[0100] Add 10 μL of Gly and quickly mix well by shaking, add 10 μL of 100 mg / mL BSA, mix well, and centrifuge to discard the supernatant.

[0101] Add 100 μL of buffer solution, sonicate to suspend the microspheres, mix well by shaking, and store at 2 - 8 °C.

[0102] Example 2: Preparation of the conjugate pad

[0103] Dilute GG - Ab1 and GG - DNP - BSA 8 - 30 times with treatment solution 1 (20 mM, pH 8.0 Tris - HCl containing 0.5% Nacl, 0.5% S9, 2% trehalose, 0.1% BSA) and spray a uniform line on glass cellulose, with a dosage of 2 - 4 μl of liquid / cm of the sample pad. Place it in an oven and dry at 37 °C overnight.

[0104] Example 3: Preparation of the chromatographic membrane

[0105] A test line and a quality control line are sequentially arranged on the nitrocellulose membrane. The test line and the quality control line are parallel to each other, with a spacing of 6 - 8 mm. Among them, the test line is close to the sample loading hole, and the quality control line is far from the sample loading hole. The test line is coated with a CRP capture antibody, and the quality control line is coated with a rabbit anti - DNP antibody. The coating concentration of the CRP capture antibody in the test line is 0.5 - 1.5 mg / ml, and the dosage is 0.5 - 1.5 μl of coating solution / cm of the membrane. The coating concentration of the rabbit anti - DNP antibody in the quality control line is 0.3 - 1.0 mg / ml, and the dosage is 0.5 - 1.5 μl of coating solution / cm of the membrane.

[0106] Adjust the concentrations of the CRP capture antibody and the rabbit anti - DNP antibody to 0.5 - 1.5 mg / ml and 0.3 - 1.0 mg / ml respectively with a coating buffer (10 mM PBS buffer containing 3.0% trehalose), with a dosage of 0.5 - 1.5 μl of coating solution / cm of the membrane. Then, draw them parallel as the test line and the quality control line on the nitrocellulose membrane for coating, and place it in an oven and dry at 55 °C overnight.

[0107] Example 4: Preparation of the sample pad

[0108] Dilute the oxidized compound to 10 - 100 mM on glass cellulose with treatment solution 2 (20 mM, pH 8.0 Tris - HCl containing 0.5% Nacl, 0.5% Zncl, 2% trehalose, 0.1% BSA) and spray a uniform line, with a dosage of 2 - 4 μl of liquid / cm of the sample pad. Place it in an oven and dry at 37 °C overnight.

[0109] Example 5: Preparation of the blood filtration membrane

[0110] The treatment solution 1 (20 mM, pH 8.0 Tris-HCl containing 0.5% Nacl, 0.5% S9, 2% trehalose, and 0.1% BSA) was evenly sprayed in parallel on the blood filtration membrane, with a dosage of 2 - 5 μl of liquid per cm of the sample pad. It was placed in an oven and dried overnight at 37°C.

[0111] Example 6. Preparation of the absorbent pad

[0112] The entire AN3 absorbent pad with dimensions of 200 mm * 300 mm was cut into pieces of 25 mm * 300 mm for standby.

[0113] Example 7. Preparation of the test strip

[0114] (1) The blood filtration membrane, sample pad, conjugate pad, chromatographic membrane, and absorbent pad prepared in Examples 2 - 6 were connected end to end and sequentially overlapped and pasted on the electrode backing to obtain the test strip as Figure 1A shown;

[0115] (2) The sample pad, conjugate pad, chromatographic membrane, and absorbent pad prepared in Examples 2 - 5 were connected end to end and sequentially overlapped and pasted on the electrode backing to obtain the test strip as Figure 1B shown;

[0116] (3) The blood filtration membrane, conjugate pad, chromatographic membrane, and absorbent pad prepared in Examples 2, 3, 5, and 6 were connected end to end and sequentially overlapped and pasted on the electrode backing to obtain the test strip as Figure 1C shown;

[0117] As Figure 2 shown, a method for assembling a light-excited lateral flow immunosensor system is provided. The test strip prepared above is placed in the lower shell buckle of the system. The sample pad layer of the test strip is aligned with the sample addition hole of the system, the detection line on the chromatographic membrane is aligned with the observation window, and the upper shell is inserted into the lower shell by pressing.

[0118] Example 8. Assembly of the kit

[0119] The prepared and cut sample pad, glass fiber conjugate pad, nitrocellulose membrane, blood filtration membrane, and absorbent pad were sequentially overlapped and pasted on the electrode backing and cut according to fixed dimensions.

[0120] The reagent card shell includes an upper shell and a lower shell. The upper shell is provided with a sample addition hole and an observation window. The sample addition hole corresponds to the sample pad of the test strip, and the observation window corresponds to the detection line on the nitrocellulose membrane. The upper shell and the lower shell are connected in a plug-in manner. The lower shell is provided with a buckle for fixing the detection reagent. After the test strip is fixed on the lower shell, it is assembled with the upper shell by pressure.

[0121] Example 9. Sample testing

[0122] (1) Sample addition: Add the CRP sample to the sample window, and it will chromatograph to the test line under the action of capillary suction. React at room temperature for 10 min, and the target analyte antigen CRP (Ag), GG-Ab1, and NC-Ab2 form a sandwich (GG-Ab1-CRP-NC-Ab2).

[0123] (2) Background signal test: Insert the reacted reagent card into the portable electrochemical analyzer to run the differential pulse voltammetry program. The program parameters are (starting potential: 0.05 V, ending potential: -0.3 V, potential increment: 0.005 V, amplitude: 0.06 V, pulse width: 0.07 s, sampling width: 0.02 s, pulse period: 0.15 s). The test target is the reduction peak signal of the product (BQ) after the oxidation of the oxidized compound (HQ). The test results are automatically recorded by the instrument.

[0124] (3) Excitation signal test: Use light with a wavelength of 680 nm to excite the electrode surface for 2 min. The photosensitive sphere is excited and energy transfer generates singlet oxygen. The singlet oxygen reacts with the oxidized compound (HQ) within the diffusion range, continuously oxidizing HQ to BQ. At this time, perform the test of the excitation signal in step (2). As Figure 4 shown, the test target analyte is the reduction peak signal of the product (BQ) after the oxidation of the oxidized compound (HQ). The reduction peak of BQ at -0.15 V belongs to the reduction peak of benzoquinone (BQ), and the reaction that occurs is BQ + e- → HQ.

[0125] (4) Subtract the background signal from the excitation signal to obtain the sample electrical signal.

[0126] (5) Performance evaluation.

[0127] Test 20 clinical samples according to the above test protocol, and at the same time use Roche immunoturbidimetry (cobas c 111) to test the above samples synchronously. Plot the test values of Roche immunoturbidimetry (cobas c 111) on the X-axis and the test results of this method on the Y-axis to calculate the correlation.

[0128] Use the test protocol described in Example 9 to test 20 clinical samples, and at the same time use Roche biochemical analyzer (COBAS e 411) to test the above samples synchronously. Plot the test values of Roche biochemical analyzer on the X-axis and the test results of this method on the Y-axis to calculate the correlation. The results are as Figure 5 shown. The test results show that the method of the present invention has good correlation with the test results of Roche biochemical analyzer, and the correlation coefficient R 2 = 0.9736.

[0129] Test the low precision of slightly inflamed and moderately inflamed samples at two medical decision levels. Using this method, test the two samples continuously for 12 times, and calculate the precision CV.

[0130] Table 2 Precision evaluation

[0131]

[0132]

[0133] As shown in the results of Table 2, the precision CV of the kit of the present invention is less than 10% for both tests. The precision of the simple chromatography method in the prior art is generally 15%, indicating that the kit of the present invention has good precision.

[0134] Comparative Example 1

[0135] According to the methodological reagents and conventional time-resolved immunofluorescence method in the above scheme, test 25 clinical samples respectively, and at the same time use Roche immunoturbidimetry (cobas c111) to test the above samples synchronously; compare the sensitivity performance of the two methodologies. The specific data is shown in Table 3:

[0136]

[0137]

[0138] As shown in Table 3, the sensitivity of the conventional immunochromatography method for detecting CRP is about 3 - 5 mg / L, and this methodology can detect up to 0.2 - 0.3 mg / L, with a sensitivity increase of 93.3% - 94.0%.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A light-excited lateral flow immunosensor reagent strip, comprising: An electrode backing, which is disposed at the bottom end of the reagent strip to provide solid support for the reagent strip; A conjugate pad, one end of which is in contact with the electrode backing, and the conjugate pad contains a first structural substance that can specifically bind to a target analyte. The first structural substance is conjugated with a photosensitive compound that can be activated under preset conditions, and the activated photosensitive compound can generate reactive oxygen species; A sample cushion layer, which is located at the front end of the reagent strip, with one end placed on the conjugate pad and the other end placed on the electrode backing. The sample cushion layer contains a second structural substance, and the second structural substance is conjugated with an oxidized compound that can accept reactive oxygen species to generate a preset electrical signal; A chromatographic membrane, which provides solid support for the specific binding of the target analyte and guides the capillary flow of the target analyte along the direction of the reagent strip; An absorbent pad, which is located at the end of the reagent strip, with one end placed on the chromatographic membrane and the other end in contact with the electrode backing, and adsorbs excess reagent to maintain lateral flow along the reagent strip.

2. The reagent strip according to claim 1, wherein the sample cushion layer comprises at least one of a sample pad and a blood filtration membrane placed on the electrode backing; (1) When the target analyte is from a whole blood sample, the sample cushion layer of the reagent strip comprises a blood filtration membrane and a sample pad. The blood filtration membrane and the sample pad are sequentially pasted on the electrode backing from left to right, and one end of the blood filtration membrane is placed above the sample pad, and the whole blood sample is filtered and separated; the second structural substance is contained in the sample pad; The blood filtration membrane, sample pad, conjugate pad, chromatographic membrane, and absorbent pad of the reagent strip are connected end to end and sequentially lapped and pasted on the electrode backing; or (2) When the target analyte is from a whole blood sample, the sample cushion layer of the reagent strip only comprises a blood filtration membrane. The blood filtration membrane and the conjugate pad are sequentially pasted on the electrode backing from left to right, and one end of the blood filtration membrane is placed above the conjugate pad, and the whole blood sample is filtered and separated; the second structural substance is contained in the blood filtration membrane; The blood filtration membrane, conjugate pad, chromatographic membrane, and absorbent pad of the reagent strip are connected end to end and sequentially lapped and pasted on the electrode backing; (3) When the target analyte is not a whole blood sample, the sample cushion layer of the reagent strip only comprises a sample pad. The sample pad and the conjugate pad are sequentially pasted on the electrode backing from left to right, and one end of the sample pad is placed above the conjugate pad; the second structural substance is contained in the sample pad; The sample pad, conjugate pad, chromatographic membrane, and absorbent pad of the reagent strip are connected end to end and sequentially lapped and pasted on the electrode backing.

3. The reagent strip according to claim 1, wherein The conjugate pad is made of cross-linked silica, glass fiber, polyester, or rayon.

4. The reagent strip according to claim 1, characterized in that, The chromatographic membrane is selected from a nitrocellulose or cellulose acetate membrane.

5. The reagent strip according to claim 1, wherein The detection line of the chromatographic membrane is coated with a third structural substance, and the third structural substance is one of an antibody or an antigen.

6. The reagent strip according to claim 1, characterized in that, The target analyte is an antigen or a hapten; The target analyte specifically binds to at least one of the first structural substance on the binding pad and the third structural substance on the chromatographic membrane; Wherein, when the first structural substance and the third structural substance contact the target analyte, the activated photosensitive compound transfers the reactive oxygen species to the oxidized compound of the second structural substance on the sample cushion layer, and the oxidized compound generates an electrical signal, and the information of the electrical signal is used to characterize the presence and / or quantity of the target analyte.

7. The reagent strip according to claim 1, wherein The photosensitive compound is rose bengal, methylene blue, phthalocyanine complex, naphthalocyanine complex or a combination thereof.

8. The reagent strip according to claim 1, characterized in that, The oxidized compound is hydroquinone, resorcinol, dopamine, paracetamol, p-aminophenol, ferrocene and its derivatives or a combination thereof.

9. The reagent strip according to claim 1, wherein, The target analyte is an antigen, and the first structural substance on the binding pad and the third structural substance on the chromatographic membrane are compositions containing polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments, and the polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments immunize the antigen; When the target analyte contacts the first structural substance on the binding pad and the third structural substance on the chromatographic membrane, the photosensitive compound is excited by light of a certain wavelength to react with the oxidized compound on the second structural substance on the sample cushion layer, generating an electrochemical signal.

10. The test strip according to claim 9, wherein the intensity of the electrochemical signal is positively correlated with the quantity of the target analyte.

11. The reagent strip according to claim 1, wherein, The target analyte is an antigen, the third structural substance on the chromatographic membrane is an antigen, and the first structural substance on the binding pad is a composition containing polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments, and the polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments immunize the antigen; When the target analyte contacts the first structural substance on the binding pad and the third structural substance on the chromatographic membrane, the target analyte competitively binds to the first structural substance on the binding pad with the third structural substance on the chromatographic membrane; The photosensitive compound is excited by light of a certain wavelength to react with the oxidized compound on the second structural substance on the sample cushion layer, generating an electrochemical signal.

12. The reagent strip according to claim 1, wherein, The target analyte is an antigen, the first structural substance on the binding pad is an antigen, and the third structural substance on the chromatographic membrane is a composition containing polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments, and the polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments immunize the antigen; When the target analyte contacts the first structural substance on the binding pad and the third structural substance on the chromatographic membrane, the target analyte and the first structural substance on the chromatographic membrane on the binding pad of the chromatographic membrane; The photosensitive compound is excited by light of a certain wavelength to react with the oxidized compound on the second structural substance on the sample cushion layer, generating an electrochemical signal.

13. The test strip according to claim 11 or 12, wherein the intensity of the electrochemical signal is negatively correlated with the quantity of the target analyte.

14. The reagent strip according to claim 1, wherein, The target analyte is a hapten, the third structural substance on the chromatographic membrane is an antigen, the first structural substance on the conjugate pad is a composition comprising a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen; When the target analyte contacts the first structural substance on the conjugate pad and the third structural substance on the chromatographic membrane, the target analyte competes with the third structural substance on the chromatographic membrane for binding to the first structural substance on the conjugate pad; The photosensitive compound is excited by light of a certain wavelength to react with the oxidized compound on the second structural substance on the sample pad, generating an electrochemical signal.

15. The reagent strip according to claim 1, wherein The target analyte is a hapten, the first structural substance on the conjugate pad is an antigen, the third structural substance on the chromatographic membrane is a composition comprising a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen; When the target analyte contacts the first structural substance on the conjugate pad and the third structural substance on the chromatographic membrane, the target analyte competes with the first structural substance on the conjugate pad for binding to the third structural substance on the chromatographic membrane; the photosensitive compound is excited by light of a certain wavelength to react with the oxidized compound on the second structural substance on the sample pad, generating an electrochemical signal.

16. The test strip according to any one of claims 14-15, wherein the intensity of the electrochemical signal is negatively correlated with the quantity of the target analyte.

17. The reagent strip according to claim 1, wherein, The target analyte is a hapten, the first structural substance on the conjugate pad and the third structural substance on the chromatographic membrane are compositions comprising a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the hapten; When the target analyte contacts the first structural substance on the conjugate pad and the third structural substance on the chromatographic membrane, the photosensitive compound is excited by light of a certain wavelength to react with the oxidized compound on the second structural substance on the sample pad, generating an electrochemical signal.

18. The test strip according to claim 17, wherein the intensity of the electrochemical signal is positively correlated with the quantity of the target analyte.

19. The reagent strip according to claim 1, characterized in that, The target analyte may also be an antibody, the first structural substance on the conjugate pad and the third structural substance on the chromatographic membrane are antigens; When the target analyte contacts the first structural substance on the conjugate pad and the third structural substance on the chromatographic membrane, the photosensitive compound is excited by light of a certain wavelength to react with the oxidized compound on the second structural substance on the sample pad, generating an electrochemical signal.

20. The test strip according to claim 19, wherein the intensity of the electrochemical signal is positively correlated with the quantity of the target analyte.

21. A photoexcited lateral flow immunosensor system, comprising: The cartridge includes an upper cartridge and a lower cartridge, and the upper shell and the lower shell are connected by plug-in; wherein, a bayonet for fixing the reagent strip is provided on the lower shell, and after the lower shell fixes the reagent strip, it is assembled with the upper shell by pressure; The sample addition hole, the upper shell is provided with a sample addition hole, the sample addition hole corresponds to the sample pad of the reagent strip described in claim 1, and the detection line on the chromatography membrane is close to the sample addition hole; The observation window, the upper shell is further provided with an observation window, and the observation window corresponds to the detection line on the chromatography membrane described in claim 1; The reagent strip is the reagent strip described in any one of claims 1-20, and a quality control line is further provided on the chromatography membrane of the reagent strip. The interval between the quality control line and the detection line is 6-8 mm, and the quality control line is far from the sample addition hole; and The electrochemical signal analysis unit, the reagent strip is connected to the electrochemical signal analysis unit through a cable or wirelessly.

22. A method for preparing the reagent strip described in any one of claims 1-20, which includes the following steps: STEP 1: Prepare photosensitive microspheres, the photosensitive microspheres contain a photosensitive compound and are conjugated with an antibody or an antigen; STEP 2: Prepare the conjugate pad. After diluting the photosensitive microspheres conjugated with the antibody or antigen in STEP 1 and the photosensitive microspheres conjugated with DNP-BSA, spray them evenly on the conjugate pad; STEP 3: Prepare the chromatography membrane, and a detection line and a quality control line are provided on the chromatography membrane; the detection line is coated with an antibody or an antigen, and the quality control line is coated with an antibody. Use the coating buffer to adjust the concentrations of the antibody or antigen on the detection line and the quality control line respectively, and dry them; STEP 4: Prepare the sample pad layer, including: STEP 4-1: Prepare the sample pad. After diluting the oxidized compound with a treatment solution, spray it evenly on the sample pad and dry it; and / or STEP 4-2: Prepare the blood filtration membrane. Spray the treatment solution evenly and parallelly on the blood filtration membrane and dry it; STEP 5: Prepare the absorbent pad. Cut the absorbent paper into a size of 25mm * 300mm; STEP 6: Prepare the reagent strip, including: STEP 6-1: Connect the sample pad, conjugate pad, chromatography membrane, and absorbent pad prepared in STEP 2, STEP 3, STEP 4-1, and STEP 5 end to end, and lap and paste them on the electrode backing in sequence; or STEP 6-2: Connect the blood filtration membrane, conjugate pad, chromatography membrane, and absorbent pad prepared in STEP 2, STEP 3, STEP 4-2, and STEP 5 end to end, and lap and paste them on the electrode backing in sequence; or STEP 6-3: Connect the blood filtration membrane, sample pad, conjugate pad, chromatography membrane, and absorbent pad prepared in STEP 2, STEP 3, STEP 4-1, STEP 4-2, and STEP 5 end to end, and lap and paste them on the electrode backing in sequence.

23. A method for assembling a photoexcited lateral flow immunosensor system, comprising placing the reagent strip prepared by the method according to claim 22 into the lower shell buckle of the lateral flow immunosensor system according to claim 21, aligning the sample pad of the reagent strip with the sample addition hole of the system, aligning the detection line on the chromatography membrane with the observation window, and inserting the upper shell into the lower shell by pressing.

24. A method for detecting a target analyte, comprising: Step 1, using the immunosensor system according to claim 21, adding a sample to the sample addition hole of the system, and chromatographing to the observation window under capillary suction; Step 2, inserting the system after the reaction in Step 1 into the electrochemical analysis unit, running a program, and testing the background electrical signal of the sample; Step 3, exciting the photosensitive compound with light of a specific wavelength and detecting the electrical signal after excitation; and Step 4, comparing the background electrical signal and the electrical signal after excitation, obtaining sample electrical signal information, and determining the presence and / or quantity of the target analyte.

25. Use (non-diagnostic) of the photoactivated lateral flow immunosensor system according to claim 21, characterized in that This system is used for quantitatively analyzing the electrochemical signals generated by various disease markers in biological body fluids such as blood, saliva, cerebrospinal fluid or urine.

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