A photoexcited lateral flow immunosensor, detection method and its application

Abstract

The present invention provides a light-activated lateral flow immunosensor reagent strip, a detection and analysis method, and its application. The reagent strip comprises an electrode substrate, a conjugate pad, a sample backing layer, a chromatographic membrane, and a water-absorbing pad. The sample backing layer, conjugate pad, chromatographic membrane, and water-absorbing pad are connected end to end and sequentially overlapped and affixed to the electrode substrate. The light-activated lateral flow immunosensor reagent strip is used for the quantitative analysis of various disease markers in biological fluids such as blood, saliva, cerebrospinal fluid, or urine.

Description

Technical Field

[0001] This invention belongs to the field of medical diagnostics, specifically relating to a photoexcited lateral flow immunosensor, a detection method, and its applications. Background Technology

[0002] Immunoassay technology has a wide range of applications in the field of medical diagnostics. Commonly used immunoassay methods include magnetic particle chemiluminescence, electrochemiluminescence, and homogeneous chemiluminescence. Magnetic particle chemiluminescence is an emerging 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. Electrochemiluminescence involves an electrochemical reaction process that can be continuously cycled, amplifying the detection signal and thus improving detection sensitivity. Magnetic particle chemiluminescence and electrochemiluminescence instruments require a liquid system, reagent refrigeration area, cleaning solution, etc., resulting in a relatively large size. Homogeneous chemiluminescence does not involve cleaning. In terms of detection principle, it can emit light as long as the distance between the photosensitive sphere and the luminescent sphere in the reagent component is <200nm. Photons are received through a photomultiplier tube (PMT). The strict design requirements of the optical module prevent the instrument from being miniaturized, resulting in relatively high instrument costs. Furthermore, this technology has background signal problems, such as (1) non-specific binding between two spheres; (2) Brownian motion between spheres causing them to approach each other. Due to the testing method, whole blood needs to be separated before testing to ensure accuracy.

[0003] Furthermore, the increased risk of cross-infection among patients with mild symptoms or those requiring long-term monitoring increases the likelihood of hospital visits. The methods mentioned above are only suitable for hospital settings and are difficult to implement at home. To address issues such as ease of home use, whole blood testing capabilities, and reduced background signal, the development of a simple, mobile-enabled, small instrument and accompanying testing reagents is urgently needed.

[0004] Electrochemical immunoassay (EIA) is based on immune recognition, achieving specific detection of specific substances through the immune recognition reaction between antigens and antibodies. The output is an electrical signal. It combines the high specificity of immunoassay with the advantages of electrochemical detection, such as low cost, high sensitivity, cost-effectiveness, and small instrument size. Compared to traditional immunoassay which uses photons as the signal output, electrochemical detection collects signals such as current and potential, eliminating the need for complex optical systems and light-gathering modules. Therefore, the instrument cost is relatively low. Sensors can use screen-printed electrodes, resulting in low-cost mass production, good repeatability, and no maintenance. Furthermore, because electrochemical detection instruments do not require complex structures and optical components, they can be miniaturized and portable. Since they detect electrical signals, whole blood samples can be used, making them more suitable for point-of-care testing (POCT) scenarios.

[0005] Electrochemical detection utilizes various techniques to acquire and analyze the electrical signals of samples. Commonly used techniques include cyclic voltammetry (CV), linear sweep voltammetry (LSV), differential pulse voltammetry (DPV), square wave voltammetry (SWV), time-current method (IT), and electrochemical impedance spectroscopy (EIS). Among these, DPV is a frequently used electrochemical analytical technique with higher detection sensitivity than conventional techniques, enabling the detection of trace substances. The principle of DPV is to superimpose a series of pulse signals onto a linear sweep, and then calculate the difference between the signals before and after each pulse to eliminate capacitive current. By continuously measuring the difference during the potential change during the sweep, a differential pulse curve can be obtained by plotting the potential versus the difference. Different substances have different electrochemical redox potentials, thus allowing for the acquisition of specific current peak curves (containing peak potential and peak current information) at specific locations. The peak potential can be used for qualitative analysis of the analyte, while the peak current height is related to the concentration of the analyte and can be used for quantitative analysis. Meanwhile, during signal acquisition, DPV only acquires signals at the end of each pulse cycle. The current generated by the oxidation-reduction of substances is affected differently from the background current by the pulse. The decay rate of the background current is much greater than that of the current generated by the oxidation-reduction of substances. Therefore, DPV can effectively reduce the background signal by acquiring signals at the end of the pulse cycle, thus having better detection sensitivity.

[0006] Lateral flow immunoassay (LFIA) is a rapid, portable, and simple technique that requires virtually no infrastructure and is typically used for rapid on-site screening. This method detects samples by allowing them to flow laterally across a membrane containing antibodies, with the signal appearing as a marker.

[0007] Photochemistry involves irradiating acceptor microspheres with excitation light of a specific wavelength to produce singlet oxygen. 1 O2), 1 O2 can only transfer energy to reducing chemicals to generate an electrochemical signal when an immune response (specific binding of antigen and antibody) occurs. This invention can effectively eliminate non-specific binding that does not occur in the immune response, thereby improving the sensitivity and detection specificity of the reagent.

[0008] The combination of electrochemical, lateral flow immunoassay, and photochemical methods integrates the high sensitivity and good signal stability of electrochemical analysis, the convenience and low cost of lateral flow immunoassay, and the high sensitivity and detection specificity of photochemical methods to meet the needs of more demanding application scenarios. Summary of the Invention

[0009] The purpose of this invention is to provide a photoexcited lateral flow electrochemical immunosensor that outputs an electrochemical signal, thereby solving the problems of high cost and inconvenience caused by complex sensors in the prior art.

[0010] Compared with currently available lateral flow electrochemical assays, this invention proposes a novel method for determining target substances. It introduces a substance that can react with reactive oxygen species to generate an electrical signal detectable by electrodes, effectively distinguishing immune binding from other background interferences and improving sensitivity. Users can collect finger-prick blood at home, add it to the sample well, and allow it to be chromatographically deposited onto the detection line of the chromatographic membrane via capillary action.

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

[0012] In a first aspect, the present invention provides a photoexcited lateral flow immunosensor strip comprising an electrode substrate, a conjugate pad, a sample pad layer, a chromatography membrane, and an absorbent pad.

[0013] The electrode substrate is located at the bottom of the reagent strip to provide solid support for the reagent strip;

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

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

[0016] The sample pad is located at the foremost end of the reagent strip, with one end placed on the conjugate pad and the other end placed on the electrode substrate. The sample pad contains a second structural material, which is coupled with an oxygen-receiving compound that can accept reactive oxygen species to generate a preset electrical signal.

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

[0018] Furthermore, the chromatography membrane is selected from nitrocellulose or cellulose acetate membranes;

[0019] The absorbent pad is located at the very end of the reagent strip, with one end placed on the chromatography membrane and the other end in contact with the electrode substrate. It adsorbs excess reagent and maintains lateral flow along the reagent strip.

[0020] Furthermore, the sample pad layer includes at least one of a sample pad and a blood filtration membrane placed on the electrode substrate;

[0021] (1) When the target analyte comes from a whole blood sample, the sample pad layer of the reagent strip includes a blood filtration membrane and a sample pad. The blood filtration membrane and the sample pad are attached to the electrode substrate from left to right, with one end of the blood filtration membrane placed above the sample pad, and the whole blood sample is filtered and separated; the second structural material is contained on the sample pad.

[0022] The blood filtration membrane, sample pad, conjugate pad, chromatography membrane, and absorbent pad of the reagent strip are connected end to end and sequentially overlapped and adhered to the electrode substrate; or

[0023] (2) When the target analyte comes from a whole blood sample, the sample pad of the reagent strip contains only a blood filtration membrane. The blood filtration membrane and the conjugate pad are attached to the electrode substrate from left to right, with one end of the blood filtration membrane placed above the conjugate pad, and the whole blood sample is filtered and separated. The second structural material is contained on the blood filtration membrane.

[0024] The blood filtration membrane, conjugation pad, chromatography membrane, and absorbent pad of the reagent strip are connected end to end and sequentially overlapped and pasted onto the electrode substrate;

[0025] (3) When the target analyte is not a whole blood sample, the sample pad layer of the reagent strip contains only the sample pad, and the sample pad and the conjugate pad are pasted on the electrode substrate from left to right with one end of the sample pad placed above the conjugate pad; the second structural material is contained on the sample pad;

[0026] The sample pad, conjugation pad, chromatography membrane, and absorbent pad of the reagent strip are connected end to end and sequentially overlapped and pasted onto the electrode substrate.

[0027] When the target analyte in the sample is an antigen or hapten, detection is performed using either a double antibody or a competitively binding single antibody. The electrode surface is untreated. The detection reagent consists of a photosensitive sphere binding pad, an oxygen-receiving compound sample pad layer, and a chromatography membrane coated with antibodies or antigens. The photosensitive sphere contains a photosensitive substance that can be excited by excitation light of a specific wavelength, such as rose bengal, methylene blue, phthalocyanine complexes, naphthol complexes, and combinations thereof. The oxygen-receiving compound does not produce electrochemical signals but can react with reactive oxygen species to generate products that induce electrochemical signals, such as hydroquinone, resorcinol, dopamine, acetaminophen, para-aminophenol, ferrocene, and their derivatives or combinations. Both the photosensitive sphere and the oxygen-receiving compound contain active groups such as amino, carboxyl, and aldehyde groups that can react with proteins. The surface of the photosensitive sphere may be coated with antibodies, antigen carriers, or avidin.

[0028] (1) The target analyte antigen, the antibody labeled on the surface of the photosensitive sphere treated on the binding pad, and the antibody coated on the detection line of the chromatography membrane form a sandwich. After excitation by light of a certain wavelength, the photosensitive sphere generates reactive oxygen species (ROS). The ROS have a limited range of propagation and can only react with oxidized compounds within 200 nm. The reaction products of the ROS and oxidized compounds generate an electrochemical signal. An electrical signal can be generated under a specific potential, and the signal becomes stronger as the analyte concentration increases. This specific potential can be controlled using different electrochemical techniques, such as CV, IT, DPV, SWV, and combinations thereof. The measured signal includes, but is not limited to, current signals, peak current signals, potential signals, and peak potential signals.

[0029] (2) The target analyte antigen competes with the antigen coated on the detection line of the chromatography membrane for binding to the antibody labeled on the surface of the photosensitive sphere treated on the pad. After excitation by light of a certain wavelength, the photosensitive sphere generates reactive oxygen species (ROS). The ROS have a limited range of transmission and can only react with oxidized compounds within 200 nm. The reaction products of the ROS and oxidized compounds generate an electrochemical signal, which can be produced under a specific potential. The generated electrical signal becomes weaker as the concentration of the target analyte increases.

[0030] (3) The target analyte antigen competes with the antigen labeled on the surface of the photosensitive sphere treated on the binding pad for the antibody coated on the detection line of the chromatographic membrane. After excitation by light of a certain wavelength, the photosensitive sphere generates reactive oxygen species (ROS). The ROS have a limited range of transmission and can only react with oxidized compounds within 200 nm. The reaction products of the ROS and oxidized compounds generate an electrochemical signal, which can be produced under a specific potential. The generated electrical signal becomes weaker as the concentration of the target analyte increases.

[0031] (4) The target analyte hapten forms a sandwich with the antibody labeled on the surface of the photosensitive sphere treated on the binding pad and the antibody coated on the detection line of the chromatography membrane. After excitation by light of a certain wavelength, the photosensitive sphere generates reactive oxygen species (ROS). The ROS have a limited range of transmission and can only react with oxidized compounds within 200 nm. The reaction products of the ROS and oxidized compounds generate an electrochemical signal, which can produce an electrical signal under a specific potential. The stronger the electrical signal, the higher the concentration of the analyte.

[0032] (5) The target analyte hapten competes with the antigen coated on the detection line of the chromatography membrane for binding to the antibody carrier labeled on the surface of the photosensitive sphere treated on the pad. After excitation by light of a certain wavelength, the photosensitive sphere generates reactive oxygen species (ROS). The ROS have a limited range of transmission and can only react with oxidized compounds within 200 nm. The reaction products of the ROS and oxidized compounds generate an electrochemical signal, which can be produced under a specific potential. The electrical signal becomes weaker as the analyte concentration increases.

[0033] (6) The hapten of the target analyte competes with the antigen carrier on the photosensitive sphere of the binding pad for binding to the antibody coated on the detection line of the chromatographic membrane. After excitation by light of a certain wavelength, the photosensitive sphere generates reactive oxygen species (ROS). The ROS have a limited range of propagation and can only react with oxidized compounds within 200 nm. The reaction products of the ROS and oxidized compounds generate an electrochemical signal, which can be produced under a specific potential. The generated electrical signal becomes weaker as the concentration of the target analyte increases.

[0034] (7) The target analyte can also form a sandwich between the antibody, the antigen on the photosensitive sphere of the binding pad, and the antigen coated on the detection line of the chromatography membrane. After excitation by light of a certain wavelength, the photosensitive sphere will generate reactive oxygen species. The reactive oxygen species have a limited transmission distance and can only react with oxygenated compounds within 200 nm. The reaction products of the reactive oxygen species and oxygenated compounds generate an electrochemical signal, which can generate an electrical signal under a specific potential. The electrical signal becomes stronger as the analyte concentration increases.

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

[0036] The cartridge includes an upper cartridge and a lower cartridge, which are connected by an interlocking mechanism. The lower cartridge has a slot for fixing a reagent strip. After the reagent strip is fixed in the lower cartridge, it is assembled with the upper cartridge by pressure.

[0037] The upper shell is provided with a sample dispensing port, which corresponds to the sample pad layer of the reagent strip, and the detection line on the chromatography membrane is close to the sample dispensing port;

[0038] An observation window is provided on the upper shell, and the observation window corresponds to the detection line on the chromatography membrane;

[0039] The chromatographic membrane of the reagent strip is also provided with a control line, the control line being 6-8 mm apart from the detection line, and the control line being located away from the sample well; and

[0040] An electrochemical signal analysis unit, wherein the reagent strip is connected to the electrochemical signal analysis unit via cable or wirelessly.

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

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

[0043] STEP 2: Prepare the conjugate pad. Dilute the photosensitive microspheres of the antibody or antigen conjugated in STEP 1 with the photosensitive microspheres conjugated with DNP-BSA, and then spray them evenly onto the conjugate pad.

[0044] STEP 3: Prepare a chromatography membrane with a detection line and a control line. The detection line is coated with an antibody or antigen, and the control line is coated with an antibody. Adjust the concentration of the antibody or antigen on the detection line and the control line with a coating buffer, and then dry them.

[0045] STEP 4: Prepare the sample pad, including:

[0046] STEP 4-1: Prepare the sample pad by diluting the oxygenated compound with the treatment solution, uniformly spraying it onto the glass fiber membrane, and drying it; and / or

[0047] STEP 4-2: Prepare the blood filtration membrane by spraying the treatment solution evenly and parallel on the blood filtration membrane and drying it; STEP 5: Prepare the absorbent pad by cutting the absorbent paper into 25mm*300mm sizes;

[0048] STEP 6: Prepare the test strips, including:

[0049] STEP 6-1: Connect the sample pad, binding pad, chromatography membrane, and absorbent pad prepared in STEP 2, STEP 3, STEP 4-1, and STEP 5 end to end and attach them sequentially to the electrode substrate;

[0050] STEP 6-2: Connect the 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 sequentially overlap and adhere them to the electrode substrate; or

[0051] 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 attach them sequentially to the electrode substrate;

[0052] Fourthly, the present invention provides a method for assembling a photoexcited lateral flow immunosensor system, wherein the reagent strip prepared above is placed in the lower shell snap-fit ​​of the sensor system, the sample pad layer of the reagent strip is aligned with the sample dispensing port of the system, the detection line on the chromatography membrane is aligned with the observation window, and the upper shell is inserted 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 the sample to the sample well of the system and chromatography it to the observation window under capillary action;

[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: Excite the photosensitive compound using light of a specific wavelength and detect the electrical signal after excitation; and

[0057] Step 4: Compare the background electrical signal and the excited electrical signal to obtain sample electrical signal information and determine the presence and / or quantity of the target analyte.

[0058] In a sixth aspect, the present invention provides the use of a photoexcited lateral flow immunosensor system (non-diagnostic) for quantitatively analyzing 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) Photo-induced chemical reaction solves the problem of poor specificity in traditional electrochemical detection methods;

[0061] 2) The back-end signal output uses electrical signals, which have significant advantages over optical signals in terms of sample types and some specific interferences, while the cost of signal acquisition is also lower.

[0062] 3) The instruments required for electrochemical detection are relatively simple, which allows for product miniaturization.

[0063] 4) Lateral flow immunoassay (LFIA) is a rapid, portable and simple technique that is typically used for rapid on-site screening and whose reagent components can be stored at room temperature.

[0064] 5) The instrument system for detecting target analytes in this invention employs screen-printed electrodes. Electrode materials include, but are not limited to, carbon, silver, gold, copper, and combinations thereof. This solves the problems of high operational difficulty and poor reproducibility associated with traditional three-electrode systems, which hinder commercialization. Furthermore, the instrument system of this invention is low-cost, disposable, and free from cross-interference, making it suitable for home and other POCT applications. Users can import test results to their mobile phones via Bluetooth, USB, TPC, etc., to monitor the significance and fluctuations of the test results in real time.

[0065] 6) The test samples of the present invention are not limited to fingertip blood; urine, saliva, etc. can also be included in the method of the present invention for testing. Attached Figure Description

[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 accompanying drawings and specific embodiments.

[0067] Figures 1A-1C This is a schematic diagram of the structure of the electrode reagent strip, in which... Figure 1A Electrode reagent strips that simultaneously contain a blood filtration membrane and a sample pad; Figure 1B Electrode reagent strips with a sample pad present but no blood filtration membrane present; Figure 1C Electrode reagent strips with the blood filtration membrane present but the sample pad absent;

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

[0069] Figure 3 This is a schematic diagram of the reaction principle;

[0070] Figure 4 This is a schematic diagram of the signal generation principle.

[0071] Figure 5 The concentration correlation plot shows a comparison between this method and Roche turbidimetric assay in 20 clinical samples.

[0072] Wherein 1-1 is the blood filtration membrane, 1-2 is the sample pad, 2 is the conjugation pad, 3 is the chromatography membrane, 4 is the absorbent pad, 5 is the electrode substrate, and 6 is the detection line. Detailed Implementation

[0073] This invention discloses a photoexcited lateral flow immunosensor, a detection method, and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0074] Terminology Explanation

[0075] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. To better understand this invention, 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, naphtholine complexes, and combinations thereof.

[0077] The term "oxygenated compound" refers to a product that can undergo a redox reaction with reactive oxygen species to produce an electrochemical signal. Oxygenated compounds themselves do not produce electrochemical signals; they can be any one or a combination of hydroquinone, resorcinol, dopamine, acetaminophen, para-aminophenol, ferrocene, and their derivatives. Oxygenated compounds can be categorized 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 naturally occurring or partially or wholly synthetic (e.g., recombinant), including any fragment that retains the binding specificity of the full-length immunoglobulin molecule, containing at least 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 group of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody group is identical to the other antibody molecules. "Polyclonal antibody" refers to a group of antibodies containing multiple different sequences.

[0080] The term "scFv fragment" refers to an antibody composed of variable regions of the antibody heavy chain and light chain linked by a short peptide linker of 5–20 amino acids. The linker length allows the two variable domains to be bridged with minimal interference.

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

[0082] The term "antigen" refers to either isolated antigens or antigens present in biological samples, typically high-molecular-weight proteins, polysaccharides, lipids, and peptides that can be detected in various immunoassay configurations.

[0083] The term "hapten" refers to a compound that can specifically bind to a corresponding antibody but does not itself act as an immunogen (or antigen) for antibody production. Haptens are typically 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" are used interchangeably to refer to a group or portion that, when conjugated to a hapten and injected into a mammal or otherwise used as an immunogen, induces an immune response and triggers the production of antibodies that bind to the hapten. Poly(amino acid) antigen carriers have molecular weights (in Daltons) ranging, 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. Poly(amino acid) antigen carriers include proteins such as, for example, albumins, serum proteins such as globulins, lens proteins, and lipoproteins. Illustrative proteins include, but are not limited to, bovine serum albumin (BSA), keyhole cyanin (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 term "specific binding" for an antibody or its antigen-binding fragment refers to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antigen's antibody-binding site. "Competitive binding" refers to the competitive binding of two or more antigens or haptens to an antibody, antigen carrier, or antigen-binding fragment in a limited number of samples.

[0086] Figures 1-5 schematically illustrate embodiments of the present invention, wherein, Figure 1A A schematic diagram of the electrode reagent strip structure where both a blood filtration membrane and a sample pad are present; Figure 1B This is a schematic diagram of the electrode reagent strip structure with the sample pad present and the blood filtration membrane absent. Figure 1C This is a schematic diagram of the electrode reagent strip structure with the blood filtration membrane present and the sample pad absent.

[0087] See Figure 1A The figure schematically illustrates a reagent strip. In one embodiment, the reagent strip includes: a blood filtration membrane 1-1, a sample pad 1-2, a conjugation pad 2, a chromatography membrane 3, and an absorbent pad 4 connected end to end and sequentially overlapped and pasted onto the electrode substrate 5.

[0088] See Figure 1B The figure schematically illustrates a reagent strip. In one embodiment, the reagent strip includes: sample pads 1-2, conjugation pads 2, chromatography membranes 3, and absorbent pads 4 connected end to end and sequentially overlapped and adhered to the electrode substrate 5.

[0089] See Figure 1C The figure schematically illustrates a reagent strip. In one embodiment, the reagent strip includes: a blood filtration membrane 1-1, a conjugate pad 2, a chromatography membrane 3, and an absorbent pad 4 connected end-to-end and sequentially overlapped and adhered to the electrode substrate 5. The chromatography membrane 3 also includes a detection line 6.

[0090] Figure 3 The schematic diagram illustrates the reaction principle, in which the target analyte contacts the photosensitive sphere on the binding pad of the reagent strip and the antibody or antigen coated on the chromatography membrane. The photosensitive sphere contains a photosensitive substance that can be excited by excitation light of a certain wavelength. The activated photosensitive compound can generate reactive oxygen species. The sample pad contains oxygenated compounds, which undergo redox reactions with the reactive oxygen species on the binding pad to produce products that can induce electrochemical signals. Under the action of a specific potential, an electrical signal can be generated.

[0091] Figure 4 The diagram shows the principle of signal generation. 其中 ， The target analyte being tested is the reduction peak signal of the product (BQ) after oxidation by an oxygen compound (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 The concentration correlation of 20 clinical samples is shown (compared to Roche turbidimetric assay).

[0093] Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used can be purchased commercially unless otherwise specified.

[0094] Table 1: Key Material Procurement Information

[0095] Material Name factory Item number Photosensitive microspheres For the sake of life 67500100 EDC sigma E7750-5G NHS sigma H1759-100MG BSA sigma SRE0096 CRP-labeled antibody Boyue Biotechnology CRP101 CRP-coated antibody Boyue Biotechnology CRP101 DNP-BSA Dongkang Bio A04501 Rabbit anti-DNP antibody Shanghai Qiming Biotechnology Co., Ltd. MAB2059 AN3 absorbent pad Shanghai Gold Standard SX42

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

[0097] Add 1 mL of microspheres, 100 μL of 50 mM MES buffer (pH 5.5), and 3.2 μL of EDC to a centrifuge tube and shake rapidly to mix.

[0098] Add 14 μL of NHS, shake quickly to mix, and discard the supernatant.

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

[0100] Add 10 μL of Gly and shake rapidly to mix. Add 10 μL of 100 mg / mL BSA, mix well, centrifuge and discard the supernatant.

[0101] Add 100 μL of buffer solution, sonicate to suspend the microspheres, shake to mix, and store at 2-8℃.

[0102] Example 2: Preparation of the conjugate pad

[0103] GG-Ab1 and GG-DNP-BSA were diluted 8–30 times with treatment solution 1 (20 mM Tris-HCl containing 0.5% NaCl, 0.5% S9, 2% trehalose, and 0.1% BSA, pH 8.0) and evenly sprayed in a line onto glass cellulose, using a volume of 2–4 μl / cm sample pad. The sample was then placed in an oven and dried overnight at 37°C.

[0104] Example 3: Preparation of Chromatographic Membranes

[0105] A test line and a control line are sequentially arranged on a nitrocellulose membrane, parallel to each other and spaced 6–8 mm apart. The test line is closer to the sample well, and the control line is further away. The test line is coated with CRP capture antibody, and the control line is coated with rabbit anti-DNP antibody. The concentration of CRP capture antibody in the test line is 0.5–1.5 mg / ml, and the volume is 0.5–1.5 μl of coating solution per cm of membrane. The concentration of rabbit anti-DNP antibody in the control line is 0.3–1.0 mg / ml, and the volume is 0.5–1.5 μl of coating solution per cm of membrane.

[0106] The concentrations of CRP capture antibody and rabbit anti-DNP antibody were adjusted to 0.5–1.5 mg / ml and 0.3–1.0 mg / ml, respectively, using coating buffer (10 mM PBS buffer containing 3.0% trehalose). The amount of coating buffer used was 0.5–1.5 μl / cm of membrane. The membrane was then streaked parallel to each other as the detection line and control line, respectively, on the nitrocellulose membrane for coating. The membrane was then placed in an oven and dried at 55°C overnight.

[0107] Example 4: Preparation of the sample pad

[0108] A line of oxygenated compound was uniformly sprayed onto glass cellulose using treatment solution 2 (20 mM containing 0.5% NaCl, 0.5% ZnCl, 2% trehalose, 0.1% BSA, and Tris-HCl at pH 8.0), diluted to 10–100 mM. The amount used was 2–4 μl / cm sample pad. The sample was then placed in an oven and dried overnight at 37°C.

[0109] Example 5: Preparation of blood filtration membrane

[0110] Spray the treatment solution 1 (containing 0.5% NaCl, 0.5% S9, 2% trehalose, 0.1% BSA in 20mM Tris-HCl at pH 8.0) evenly and parallelly onto the blood filtration membrane at a volume of 2-5 μl / cm sample pad. Place the sample pad in an oven and dry it overnight at 37°C.

[0111] Example 6: Preparation of the absorbent pad

[0112] Cut the AN3 absorbent pad, which measures 200mm*300mm, into 25mm*300mm pieces for later use.

[0113] Example 7: Preparation of reagent strips

[0114] (1) Connect the blood filtration membrane, sample pad, conjugate pad, chromatography membrane, and absorbent pad prepared in Examples 2-6 end to end, and then sequentially overlap and adhere them to the electrode substrate to obtain a reagent strip as shown in the figure. Figure 1A As shown;

[0115] (2) Connect the sample pad, binding pad, chromatography membrane, and absorbent pad prepared in Examples 2-5 end to end, and then overlap them sequentially on the electrode substrate to obtain a reagent strip as shown in the figure. Figure 1B As shown;

[0116] (3) The blood filtration membrane, conjugate pad, chromatography membrane, and absorbent pad prepared in Examples 2, 3, 5, and 6 are connected end to end and sequentially overlapped and pasted onto the electrode substrate to obtain a reagent strip as shown. Figure 1C As shown;

[0117] like Figure 2 As shown, a method for assembling a photoexcited lateral flow immunosensor system is provided, wherein the reagent strip prepared above is placed in the lower shell snap-fit ​​of the system, the sample pad layer of the reagent strip is aligned with the sample dispensing well of the system, the detection line on the chromatography 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 reagent kit

[0119] The prepared and cut sample pad, glass fiber conjugate pad, nitrocellulose membrane, blood filtration membrane and absorbent pad are sequentially overlapped and pasted onto the electrode substrate and cut to a fixed size.

[0120] The reagent cartridge includes an upper shell and a lower shell. The upper shell is provided with a sample application hole and an observation window. The sample application 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 by a splice. The lower shell is provided with a slot for fixing the test reagent. After the test strip is fixed in the lower shell, it is assembled with the upper shell by pressure.

[0121] Example 9, Sample Testing

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

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

[0124] (3) Excitation signal test: The electrode surface is excited using light with a wavelength of 680nm for 2 minutes. The photosensitive sphere is excited and energy transfer occurs, producing singlet oxygen. The singlet oxygen reacts with the oxygenated compound (HQ) within the diffusion range, continuously oxidizing HQ to BQ. At this time, step (2) is run to test the excitation signal. Figure 4 As shown, the target analyte tested is the reduction peak signal of the product (BQ) after oxidation by oxygen compound (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.

[0125] (4) The sample electrical signal can be obtained by subtracting the background signal from the excitation signal.

[0126] (5) Performance evaluation.

[0127] Twenty clinical samples were tested according to the above test protocol. The samples were also tested simultaneously using Roche immunoturbidimetry (cobas c 111). The Roche immunoturbidimetry (cobas c 111) test values ​​were plotted on the X-axis and the test results of this method were plotted on the Y-axis to calculate the correlation.

[0128] The test protocol described in Example 9 was used to test 20 clinical samples. Simultaneously, the samples were tested using a Roche biochemical analyzer (COBAS e 411). The Roche biochemical analyzer test values ​​were plotted on the X-axis, and the test results of this method were plotted on the Y-axis to calculate the correlation. The results are as follows: Figure 5 As shown, the test results indicate that the method of this invention has a good correlation with the test results of the Roche biochemical analyzer, with a correlation coefficient R0. 2 =0.9736.

[0129] To test the low precision of samples with mild and moderate inflammation at two medical decision levels, this method was used to test the two samples 12 times consecutively, and the precision CV was calculated.

[0130] Table 2 Precision Evaluation

[0131]

[0132]

[0133] As shown in Table 2, the precision (CV) of the kit of the present invention is less than 10%, while the precision of the existing simple chromatography method is generally 15%, indicating that the kit of the present invention has better precision.

[0134] Comparative Example 1

[0135] Following the methodological reagents described above and the conventional time-resolved immunofluorescence assay, 25 clinical samples were tested for each method. Simultaneously, the samples were tested using the Roche immunoturbidimetric assay (cobas c111). The sensitivity performance of the two methods was compared; specific data are shown in Table 3.

[0136]

[0137]

[0138] As shown in Table 3, the sensitivity of conventional immunochromatographic assay for CRP is about 3-5 mg / L, while this method can detect 0.2-0.3 mg / L, improving the sensitivity by 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 are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A photoexcited lateral flow immunosensor strip, comprising: An electrode substrate is disposed at the bottom of the reagent strip to provide solid support for the reagent strip. A binding pad, one end of which is in contact with the electrode substrate, and the binding pad contains a first structural material that can specifically bind to the target analyte. The first structural material is coupled with a photosensitive compound that can be activated under preset conditions. The activated photosensitive compound can generate reactive oxygen species. The sample pad is located at the foremost end of the reagent strip, with one end placed on the conjugate pad and the other end placed on the electrode substrate. The sample pad contains a second structural material, which is coupled with an oxygen-receiving compound that can accept reactive oxygen species to generate a preset electrical signal. A chromatography membrane that provides solid support for the specific binding of the target analyte and guides the target analyte to capillary flow along the reagent strip direction; An absorbent pad is located at the very end of the reagent strip, with one end placed on the chromatography membrane and the other end in contact with the electrode substrate, and it adsorbs excess reagent to maintain lateral flow along the reagent strip. The chromatography membrane is provided with a detection line, and the detection line of the chromatography membrane is coated with a third structural substance, which is either an antibody or an antigen. The target analyte is an antigen or a hapten; The target analyte specifically binds to at least one of the first structural material on the binding pad and the third structural material on the chromatography membrane; When the first and third structural materials come into contact with the target analyte, the activated photosensitive compound transfers reactive oxygen species to the oxygen-receiving compound of the second structural material on the sample pad. The oxygen-receiving 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. The photosensitive compound is rose bengal, methylene blue, phthalocyanine complex, naphtholine complex, or a combination thereof; The oxygenated compound is hydroquinone, resorcinol, dopamine, acetaminophen, para-aminophenol, ferrocene and its derivatives or combinations thereof.

2. The reagent strip according to claim 1, wherein the sample pad layer comprises at least one of a sample pad and a blood filtration membrane disposed on the electrode substrate; (1) When the target analyte comes from a whole blood sample, the sample pad layer of the reagent strip includes a blood filtration membrane and a sample pad. The blood filtration membrane and the sample pad are attached to the electrode substrate from left to right, with one end of the blood filtration membrane placed above the sample pad, and the whole blood sample is filtered and separated; the second structural material is contained on the sample pad. The blood filtration membrane, sample pad, conjugate pad, chromatography membrane, and absorbent pad of the reagent strip are connected end to end and sequentially overlapped and adhered to the electrode substrate; or (2) When the target analyte comes from a whole blood sample, the sample pad of the reagent strip contains only a blood filtration membrane. The blood filtration membrane and the conjugate pad are pasted on the electrode substrate from left to right, with one end of the blood filtration membrane placed above the conjugate pad, and the whole blood sample is filtered and separated. The second structural material is contained on the blood filtration membrane. The blood filtration membrane, conjugation pad, chromatography membrane, and absorbent pad of the reagent strip are connected end to end and sequentially overlapped and adhered to the electrode substrate; or (3) When the target analyte is not a whole blood sample, the sample pad layer of the reagent strip contains only the sample pad, and the sample pad and the conjugate pad are pasted on the electrode substrate from left to right with one end of the sample pad placed above the conjugate pad; the second structural material is contained on the sample pad; The sample pad, conjugation pad, chromatography membrane, and absorbent pad of the reagent strip are connected end to end and sequentially overlapped and pasted onto the electrode substrate.

3. The reagent strip according to claim 1, characterized in that, The bonding pad is made of cross-linked silica, glass fiber, polyester, or rayon.

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

5. The reagent strip according to claim 1, wherein, The target analyte is an antigen, and the first structural material on the binding pad and the third structural material on the chromatography membrane are a composition containing polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments, wherein the polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments immunize the antigen. When the target analyte comes into contact with the first structural material on the binding pad and the third structural material on the chromatography membrane, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound on the second structural material on the sample pad to generate an electrochemical signal.

6. The reagent strip according to claim 5, wherein the intensity of the electrochemical signal is positively correlated with the amount of the target analyte.

7. The reagent strip according to claim 1, wherein, The target analyte is an antigen, the third structural material on the chromatography membrane is an antigen, and the first structural material on the binding pad is a composition containing a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment, wherein the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the antigen. When the target analyte comes into contact with the first structural material on the binding pad and the third structural material on the chromatography membrane, the target analyte and the third structural material on the chromatography membrane competitively bind to the first structural material on the binding pad. The photosensitive compound is excited by light of a certain wavelength and reacts with the oxygen-receiving compound on the second structural material on the sample pad to generate an electrochemical signal.

8. The reagent strip according to claim 1, wherein, The target analyte is an antigen, the first structural material on the binding pad is the antigen, and the third structural material on the chromatography membrane is a composition containing polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments, wherein the polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments immunize the antigen. When the target analyte comes into contact with the first structural material on the binding pad and the third structural material on the chromatography membrane, the target analyte and the first structural material on the binding pad competitively bind to the third structural material on the chromatography membrane; the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygen-receiving compound on the second structural material on the sample pad to generate an electrochemical signal.

9. The reagent strip according to claim 7 or 8, wherein the intensity of the electrochemical signal is negatively correlated with the amount of the target analyte.

10. The reagent strip according to claim 1, wherein, The target analyte is a hapten, the third structural material on the chromatographic membrane is an antigen, and the first structural material on the binding pad is a composition containing a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment. The polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the hapten. When the target analyte comes into contact with the first structural material on the binding pad and the third structural material on the chromatography membrane, the target analyte and the third structural material on the chromatography membrane competitively bind to the first structural material on the binding pad. The photosensitive compound is excited by light of a certain wavelength and reacts with the oxygen-receiving compound on the second structural material on the sample pad to generate an electrochemical signal.

11. The reagent strip according to claim 1, wherein, The target analyte is a hapten, the first structural material on the binding pad is an antigen, and the third structural material on the chromatography membrane is a composition containing a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment, wherein the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the hapten. When the target analyte comes into contact with the first structural material on the binding pad and the third structural material on the chromatography membrane, the target analyte and the first structural material on the binding pad competitively bind to the third structural material on the chromatography membrane; the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygen-receiving compound on the second structural material on the sample pad to generate an electrochemical signal.

12. The reagent strip according to claim 10 or 11, wherein the intensity of the electrochemical signal is negatively correlated with the amount of the target analyte.

13. The reagent strip according to claim 1, wherein, The target analyte is a hapten, and the first structural material on the binding pad and the third structural material on the chromatography membrane are a composition containing a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment, wherein the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the hapten. When the target analyte comes into contact with the first structural material on the binding pad and the third structural material on the chromatography membrane, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound on the second structural material on the sample pad to generate an electrochemical signal.

14. The reagent strip according to claim 13, wherein the intensity of the electrochemical signal is positively correlated with the amount of the target analyte.

15. The reagent strip according to claim 1, characterized in that, The target analyte may also be an antibody, and the first structural material on the binding pad and the third structural material on the chromatography membrane may be antigens. When the target analyte comes into contact with the first structural material on the binding pad and the third structural material on the chromatography membrane, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound on the second structural material on the sample pad to generate an electrochemical signal.

16. The reagent strip according to claim 15, wherein the intensity of the electrochemical signal is positively correlated with the amount of the target analyte.

17. A photoexcited lateral flow immunosensor system, comprising: The casing includes an upper shell and a lower shell, which are connected by an interlocking mechanism; wherein, the lower shell is provided with a slot for fixing the reagent strip, and after the reagent strip is fixed in the lower shell, it is assembled with the upper shell by pressure; The upper shell is provided with a sample dispensing hole, which corresponds to the sample pad of the reagent strip according to claim 1, and the detection line on the chromatography membrane is close to the sample dispensing hole; An observation window is provided on the upper shell, and the observation window corresponds to the detection line on the chromatography membrane of claim 1. The reagent strip is the reagent strip according to any one of claims 1-16, and the chromatographic membrane of the reagent strip is further provided with a control line, the interval between the control line and the detection line is 6-8 mm, and the control line is far away from the sample well; and An electrochemical signal analysis unit, wherein the reagent strip is connected to the electrochemical signal analysis unit via cable or wirelessly.

18. A method for preparing a reagent strip according to any one of claims 1-16, comprising the following steps: STEP 1: Prepare photosensitive microspheres, wherein the photosensitive microspheres contain a photosensitive compound and are conjugated with an antibody or antigen; STEP 2: Prepare the conjugate pad. Dilute the photosensitive microspheres of the antibody or antigen conjugated in STEP 1 with the photosensitive microspheres conjugated with DNP-BSA, and then spray them evenly onto the conjugate pad. STEP 3: Prepare a chromatography membrane with a detection line and a control line. The detection line is coated with an antibody or antigen, and the control line is coated with an antibody. Adjust the concentration of the antibody or antigen on the detection line and the control line with a coating buffer, and then dry them. STEP 4: Prepare the sample pad, including: STEP 4-1: Prepare the sample pad by diluting the oxygenated compound with the treatment solution, spraying it evenly onto the sample pad, and drying; and / or STEP 4-2: Prepare the blood filtration membrane by spraying the treatment solution evenly and parallel onto the blood filtration membrane and then drying it. STEP 5: Prepare the absorbent pad by cutting the absorbent paper into 25mm*300mm sizes; STEP 6: Prepare the test strips, including: STEP 6-1: Connect the sample pad, binding pad, chromatography membrane, and absorbent pad prepared in STEP 2, STEP 3, STEP 4-1, and STEP 5 end to end, and sequentially overlap and attach them to the electrode substrate; or STEP 6-2: Connect the 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 sequentially overlap and adhere them to the electrode substrate; 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 attach them sequentially to the electrode substrate.

19. A method for detecting a target analyte, comprising: Step 1: Using the immunosensor system of claim 17, add the sample to the sample well of the system and chromatography it to the observation window under the action of capillary suction. Step 2: Insert the system after the reaction in Step 1 into the electrochemical signal analysis unit, run the program, and test the background electrical signal of the sample; Step 3: Excite the photosensitive compound with light of a specific wavelength and detect the electrical signal after excitation; as well as Step 4: Compare the background electrical signal and the excited electrical signal to obtain sample electrical signal information and determine the presence and / or quantity of the target analyte.

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