Homogeneous immunodetection method, reagent and instrument system

Abstract

The invention provides a novel homogeneous immune detection method and a reagent and an instrument system thereof, the method adopts an active oxygen substance which can react with an oxygen-receiving substance to generate an electrical signal capable of being detected by an electrode, and the active oxygen substance is generated by exciting a photosensitive compound; the problems that in the prior art, a complex instrument is high in cost, not portable and the like are solved. By adopting the method provided by the invention, a user can complete detection by collecting fingertip blood, urine, saliva and the like at home, so that integrated miniaturization and convenience in operation of an instrument are realized, and the method is suitable for POCT application scenes such as home use and the like.

Description

Technical Field

[0001] The present invention belongs to the field of medical diagnosis, and particularly relates to a homogeneous immunoassay method, reagent and instrument system. 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. Since magnetic particle chemiluminescence integrates two analytical technologies, it has relatively high requirements in terms of cost and technology, which causes difficulties for the subsequent marketization of products. The principle of electrochemiluminescence involves an electrochemical reaction process. The entire 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 their appearance is relatively large, which is not suitable for POCT usage scenarios such as home use. 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, and the instrument cost is relatively high. Moreover, this technology has problems with background signals. 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, and the operation is complex, making it difficult to be used in POCT usage scenarios such as home use.

[0003] In addition, patients with mild illnesses or those who need long-term monitoring of test items going 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. In order to solve problems such as being suitable for home use, being able to perform whole blood testing, and reducing background signals, it is urgently necessary to develop a small instrument with a simple system that can monitor disease levels via mobile phone and supporting detection reagents.

[0004] Electrochemical immunoassay is based on immune recognition and realizes the specific detection of specific substances through the immune recognition reaction of antigen-antibody. The backend uses electrical signals for output. Traditional immuno-electrochemistry generally requires a multi-step cleaning process to wash away the excess antibodies before accurate testing can be carried out. Compared with traditional immuno-electrochemistry, electrochemical immunoassay can omit the intermediate cleaning steps for accurate testing, and has the high specificity of immune technology and the advantages of electrochemical detection, such as low cost, high sensitivity, good cost performance, and small detection instruments. Compared with traditional immunoassay that uses photons as signal output, electrochemical immunoassay 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. Moreover, 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 technologies to collect and analyze the electrical signals of samples. Differential pulse voltammetry (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. At the same time, during the signal acquisition process of DPV, signal acquisition is only carried out 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. In the patent CN113252749B, differential pulse voltammetry, current-time method or detection of impedance-frequency curve are used to determine whether there are trace organic substances in ice crystals. Summary of the Invention

[0006] The object of the present invention is to provide a new homogeneous immunoassay method, its reagents and instrument system. This method removes the currently commonly used luminescent beads and uses reactive oxygen species that can react with oxygen-receiving substances to generate electrical signals that can be detected by electrodes. The reactive oxygen species are generated by the excitation of photosensitive compounds, so as to solve the problems of high cost and inconvenience in portability caused by complex instruments in the prior art.

[0007] Using the instrument of the present invention, users can collect fingertip blood, urine, saliva, etc. at home to complete the detection, realizing the integration and miniaturization of the instrument and the convenience of operation, which is suitable for POCT use scenarios such as home use.

[0008] To achieve the above object, the main technical solutions of the present invention are as follows:

[0009] In a first aspect, the present invention provides a kit for detecting a target analyte, comprising a first structural substance and a second structural substance capable of binding 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; the second structural substance is conjugated with an oxygen-receiving composition that can receive the reactive oxygen species to generate a preset electrical signal; the target analyte specifically binds to at least one of the first structural substance and the second structural substance; wherein, when the first structural substance and the second structural substance contact the target analyte, the activated photosensitive compound transfers the reactive oxygen species to the oxygen-receiving composition, and the oxygen-receiving composition generates an electrical signal, and the information of the electrical signal is used to characterize the presence and / or quantity of the target analyte.

[0010] Compared with the currently available LOCI assays, the present invention proposes a new method for determining a target substance, removing the currently commonly used luminescent beads, and using reactive oxygen species that can react with an oxygen-receiving substance to generate an electrical signal that can be detected by an electrode, wherein the reactive oxygen species are generated by the excitation of a photosensitive compound. The present invention can add or apply fingertip blood, urine or saliva collected at home to the outside of the electrode sheet, and fill the reaction chamber through capillary action to react with the reagent. The target analyte in the sample is detected in the form of binding to a double antibody or competitively binding to a single antibody.

[0011] Further, the target analyte is an antigen, and the first structural substance and the second structural substance are a composition comprising a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, and the polyclonal antibody, the monoclonal antibody, the ScFv or the antibody fragment immunizes the antigen; the composition that the second structural substance is conjugated with and can receive the reactive oxygen species to generate a preset electrical signal is an oxygenated compound.

[0012] Further, when the first structural substance and the second structural substance contact the target analyte, the photosensitive compound is excited by light of a certain wavelength to react with the oxygenated compound, generating an electrical signal.

[0013] Further, the first structural substance is not directly conjugated with the photosensitive compound, and the kit further comprises a third structural substance conjugated with the photosensitive compound. When the third structural substance contacts the first structural substance, the third structural substance and the first structural substance undergo receptor-ligand affinity adsorption, and the photosensitive compound is excited by light of a certain wavelength to react with the oxygen-receiving composition, generating an electrical signal.

[0014] Further, the intensity of the electrical signal is positively correlated with the quantity of the target analyte.

[0015] Further, the target analyte is a hapten, the second structural substance is an antigen, the first structural substance is 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 or hapten, wherein:

[0016] When the target analyte contacts the first structural substance and the second structural substance, the target analyte competes with the second structural substance for binding to the first structural substance, wherein:

[0017] The photosensitive compound on the first structural substance bound to the second structural substance is excited by light of a certain wavelength to react with the oxygen-containing composition, generating an electrical signal.

[0018] Further, the target analyte is a hapten, the first structural substance is an antigen, the second structural substance is 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 or hapten, wherein:

[0019] When the target analyte contacts the first structural substance and the second structural substance, the target analyte competes with the first structural substance for binding to the second structural substance, and the photosensitive compound on the first structural substance bound to the second structural substance is excited by light of a certain wavelength to react with the oxygen-containing composition, generating an electrical signal.

[0020] Further, the target analyte is a hapten, the second structural substance is an antigen, and the first structural substance is not directly conjugated with the photosensitive compound;

[0021] The kit of reagents further comprises a third structural substance conjugated with the photosensitive compound, the third structural substance is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, and the third structural substance can undergo receptor-ligand affinity adsorption with the first structural substance, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten;

[0022] When the target analyte contacts the second structural substance and the third structural substance, the target analyte competes with the second structural substance for binding to the third structural substance, wherein the oxygen-containing composition on the second structural substance bound to the third structural substance reacts with the photosensitive compound excited by light of a certain wavelength, generating an electrical signal.

[0023] Further, the target analyte is a hapten, the second structural substance is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten, and the first structural substance is not directly conjugated to the photosensitive compound;

[0024] The kit of reagents further comprises the third structural substance conjugated to the photosensitive compound, the third structural substance is an antigen or an antigen carrier, and the third structural substance can undergo receptor-ligand affinity adsorption with the first structural substance;

[0025] When the target analyte contacts the second structural substance and the third structural substance, the target analyte competes with the second structural substance for binding to the third structural substance. Among them, the oxygen-containing composition on the second structural substance bound to the third structural substance reacts with the photosensitive compound excited by light of a certain wavelength to generate an electrical signal.

[0026] Further, the intensity of the electrical signal is negatively correlated with the quantity of the target analyte.

[0027] In a second aspect, the present invention provides a method for detecting a target analyte, comprising using the kit of reagents of the present invention to incubate with a sample to be detected; using an electrode to detect the background electrical signal of the incubated sample; using light of a specific wavelength to excite the photosensitive compound of the kit of reagents, and detecting the electrical signal after excitation; and comparing the background electrical signal and the electrical signal after excitation to obtain sample electrical signal information and determine the presence and / or quantity of the target analyte.

[0028] In a third aspect, the present invention provides an instrument system for detecting a target analyte, comprising a liquid reaction module configured to incubate a sample to be detected and the kit of reagents of the present invention; an electrode module configured to contact the liquid in the liquid reaction module and detect an electrical signal; a light module configured to emit light of a certain wavelength to the liquid reaction module; and a processor configured to receive the signal of the electrode module and process it to determine the presence and / or quantity of the target analyte;

[0029] Optionally, the liquid reaction module is a container capable of accommodating liquid, preferably a cuvette, a cuvette array, or a microplate;

[0030] Optionally, the liquid reaction module is a test strip coated with the kit of reagents of the present invention.

[0031] Fourthly, the present invention provides a target analyte detection system, comprising an electrode that adsorbs or couples with an immune molecule; a photosensitive microsphere and an oxidizable compound reagent. Wherein, the photosensitive sphere contains a photosensitive compound, and the photosensitive compound generates reactive oxygen species capable of reacting with the oxidizable compound reagent under the excitation of light of a certain wavelength, thereby generating an electrical signal that can be detected by the electrode; wherein, the photosensitive sphere further contains a binding ligand.

[0032] Further, the target analyte is an antigen, the binding ligand is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, and the immune molecule adsorbed or coupled by the electrode is 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;

[0033] When the target analyte contacts the electrode and the photosensitive microsphere, the photosensitive compound is excited by light of a certain wavelength to react with the oxidizable compound reagent.

[0034] Further, the target analyte is a hapten, the binding ligand is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, and the immune molecule adsorbed or coupled by the electrode is an antigen, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten;

[0035] When the target analyte contacts the photosensitive microsphere and the electrode, the target analyte and the antigen on the electrode competitively bind to the photosensitive microsphere, and the photosensitive compound is excited by light of a certain wavelength to react with the oxidizable compound reagent.

[0036] Further, the target analyte is an antigen, and the immune molecule adsorbed or coupled by the electrode is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment. The detection system further includes an antibody labeled with receptor-ligand affinity adsorption, and the antibody labeled with receptor-ligand affinity adsorption can undergo affinity adsorption with the photosensitive sphere, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten;

[0037] When the target analyte contacts the electrode and the antibody labeled with receptor-ligand affinity adsorption, the photosensitive compound is excited by light of a certain wavelength to react with the oxidizable compound reagent.

[0038] Further, the target analyte is a hapten, the immune molecule adsorbed or conjugated to the electrode is an antigen, the detection system further includes an antibody labeled with receptor-ligand affinity adsorption, and the antibody labeled with receptor-ligand affinity adsorption is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment. The antibody labeled with receptor-ligand affinity adsorption can undergo affinity adsorption with the photosensitive sphere, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten.

[0039] When the target analyte contacts the photosensitive microsphere and the electrode, the target analyte and the antigen on the electrode competitively bind to the antibody labeled with receptor-ligand affinity adsorption, and the photosensitive compound is excited by light of a certain wavelength to react with the oxidized compound reagent.

[0040] In a fifth aspect, the present invention provides a method for detecting a target analyte, including incubating the detection system of the present invention with a sample to be detected; using an electrode to detect the background electrical signal of the incubated sample; using light of a specific wavelength to excite the photosensitive compound and detecting the electrical signal after excitation; and comparing the background electrical signal and the electrical signal after excitation to obtain sample electrical signal information and determine the presence and / or quantity of the target analyte.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] 1) The homogeneous immunoassay of the present invention can solve the problem of poor detection specificity caused by the need for an intermediate cleaning process in traditional electrochemical methods.

[0043] 2) The backend signal output and detection of the present invention use electrical signals, which reduces the volume in the structure of the instrument detection part compared with the optical signals of the prior art, making it possible to miniaturize the instrument and making it portable. At the same time, the cost of signal acquisition is relatively low.

[0044] 3) The detection method of the present invention uses electrochemical detection, and the required instrument is relatively simple, and product integration and miniaturization can be achieved.

[0045] 4) The instrument system for detecting the target analyte of the present invention uses a screen-printed electrode, and the electrode material includes but is not limited to carbon, silver, gold, copper and their combinations, which solves the problems of high operation difficulty and poor reproducibility of traditional three electrodes, resulting in difficulty in commercialization. At the same time, the instrument system of the present invention has a low cost, and there is no cross-interference in single use, and it is suitable for POCT usage scenarios such as home use. Users can import the detection results into a mobile phone through Bluetooth, USB, TPC and other methods to monitor the significance and fluctuations of the detection results in real time.

[0046] 5) 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

[0047] Figure 1 This is a schematic diagram of the reaction process and testing of the present invention;

[0048] Figure 2 This is a typical test result graph (reduction peak of BQ). Among them, Figure 2 This is a typical peak shape graph of the relationship between current and concentration

[0049] Figure 3 This is a graph of the gradient of the photoexcitation signal of current with different concentrations of luteinizing hormone (LH);

[0050] Figure 4 This is a graph of the linear relationship of the current signal generated by luteinizing hormone (LH) after the electrode is photoexcited;

[0051] Figure 5 This is a graph for evaluating the consistency of the methodology of the present invention with that of Roche biochemical analyzer. Detailed Embodiments

[0052] The present invention discloses a new homogeneous immunoassay method, its reagents and instruments. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. 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 methods and applications 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.

[0053] Term Explanation

[0054] In the present invention, unless otherwise specified, the 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.

[0055] 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 complex, naphthalocyanine complex and their combinations.

[0056] The term "oxidizable compound" refers to a compound that can undergo an oxidation-reduction 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 form of the oxidizable compound can be divided into chemical substances, silica microspheres containing oxidizable chemical substances, and polystyrene microspheres containing oxidizable chemical substances.

[0057] The term "antibody" refers to immunoglobulins and immunoglobulin fragments, whether produced naturally or in part or in whole synthetically (e.g., recombinantly), including any fragment that retains the binding specificity of a full-length immunoglobulin and that comprises at least a portion of the variable region of an immunoglobulin molecule. The term antibody includes polyclonal antibodies, monoclonal antibodies, ScFv, or antibody fragments.

[0058] The term "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to the other antibody molecules. "Polyclonal antibodies" refer to a population of antibodies that contain antibodies with multiple different sequences.

[0059] The term "scFv fragment" refers to an antibody formed by linking the variable region of the antibody heavy chain and the variable region of the light chain by a short peptide (linker) of 5 to 20 amino acids. The linker length is such that the two variable domains are bridged without substantial interference.

[0060] The "antibody fragment" of an antibody refers to any part of a full-length antibody that is less than full-length but that comprises at least a portion of the variable region of the antibody that binds 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.

[0061] The term "antigen" refers to an antigen that can be isolated or present in a biological sample.

[0062] The term "hapten" is a compound that is capable of specifically binding to a corresponding antibody but that does not itself serve as an immunogen (or antigen) for the preparation of an antibody. Haptens are typically conjugated to an antigen carrier used to generate the antibody.

[0063] The terms "antigen carrier" or "immunogenic carrier" are 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 a 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, eye lens proteins, and lipoproteins. Illustrative 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).

[0064] The term "specific binding" with respect to an antibody or an antigen-binding fragment thereof refers to the ability of the antibody or antigen-binding fragment to form one or more non-covalent bonds with the same 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, an antigen carrier, or an antigen-binding fragment in the case of limited amounts of the antibody, antigen carrier, or antigen-binding fragment.

[0065] The term "biotin" refers to a substance that can be conjugated with an antigen / antibody and is essentially a vitamin.

[0066] The term "avidin" refers to a glycoprotein that can be extracted from egg white. Avidin includes avidin, neutravidin, streptavidin, etc.

[0067] The term "receptor-ligand affinity adsorption label" refers to a specific molecule or particle with a certain label (such as a fluorophore, an enzyme, a radioisotope, etc.).

[0068] Figure 1 For the reaction process and test schematic diagram of the present invention, the target analyte contacts the photosensitive sphere, and 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 reactive oxygen species undergo an oxidation-reduction reaction with the oxidized compound to produce a product that can cause an electrochemical signal, and an electrical signal can be generated under the action of a specific potential.

[0069] Figure 2 It is a typical test result graph (reduction peak of BQ), Figure 2 It is a peak-shaped graph of the relationship between current and LH concentration, that is, a graph of the relationship between luteinizing hormone (LH) and the electrical signal generated by the product (BQ) after oxidation of the oxidized compound (HQ); Figure 3 It is a graph of the light excitation signal gradient of current versus different concentrations of luteinizing hormone (LH).

[0070] Figure 4 It is a linear relationship graph of the current signal generated by luteinizing hormone LH after the electrode is photoexcited.

[0071] Figure 5 It is a graph for evaluating the methodological consistency between the method of the present invention and the test method of Roche biochemical analyzer.

[0072] In the examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can be purchased from commercial channels.

[0073] In the following examples, luteinizing hormone (LH) is the target analyte contained in the plasma sample; silicon dioxide (SiO2) is synthesized in step 1.1 of Example 1; tetraethyl orthosilicate (TEOS) is purchased from Sigma-Aldrich with a catalog number of 8006580250; 3-aminopropyltrimethoxysilane (APTMS) is purchased from Sigma-Aldrich with a catalog number of 281778-5mL; hydroquinone oxygenated compound (HQ) is purchased from Sigma-Aldrich with a catalog number of H9003-1 00G; p-benzoquinone (BQ) is the oxidation product of HQ; LH antibody 1 (Ab1) and LH antibody 2 (Ab2) were purchased from Hangzhou Boyue Biotechnology Co., Ltd.; SiO2-HQ-Ab1 is SiO2 microspheres coated with HQ coupled to LH-specific monoclonal antibody 1; photosensitive microspheres (GG) were purchased from Xi'an Qiyue Biotechnology Co., Ltd.; streptavidin (SA) was purchased from Sigma-Aldrich, catalog number S6402-1ML; biotin coupled to LH-specific monoclonal antibody 2 (Bio-Ab2).

[0074] The present invention will be further described below in conjunction with embodiments:

[0075] Example 1. Synthesis of SiO2 microspheres

[0076] (1) Take 60 mL of cyclohexane, 5 mL of n-hexanol, 20 mL of Triton X-100, and 5 mL of purified water and add them to a 250 mL clean conical flask. Place a 2 cm diameter magnetic stirrer and adjust the speed to 100 RPM for stirring for 20 min. Add 0.5 mL of ammonia and 1 mL of TEOS and continue stirring for 24 h. After stirring, aspirate the solution from the conical flask and add it to two 50 mL centrifuge tubes for centrifugation. Aspirate the supernatant.

[0077] (2) Add 10 mL of anhydrous ethanol and 10 mL of purified water respectively, resuspend by ultrasonication, centrifuge at 10,000 RPM for 25 minutes, and remove the supernatant.

[0078] Repeat step (2).

[0079] (3) Add 6 mL of anhydrous ethanol and 6 mL of purified water again, resuspend by ultrasonication, centrifuge at 10,000 RPM for 25 minutes, and remove the supernatant.

[0080] Repeat step (3)

[0081] (4) Add 3 mL of anhydrous ethanol to each sample and resuspend to prepare SiO2 microspheres for later use.

[0082] Example 2. Synthesis of SiO2-HQ microspheres

[0083] (1) Take out a 10 ml round-bottom flask, clean it, and rinse it once with pure water and ethanol respectively.

[0084] (2) Take 2 ml of the synthesized SiO2 microspheres in Example 1 and add them to the round-bottom flask. Add 0.5 ul of ATPMS, and stir magnetically in the dark for 2 hours.

[0085] (3) Water bath at 65 °C for 30 minutes.

[0086] (4) Wash 3 times with absolute ethanol, 12800 RPM, for 5 minutes.

[0087] (5) Add hydroquinone (4 mg / ml), 400 uL, and stir magnetically in the dark for 2 hours.

[0088] (6) Wash and centrifuge 3 times with absolute ethanol at 12800 RPM for 5 min and resuspend in 2 mL of absolute ethanol.

[0089] (7) Add 4 uL of TEOS and react overnight for 15 hours. Wash and centrifuge once with absolute ethanol at 12800 RPM for 5 min.

[0090] Repeat steps (2)-(7) 4 times.

[0091] (8) Take 2 mL of silica microspheres and add them to the round-bottom flask. Add 6 uL of ATPMS, and stir magnetically in the dark for 2 h.

[0092] (9) Water bath at 65 °C for 5 minutes. Wash 3 times with absolute ethanol, 12800 RPM, for 5 minutes, and resuspend in 2 mL of absolute ethanol.

[0093] Example 3. Synthesis of SiO2-HQ-Ab1 microspheres, i.e., SiO2 microspheres coated with HQ and coupled with LH-specific monoclonal antibody 1

[0094] Take SiO2-HQ microspheres (12 mg / mL, 208.3 μL) in a centrifuge tube. Add 100 μL of 2 mg / mL glutaraldehyde to the microsphere solution and react at room temperature at 1300 RPM for 2 h. After the reaction, wash 5 times with 10 mM PBS pH7.4 and dialyze overnight; centrifuge and add 76.39 μL of 10 mM PBS (pH7.2) buffer solution, and sonicate.

[0095] Add 17.61 μL of LH antibody (7.1 mg / mL) to the microspheres, mix well, then add 4 μL of sodium cyanoborohydride (50 mg / mL), and react at 1300 rpm at 37 °C in a thermostatic mixer for 1 h. Then add 2 μL of TW-20 and continue to react for 23 h.

[0096] Add 10 μL of 150 mg / mL Gly and oscillate for 2 h, then add 20 μL of PBS pH 7.2 and react for 2 hours.

[0097] Add 100 μL of 10 mM PBS pH 7.4, take 80 μL and add it to the bottom A of the centrifuge tube, then perform lyophilization. After lyophilization, store it at -20 °C to prepare SiO2-HQ-Ab1 microspheres for later use.

[0098] Example 4. Synthesis of GG-SA microspheres, i.e., photosensitive microspheres conjugated with streptavidin

[0099] Add 1 mL of carboxyl-containing photosensitive microspheres to centrifuge tubes respectively, add 100 μL of 50 mM MES buffer (pH 5.5), and quickly oscillate and mix well with 3.2 μL of EDC. Then add 14 μL of TW-20 (0.5%, v / v) and quickly oscillate and mix well, then discard the supernatant. Add 100 μL of (pH 6, 50 mM MES) buffer, resuspend by ultrasound, and add 500 μg of LH-specific monoclonal antibody.

[0100] Place the above reaction solution at 37 °C and react for 2 hours. Add 10 μL of Gly and quickly oscillate and mix well, add 10 μL of 100 mg / mL BSA, mix well, centrifuge and discard the supernatant. Add 100 μL of (pH 6, 50 mM MES) buffer solution, suspend the microspheres by ultrasound, oscillate and mix well, and add all of it to the bottom B of the centrifuge tube, then perform lyophilization. After lyophilization, store it at -20 °C to prepare GG-SA microspheres for later use.

[0101] Example 5. Synthesis of Bio-Ab2, i.e., biotin conjugated with LH-specific monoclonal antibody 2

[0102] Add 36.3 μL of 10 mM PBS pH 7.2 buffer to centrifuge tubes respectively, add 11.8 μL of LH-specific monoclonal antibody, and mix well.

[0103] Add 1.8 μL of biotin, mix well, oscillate and incubate for 2 hours. After adding 1 μL of Gly and reacting at room temperature for 5 hours, add all of it to the bottom C of the centrifuge tube, then perform lyophilization. After lyophilization, store it at 2-8 °C to prepare Bio-Ab2 for later use.

[0104] Example 6. Establishment of standard curve

[0105] (1) Incubation

[0106] Add 150 μL of purified water to the centrifuge tube containing the freeze-dried substance prepared in Example 5 to obtain LH samples with different concentration gradients. Determine the concentration using a Roche biochemical analyzer. Add the above samples with different concentrations to the centrifuge tubes (the sample concentrations are 0, 5.5, 11.71, 19.19, 38.33, 53.55 mIU / mL), invert and shake gently to mix evenly, and incubate at 37 °C for 10 min. The target analyte antigen LH (Ag), GG-SA, Bio-Ab2, and SiO2-HQ-Ab1 form a sandwich (GG-SA-Bio-Ab2-Ag-Ab1-HQ-SiO2).

[0107] (2) Sample loading

[0108] Refer to the method of CN114216949B. Insert the screen-printed electrode into an electrochemical analyzer such as CHI660 or a portable electrochemical detector. Use a dropper to suck an appropriate amount of the reacted solution and drop it onto the working surface of the screen-printed electrode. The solution needs to cover all three electrodes to ensure circuit conduction.

[0109] (3) Background signal test

[0110] After sample loading, the electrochemical analyzer runs 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. As Figure 2 shown is a typical test result graph, that is, the reduction peak of BQ at -0.15 V. At this time, the reaction that occurs is BQ + e- → HQ.

[0111] (4) Excitation signal test

[0112] Irradiate the electrode surface with light at a wavelength of 680 nm 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 and continuously oxidizes HQ to BQ.

[0113] (5) Subtracting the background signal from the excitation signal can obtain the sample electrical signal. Plotting the sample electrical signal against the standard concentration can obtain the standard curve of luteinizing hormone LH.

[0114] Example 7, Sample testing

[0115] (1) Synthesis: Refer to the reagent synthesis in the establishment of the standard curve.

[0116] (2) Incubation: Add 150 μL of purified water to the centrifuge tube containing the above freeze-dried substance. Take 10 μL - 20 μL of plasma sample and add it to the reaction cup (the sample can be plasma, serum, whole blood, urine, saliva). Invert and shake well. Incubate at 37 °C for 10 min and then wait for measurement. The target analyte antigens LH (Ag), GG-SA, Bio-Ab2, and SiO2-HQ-Ab1 form a sandwich (GG-SA-Bio-Ab2-Ag-Ab1-HQ-SiO2).

[0117] (3) Sampling: Referring to the method of CN114216949B, insert the screen-printed electrode into an electrochemical analyzer such as CHI660 or a portable electrochemical detector. Use a dropper to suck an appropriate amount of the reacted solution and drop it onto the working surface of the screen-printed electrode. The solution needs to cover all three electrodes to ensure circuit conduction.

[0118] (4) Background signal test: After sampling, the electrochemical analyzer runs 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.

[0119] (5) 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, run the differential pulse voltammetry program with the parameters described in step (4) to test the excitation signal. Figure 3 The shown is the gradient diagram of the light excitation signal at different concentrations.

[0120] (6) Subtracting the background signal from the excitation signal can obtain the electrical signal of the sample. According to the standard curve, the concentration of LH in the sample can be obtained. The relationship between its concentration and the current gradient is as Figure 4 shown, and the correlation coefficient R 2 = 0.978.

[0121] Example 8. Performance evaluation

[0122] The test scheme described in Example 7 was used to test 20 clinical samples. At the same time, a Roche biochemical analyzer (COBAS e 411) was used to test the above samples synchronously. Using the test value of the Roche biochemical analyzer as the X-axis and the test result of this method as the Y-axis to plot a graph and calculate the correlation. The results are as Figure 4As shown, the test results indicate that the method of the present invention has a good correlation with the test results of Roche biochemical analyzer, and the correlation coefficient R 2 = 0.9814.

[0123] Taking the luteinizing hormone LH item as an example, the precision of slightly inflamed and moderately inflamed samples at two medical decision levels was tested. Using this method, the two samples were continuously tested 11 times, and the precision CV was calculated.

[0124] Table 1 Precision evaluation

[0125] Serial number Low value High value 1 3.69 35.14 2 3.21 34.83 3 3.21 36.39 4 3.69 36.39 5 3.43 32.03 6 3.75 33.28 7 3.24 34.83 8 3.15 35.14 9 3.18 37.01 10 3.69 33.28 11 3.69 36.70 CV 7％ 5％

[0126] As shown in the results of Table 1, the precision CV of the test kits of the present invention is less than 10%, indicating good precision.

[0127] In this embodiment, the i-t current curve (constant initial voltage, measuring the change of current with time) is used for the test detection method, but it is not limited to this method. Similar to differential pulse voltammetry, square wave pulse method SMV or other common electrochemical detection methods can be used for the electrochemical detection method of the present invention.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than 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 kit for detecting a target analyte, comprising: A first structural substance capable of binding to the target analyte, the first structural substance being conjugated with a photosensitive compound that can be activated under preset conditions, and the activated photosensitive compound can generate reactive oxygen species; A second structural substance capable of binding to the target analyte, the second structural substance being conjugated with an oxygen acceptor composition that can accept reactive oxygen species to generate a preset electrical signal; The target analyte specifically binds to at least one of the first structural substance and the second structural substance; Wherein, when the first structural substance and the second structural substance contact the target analyte, the activated photosensitive compound transfers the reactive oxygen species to the oxygen acceptor composition, and the oxygen acceptor composition generates an electrical signal, and the information of the electrical signal is used to characterize the presence and / or quantity of the target analyte.

2. The kit of reagents according to claim 1, wherein The target analyte is an antigen, and the first structural substance and the second structural substance are compositions comprising polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments, and the polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments immunize the antigen; The composition that the second structural substance is conjugated with and can accept reactive oxygen species to generate a preset electrical signal is an oxygen acceptor compound.

3. The kit of reagents according to claim 2, wherein, When the first structural substance and the second structural substance contact the target analyte, the photosensitive compound is excited by light of a certain wavelength to react with the oxygen acceptor compound, generating an electrical signal.

4. The kit according to claim 2, wherein the first structural substance is not directly conjugated with the photosensitive compound, and the kit further comprises a third structural substance conjugated with the photosensitive compound. When the third structural substance contacts the first structural substance, the third structural substance and the first structural substance undergo receptor-ligand affinity adsorption, and the photosensitive compound is excited by light of a certain wavelength to react with the oxygen acceptor composition, generating an electrical signal.

5. The kit according to claim 3 or 4, wherein the intensity of the electrical signal is positively correlated with the quantity of the target analyte.

6. The kit according to claim 1, wherein the target analyte is a hapten, the second structural substance is an antigen, and the first structural substance is a polyclonal antibody, monoclonal antibody, ScFv or antibody fragment, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten, wherein: When the target analyte contacts the first structural substance and the second structural substance, the target analyte competitively binds to the first structural substance with the second structural substance, and the photosensitive compound on the first structural substance bound to the second structural substance is excited by light of a certain wavelength to react with the oxygen acceptor composition, generating an electrical signal.

7. The kit of reagents according to claim 1, wherein, The target analyte is a hapten, the first structural substance is an antigen, and the second structural substance is a polyclonal antibody, monoclonal antibody, ScFv or antibody fragment, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten, wherein: When the target analyte contacts the first structural substance and the second structural substance, the target analyte competes with the first structural substance for binding to the second structural substance. Among them, the photosensitive compound on the first structural substance that binds to the second structural substance is excited by light of a certain wavelength to react with the oxygen acceptor combination, generating an electrical signal.

8. The kit of reagents according to claim 1, wherein, The target analyte is a hapten, the second structural substance is an antigen, and the first structural substance is not directly conjugated with the photosensitive compound; The kit of reagents further comprises a third structural substance conjugated with the photosensitive compound. The third structural substance is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, and the third structural substance can undergo receptor-ligand affinity adsorption with the first structural substance. The polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten; When the target analyte contacts the second structural substance and the third structural substance, the target analyte competes with the second structural substance for binding to the third structural substance. Among them, the oxygen acceptor composition on the second structural substance that binds to the third structural substance reacts with the photosensitive compound excited by light of a certain wavelength, generating an electrical signal.

9. The kit of reagents according to claim 1, wherein The target analyte is a hapten, the second structural substance is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment. The polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten, and the first structural substance is not directly conjugated with the photosensitive compound; The kit of reagents further comprises a third structural substance conjugated with the photosensitive compound. The third structural substance is an antigen or an antigen carrier, and the third structural substance can undergo receptor-ligand affinity adsorption with the first structural substance; When the target analyte contacts the second structural substance and the third structural substance, the target analyte competes with the second structural substance for binding to the third structural substance. Among them, the oxygen acceptor composition on the second structural substance that binds to the third structural substance reacts with the photosensitive compound excited by light of a certain wavelength, generating an electrical signal.

10. The kit of reagents according to any one of claims 6 to 9, wherein the intensity of the electrical signal has a negative correlation with the quantity of the target analyte.

11. A method for detecting a target analyte, comprising: using the kit of reagents according to any one of claims 1 to 10 to incubate with a sample to be detected; using an electrode to detect the background electrical signal of the incubated sample; using light of a specific wavelength to excite the photosensitive compound of the kit of reagents and detecting the electrical signal after excitation; and comparing the background electrical signal and the electrical signal after excitation to obtain sample electrical signal information and determining the presence and / or quantity of the target analyte.

12. An instrument system for detecting a target analyte, comprising: a liquid reaction module configured to incubate a sample to be detected and the kit of reagents according to any one of claims 1 to 10; an electrode module configured to contact the liquid in the liquid reaction module and detect an electrical signal; An optical module, configured to emit light of a certain wavelength to the liquid reaction module; And A processor, configured to receive signals from the electrode module and process them to determine the presence and / or quantity of the target analyte; Optionally, the liquid reaction module is a container capable of accommodating liquid, preferably a cuvette, a cuvette array, or a microplate; Optionally, the liquid reaction module is a test strip coated with the kit of reagents according to any one of claims 1 to 10.

13. A target analyte detection system, comprising: An electrode that adsorbs or couples an immune molecule; Photosensitive microspheres and an oxidizable compound reagent, wherein the photosensitive spheres contain a photosensitive compound that generates reactive oxygen species capable of reacting with the oxidizable compound reagent under excitation by light of a certain wavelength, thereby generating an electrical signal that can be detected by the electrode; Wherein the photosensitive spheres further contain a binding ligand.

14. The detection system according to claim 13, wherein the target analyte is an antigen, the binding ligand is a polyclonal antibody, a monoclonal antibody, a ScFv, or an antibody fragment, and the immune molecule adsorbed or coupled by the electrode is a polyclonal antibody, a monoclonal antibody, a ScFv, or an antibody fragment that immunizes the antigen; When the target analyte contacts the electrode and the photosensitive microspheres, the photosensitive compound is excited by light of a certain wavelength to react with the oxidizable compound reagent.

15. The detection system according to claim 13, wherein the target analyte is a hapten, the binding ligand is a polyclonal antibody, a monoclonal antibody, a ScFv, or an antibody fragment, and the immune molecule adsorbed or coupled by the electrode is an antigen, and the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the antigen or hapten; When the target analyte contacts the photosensitive microspheres and the electrode, the target analyte and the antigen on the electrode competitively bind to the photosensitive microspheres, and the photosensitive compound is excited by light of a certain wavelength to react with the oxidizable compound reagent.

16. The detection system according to claim 13, wherein the target analyte is an antigen, the immune molecule adsorbed or coupled by the electrode is a polyclonal antibody, a monoclonal antibody, a ScFv, or an antibody fragment, and the detection system further comprises an antibody labeled with receptor-ligand affinity adsorption that can undergo affinity adsorption with the photosensitive spheres, and the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the antigen or hapten; When the target analyte contacts the electrode and the antibody labeled with receptor-ligand affinity adsorption, the photosensitive compound is excited by light of a certain wavelength to react with the oxidizable compound reagent.

17. The detection system according to claim 13, wherein the target analyte is a hapten, the immune molecules adsorbed or coupled to the electrode are antigens, the detection system further comprises an antibody labeled with receptor-ligand affinity adsorption, the antibody labeled with receptor-ligand affinity adsorption is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, the antibody labeled with receptor-ligand affinity adsorption can undergo affinity adsorption with the photosensitive microsphere, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten; When the target analyte contacts the photosensitive microsphere and the electrode, the target analyte and the antigen on the electrode competitively bind to the antibody labeled with receptor-ligand affinity adsorption, and the photosensitive compound is excited by light of a certain wavelength to react with the oxidized compound reagent.

18. A method for detecting a target analyte, comprising: using the detection system according to any one of claims 13 to 17 and incubating with a sample to be detected; detecting the background electrical signal of the incubated sample using an electrode; exciting the photosensitive compound with light of a specific wavelength and detecting the electrical signal after excitation; and comparing the background electrical signal and the electrical signal after excitation to obtain sample electrical signal information and determine the presence and / or quantity of the target analyte.

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