Method, reagent and instrument system for homogeneous immunodetection of target antibody analyte

Abstract

The invention provides a method for homogeneous immunodetection of a target antibody analyte as well as a reagent and an instrument system thereof. According to the method, a luminous ball conventionally used at present is removed, an active oxygen substance which can react with an oxygen-receiving substance to generate an electrical signal capable of being detected by an electrode is adopted, the active oxygen substance is generated by exciting a photosensitive compound, complex instrument module setting is not needed, the reaction of an antibody target antibody analyte and an antigen is more facilitated, and the detection sensitivity is improved. The reaction time is shortened, and the utilization rate of the antibody is improved, so that the detection instrument has higher sensitivity and accuracy.

Description

Technical Field

[0001] The present invention belongs to the field of medical diagnosis, and in particular relates to a method, reagent and instrument system for homogeneous immunoassay of target antibody analyte. Background Art

[0002] Immunoassay technology has a wide range of applications in the field of medical diagnosis. Commonly used immunoassay methods include enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay, homogeneous immunochemiluminescence, etc. Antibody detection technology has developed a variety of improved forms in recent years, and the detection performance has been further improved through rapid detection and point-of-care testing (POCT).

[0003] Since homogeneous chemiluminescence does not involve cleaning, the instrument cannot be miniaturized, the instrument cost is relatively high, and the technology has problems such as background signal problems. Due to the detection method, the whole blood needs to be separated before testing to ensure accuracy. The operation is complicated and difficult to use in POCT scenarios such as home use. In addition, patients with mild symptoms or who need long-term monitoring and testing items go to the hospital, which increases the cross-infection rate of the disease. The above-mentioned methods are only applicable to hospital scenarios and are difficult to promote for home use. In order to solve the problems of home use, whole blood testing, and background signal reduction, it is urgently needed to develop a small instrument with a simple system that can monitor disease levels on a mobile phone and supporting detection reagents.

[0004] The target antibody analyte of electrochemical immunoassay is based on immune recognition. It realizes specific detection of specific substances through the immune recognition reaction of antigens and antibodies, and uses electrical signals for output at the back end. Traditional immunoelectrochemistry generally requires a multi-step cleaning process to clean up the excess antibodies before accurate testing can be performed. Compared with traditional immunoelectrochemistry, electrochemical immunoassay can omit the intermediate cleaning steps for accurate testing. It has the high specificity of immunoassay technology and the advantages of electrochemical detection, such as low cost, high sensitivity, cost-effectiveness, and small detection instrument. Electrochemical detection instruments do not require complex structures and optical path components, which can realize the miniaturization and portability of the instrument. At the same time, since the detection is an electrical signal, whole blood samples can be used for detection, which is more suitable for point-of-care testing (POCT) scenarios. Summary of the invention

[0005] The object of the present invention is to provide a method for detecting a target antibody analyte, as well as its reagent and instrument system, by using homogeneous immunoassay technology. This method removes the currently commonly used luminescent beads and adopts 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, without the need for complex instrument module settings, which is more conducive to the reaction between the target antibody analyte and the antigen, shortens the reaction time, improves the utilization rate of antibodies, and enables the detection instrument of the present invention to have higher sensitivity and accuracy.

[0006] To achieve the above object, the main technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides a set of reagents for detecting a target antibody analyte, characterized in that the target antibody analyte is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment; the set of reagents includes a first structural substance and a second structural substance that can bind to the target antibody 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 first structural substance and the second structural substance are not both one of an antigen, a hapten, a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment; the target antibody 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 antibody 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 antibody analyte.

[0008] Further, the first structural substance and the second structural substance are an antigen or a hapten, and the antigen or hapten can specifically bind to the polyclonal antibody, the monoclonal antibody, the ScFv or the antibody fragment; when the first structural substance and the second structural substance contact the target antibody analyte, the photosensitive compound is excited by light of a certain wavelength to react with the oxygen-receiving compound, generating an electrical signal.

[0009] Further, the set of reagents further includes 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.

[0010] Further, the intensity of the electrical signal is positively correlated with the quantity of the target antibody analyte.

[0011] Further, the second structural substance is an antigen or a hapten, 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:

[0012] When the target antibody analyte contacts the first structural substance and the second structural substance, the target antibody analyte competitively binds to the second structural substance with the first structural substance. The photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxidized compound, which reacts with the oxygen-containing composition of the second structural substance to generate an electrical signal.

[0013] Further, the first structural substance is an antigen or a hapten, 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:

[0014] When the target antibody analyte contacts the first structural substance and the second structural substance, the target antibody analyte competitively binds to the first structural substance with the second structural substance. The photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxidized compound, which reacts with the oxygen-containing composition of the second structural substance to generate an electrical signal.

[0015] Further, the second structural substance is an antigen or a hapten, and the first structural substance is not directly conjugated with the photosensitive compound;

[0016] 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;

[0017] When the target antibody analyte contacts the second structural substance and the third structural substance, the target antibody analyte competitively binds to the second structural substance with the third structural substance. Wherein, the photosensitive compound on the third structural substance is excited by light of a certain wavelength to generate an oxidized compound, which reacts with the oxygen-containing composition of the second structural substance to generate an electrical signal.

[0018] Further, the intensity of the electrical signal has a negative correlation with the quantity of the target antibody analyte.

[0019] In a second aspect, the present invention provides a method for detecting a target antibody analyte, comprising incubating a sample to be detected with the kit of reagents described in the present invention; detecting the background electrical signal of the incubated sample using an electrode; exciting the photosensitive compound of the kit of reagents 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 antibody analyte.

[0020] In a third aspect, the present invention provides an instrument system for detecting an antibody target antibody analyte, comprising a liquid reaction module configured to incubate a sample to be detected and the kit of reagents described in 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 from the electrode module and process it to determine the presence and / or quantity of the target antibody analyte;

[0021] Optionally, the liquid reaction module is a container capable of accommodating liquid, preferably a cuvette, a cuvette array, or a microplate;

[0022] Optionally, the liquid reaction module is a test strip coated with the kit of reagents described in the present invention.

[0023] In a fourth aspect, the present invention provides a target antibody analyte detection system, comprising an electrode that adsorbs or couples an immune molecule; a photosensitive microsphere and an oxidizable compound reagent, wherein the photosensitive sphere contains 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; and wherein the photosensitive sphere further contains a binding ligand.

[0024] Further, the binding ligand is an antigen or a hapten, the immune molecule adsorbed or coupled by the electrode is an antibody, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten;

[0025] When the target antibody analyte contacts the photosensitive microsphere and the electrode, the target antibody analyte and the antibody 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.

[0026] Further, the binding ligand is an antigen or a hapten, 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;

[0027] When the target antibody analyte contacts the electrode and the photosensitive microsphere, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxidizable compound reagent.

[0028] Further, the immune molecule adsorbed or coupled to the electrode is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment. The detection system further includes an antigen labeled with receptor-ligand affinity adsorption. The antigen 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.

[0029] When the target antibody analyte contacts the photosensitive microsphere and the electrode, the target antibody analyte and the antibody on the electrode competitively bind to the antigen labeled with receptor-ligand affinity adsorption, and the photosensitive compound is excited by light of a certain wavelength and reacts with the oxidizable compound reagent.

[0030] Further, the immune molecule antigen adsorbed or coupled to the electrode, the detection system further includes an antigen labeled with receptor-ligand affinity adsorption. 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.

[0031] When the target antibody analyte contacts the electrode and the antigen labeled with receptor-ligand affinity adsorption, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxidizable compound reagent.

[0032] In a fifth aspect, the present invention provides a method for detecting a target antibody analyte of an antibody, 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 antibody analyte.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1) The homogeneous immune reaction 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.

[0035] 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 at the same time reducing the cost of signal acquisition.

[0036] 3) The detection method of the present invention uses electrochemical detection, and the required instruments are relatively simple, enabling the integration and miniaturization of products.

[0037] 4) The instrument system for detecting the target analyte of the present invention uses screen-printed electrodes, and the electrode materials include but are not limited to carbon, silver, gold, copper and their combinations, solving the problems of high operation difficulty and poor reproducibility of traditional three-electrode systems, which are not conducive to commercialization. At the same time, the instrument system of the present invention has a low cost, is disposable and has no cross-interference, and is suitable for POCT usage scenarios such as home use. Users can import the detection results into the mobile phone via Bluetooth, USB, TPC, etc., and monitor the significance and fluctuations of the detection results in real time.

[0038] 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

[0039] Figure 1 It is a schematic diagram of the reaction process and test of the present invention;

[0040] Figure 2 It is a peak-shaped diagram of the relationship between current and antibody concentration, that is, a diagram of the relationship between current and the concentration of hepatitis B surface antibody (HbsAb);

[0041] Figure 3 It is a gradient diagram of the photoexcitation signal of the concentration of different concentrations of hepatitis B surface antibody (HbsAb);

[0042] Figure 4 It is a diagram of the relationship between current and the concentration of hepatitis B surface antibody (HbsAb);

[0043] Figure 5 It is a diagram for evaluating the methodological consistency between the method of the present invention and the Roche biochemical analyzer. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention discloses a new homogeneous immunoassay detection method and 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. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0045] TERM EXPLANATION

[0046] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. To better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0047] The term "photosensitive compound" refers to a photosensitive substance that can be excited by excitation light of a certain wavelength, such as rose bengal, methylene blue, phthalocyanine complexes, naphthalocyanine complexes, and combinations thereof.

[0048] The term "oxidizable compound" refers to a compound that can undergo a redox reaction with reactive oxygen species to produce a product that can cause an electrochemical signal. The oxidizable compound itself does not produce an electrochemical signal substance, and it can be any one or combination of hydroquinone, resorcinol, dopamine, paracetamol, p-aminophenol, ferrocene, and its derivatives. The forms of oxidizable compounds can be divided into chemical substances, silica microspheres containing oxygenated chemical substances, and polystyrene microspheres containing oxygenated chemical substances.

[0049] The term "antibody" refers to immunoglobulins and immunoglobulin fragments, whether produced naturally or partially or fully synthetically (e.g., recombinantly), including any fragment that retains the binding specificity of the full-length immunoglobulin and at least contains a portion of the variable region of the immunoglobulin molecule. The term antibody includes polyclonal antibodies, monoclonal antibodies, ScFv, or antibody fragments.

[0050] The term "monoclonal antibody" refers to a population of identical antibodies, indicating that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. "Polyclonal antibody" refers to a population of antibodies containing antibodies with multiple different sequences.

[0051] The term "scFv fragment" refers to an antibody formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody through a short peptide (linker) of 5 to 20 amino acids. The length of the linker allows the two variable domains to bridge without substantial interference.

[0052] The "antibody fragment" of an antibody refers to any part of the full-length antibody that is less than the full-length but at least contains a portion of the variable region of the antibody that binds to the antigen (e.g., one or more CDRs and / or one or more antibody binding sites), and thus retains the binding specificity and at least part of the specific binding ability of the full-length antibody.

[0053] The term "antigen" refers to an antigen that can be isolated or present in a biological sample.

[0054] The term "hapten" is a compound that can specifically bind to a corresponding antibody but does not itself act as an immunogen (or antigen) for the preparation of antibodies. Haptens are usually linked to an antigen carrier used to generate antibodies.

[0055] The terms "antigen carrier" or "immunogenic carrier" may be used interchangeably and refer to such a group or moiety that, when conjugated to a hapten and injected into a mammal or otherwise used as an immunogen, induces an immune response and elicits the production of antibodies that bind the hapten. The molecular weight range (in daltons) of the 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 a molecular weight of from about 25,000 to about 250,000. 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 (biomaterials and synthetic materials).

[0056] 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 homologous antigen through non-covalent interactions between the antibody-binding site of the antibody and the antigen. "Competitive binding" refers to the competition of two or more antigens or haptens for binding to an antibody, antigen carrier, or antigen-binding fragment in the case of a limited amount of antibody, antigen carrier, or antigen-binding fragment.

[0057] The term "biotin" refers to a substance that can be conjugated to an antigen / antibody and is essentially a vitamin.

[0058] The term "avidin" refers to a glycoprotein that can be extracted from egg white. Avidin includes avidin, neutravidin, streptavidin, etc.

[0059] The term "receptor-ligand affinity adsorption label" refers to a specific molecule or particle having a certain label (such as a fluorophore, an enzyme, a radioisotope, etc.).

[0060] Figure 1 The reaction process and test schematic diagram of the present invention are shown. The target antibody 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 a redox 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.

[0061] Figure 2A typical test result diagram (reduction peak of BQ) is shown, that is, a peak shape diagram of the relationship between current and antibody concentration, that is, the reduction peak signal of the target antibody being the oxidation product (BQ) of the oxygen-accepting compound (HQ); specifically, a linear diagram of the current and the concentration of the hepatitis B surface antibody (HbsAb) verified in an embodiment of the present invention, that is, the electrical signal of the target antibody analyte antibody being the oxidation product (BQ) of the oxygen-accepting compound (HQ) is plotted against the standard concentration, and the concentration standard curve of the hepatitis B surface antibody (HbsAb) is obtained.

[0062] Figure 3 The light excitation signal gradient diagram shows different concentrations of hepatitis B surface antibody (HbsAb).

[0063] Figure 4 A graph showing the relationship between current and hepatitis B surface antibody (HbsAb) concentration.

[0064] Figure 5 The diagram showing the consistency evaluation of the method of the present invention and the test methodology of the Roche biochemical analyzer is shown.

[0065] In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used can be purchased from commercial channels unless otherwise specified.

[0066] In the following examples, hepatitis B surface antibody (HbsAb) was used as the target antibody analyte; silicon dioxide (SiO2) was synthesized in step 1.1 of Example 1; tetraethyl orthosilicate (TEOS) was purchased from Sigma-Aldrich with a catalog number of 8006580250; 3-aminopropyltrimethoxysilane (APTMS) was purchased from Sigma-Aldrich with a catalog number of 281778-5mL; hydroquinone (HQ) was purchased from Sigma-Aldrich with a catalog number of H9003-100G; and p-benzoquinone (B Q) is the oxidation product of HQ; hepatitis B surface antibody (Ab) and hepatitis B surface antibody antigen (Ag) were purchased from Hangzhou Boyue Biotechnology Co., Ltd.; SiO2-HQ-Ab1 is a SiO2 microsphere coated with HQ coupled to hepatitis B surface antibody (HbsAb)-specific antigen Ag1; photosensitive microspheres (GG) were purchased from Xi'an Qiyue Biotechnology Co., Ltd.; streptavidin (SA) was purchased from Sigma-Aldrich, product number S6402-1ML; biotin coupled to hepatitis B surface antibody (HbsAb)-antigen (Bio-Ag2).

[0067] The present invention will be further described below in conjunction with embodiments:

[0068] Example 1. Synthesis of SiO2 microspheres

[0069] (1) Add 60 mL of cyclohexane, 5 mL of n-hexanol, 20 mL of Triton X-100, and 5 mL of purified water into a 250 mL washed conical flask. Place a magnetic stir bar with a diameter of 2 cm, adjust the rotation speed to 100 RPM, and stir for 20 min. Add 0.5 mL of ammonia water and 1 mL of TEOS, and continue stirring for 24 h. After stirring is completed, suck out the solution from the conical flask, add it into 2 50 mL centrifuge tubes respectively for centrifugation, and suck out the supernatant.

[0070] (2) Add 10 mL of absolute ethanol and 10 mL of purified water respectively, resuspend by ultrasound, centrifuge at a rate of 10000 RPM for 25 minutes, and take out the supernatant.

[0071] Repeat step (2).

[0072] (3) Add 6 mL of absolute ethanol and 6 mL of purified water respectively again, resuspend by ultrasound, centrifuge at a rate of 10000 RPM for 25 minutes, and take out the supernatant.

[0073] Repeat step (3)

[0074] (4) Add 3 mL of absolute ethanol to each for resuspension to prepare SiO2 microspheres for standby.

[0075] Example 2. Synthesis of SiO2-HQ microspheres

[0076] (1) Take out a 10 ml round-bottom flask, clean it, and rinse it once with pure water and ethanol respectively.

[0077] (2) Take 2 ml of the SiO2 microspheres synthesized in Example 1 and add them into the round-bottom flask, add 0.5 ul of ATPMS, and stir magnetically in the dark for 2 hours.

[0078] (3) Water bath at 65 °C for 30 minutes.

[0079] (4) Wash 3 times with absolute ethanol, 12800 RPM, 5 minutes.

[0080] (5) Add hydroquinone (4 mg / ml), 400 uL, and stir magnetically in the dark for 2 hours.

[0081] (6) Wash and centrifuge 3 times with absolute ethanol at 12800 RPM for 5 min and resuspend in 2 mL of absolute ethanol.

[0082] (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.

[0083] Repeat steps (2)-(7) 4 times.

[0084] (8) Add 2 mL of silica microspheres to a round-bottom flask, add 6 μL of ATPMS, and stir magnetically in the dark for 2 h.

[0085] (9) Heat in a water bath at 65 °C for 5 minutes, wash 3 times with absolute ethanol, centrifuge at 12800 RPM for 5 minutes, and resuspend in 2 mL of absolute ethanol.

[0086] Example 3: Synthesis of SiO2-HQ-Ag1 microspheres, i.e., SiO2 microspheres coated with HQ and hepatitis B surface antibody (HbsAb)-specific antigen Ag1

[0087] 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 pH 7.4 and dialyze overnight; centrifuge and add 76.39 μL of 10 mM PBS (pH 7.2) buffer solution, and sonicate.

[0088] Add 22.4 μL of hepatitis B surface antibody (HbsAb)-specific antigen Ag1 (5.0 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.

[0089] 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.

[0090] Add 100 μL of 10 mM PBS pH 7.4, take 80 μL and add it to the bottom A of a centrifuge tube, then freeze-dry, and store at -20 °C after freeze-drying to obtain SiO2-HQ-Ag1 microspheres for standby.

[0091] Example 4: Synthesis of GG-SA microspheres, i.e., photosensitive microspheres conjugated with streptavidin

[0092] Add 1 mL of photosensitive microspheres containing carboxyl groups 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 to remove the supernatant. Add 100 μL of (pH 6 50 mM MES) buffer, sonicate and resuspend, and add the antigen of 500 μg of hepatitis B surface antibody (HbsAb).

[0093] Place the above reaction solution at 37 °C for reaction for 2 hours. Add 10 μL of Gly and mix well by rapid oscillation. Then add 10 μL of 100 mg / mL BSA, mix well, and centrifuge to discard the supernatant. Add 100 μL of buffer solution (pH 6.5, 0 mM MES), sonicate to suspend the microspheres, mix well by oscillation, add all of it to the bottom B of the centrifuge tube, then perform lyophilization, and store it at -20 °C after lyophilization to obtain GG-SA microspheres for standby.

[0094] Example 5. Synthesis of Bio-Ag2, that is, biotinylation of hepatitis B surface antibody (HbsAb)-antigen

[0095] Add 36.3 μL of 10 mM PBS pH 7.2 buffer solution to a centrifuge tube respectively, add 11.8 μL of hepatitis B surface antibody (HbsAb)-antigen, and mix well.

[0096] Add 1.8 μL of biotin, mix well, and incubate with oscillation 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, and store it at 2 - 8 °C after lyophilization to obtain Bio-Ag2 for standby.

[0097] Example 6. Establishment of standard curve

[0098] (1) Incubation

[0099] Add 150 μL of purified water to the centrifuge tube containing the lyophilized substance prepared in Example 5 to obtain HbsAb antibody samples with different concentration gradients, determine the concentration using a Roche instrument, and add the above samples with different concentrations to the centrifuge tube respectively (sample concentrations are 0, 5.5, 11.71, 19.19, 38.33, 53.55 mIU / mL), invert and mix well up and down, incubate at 37 °C for 10 min, and the target antibody analyte antibody hepatitis B surface antibody (HbsAb, Ab), GG-SA, Bio-Ag2, SiO2-HQ-Ag1 form a sandwich sandwich (GG-SA-Bio-Ag2-Ab-Ag1-HQ-SiO2).

[0100] (2) Sampling

[0101] Refer to the method of CN114216949B, insert the screen-printed electrode into an electrochemical analyzer such as CHI660 or a portable electrochemical detector, suck an appropriate amount of the reacted solution with a dropper, and drop it onto the working surface of the screen-printed electrode. The solution needs to cover all three electrodes to ensure circuit conduction.

[0102] (3) Background signal test

[0103] After sample loading, the electrochemical analyzer runs the differential pulse voltammetry program with the following parameters: (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.

[0104] (4) Excitation signal test

[0105] The electrode surface is excited with light of wavelength 680 nm for 2 min. The photosensitive sphere undergoes excitation energy transfer to generate singlet oxygen, and the singlet oxygen reacts with the oxidized compound (HQ) within the diffusion range, continuously oxidizing HQ to BQ.

[0106] (5) Test the excitation signal

[0107] 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 hepatitis B surface antibody (HbsAb).

[0108] Example 7. Sample test

[0109] (1) Synthesis: Refer to the reagent synthesis in the establishment of the standard curve.

[0110] (2) Incubation: Add 150 μL of purified water to the centrifuge tube containing the above freeze-dried substance. Take 10 μL of the antibody sample and add it to the reaction cup. Invert and shake well. After incubating at 37 °C for 10 min, it is ready for testing. The target antibody analyte hepatitis B surface antibody (HbsAb), GG-SA, Bio-Ag2, and SiO2-HQ-Ag1 form a sandwich structure (GG-SA-Bio-Ag2-Ab-Ag1-HQ-SiO2)

[0111] (3) Sample loading: 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.

[0112] (4) Background signal test: After sample loading, the electrochemical analyzer runs the differential pulse voltammetry program with the following parameters (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.

[0113] (5) Excitation signal test: The electrode surface is excited with light at a wavelength of 680 nm for 2 min. The photosensitive sphere undergoes energy transfer upon excitation to generate singlet oxygen, which reacts with the oxidized compound (HQ) within the diffusion range to continuously oxidize HQ to BQ. At this time, the differential pulse voltammetry program with the parameters described in step (4) is run to test the excitation signal. Figure 3 The shown is the gradient diagram of light excitation signals at different concentrations.

[0114] (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 hepatitis B surface antibody (HbsAb) 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 = 0.9976.

[0115] Example 8. Performance evaluation

[0116] The test scheme described in Example 7 is used to test 20 clinical samples. At the same time, a Roche instrument (Roche E11) is used to synchronously test the above samples. Taking the test values of the Roche instrument as the X-axis and the test results of this method as the Y-axis, a graph is plotted to calculate the correlation. The results are as Figure 5 shown. The test results show that the method of the present invention has a good correlation with the test results of the Roche instrument, and the correlation coefficient R 2 = 0.9841.

[0117] Taking the hepatitis B surface antibody (HbsAb) item as an example, the precision of slightly inflamed and moderately inflamed samples at two medical decision levels is tested. This method is used to continuously test the two samples 10 times, and the precision CV is calculated.

[0118] Table 1 Precision evaluation

[0119]

[0120]

[0121] As shown in the results of Table 1, the precision CV of the hepatitis B surface antibody (HbsAb) at three concentrations tested by the kit of the present invention is less than 10%, indicating that the kit of the present invention has good precision.

[0122] 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 methods such as 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.

[0123] 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, characterized in that, The target analyte is a target antibody analyte, preferably a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment; The kit contains a first structural substance and a second structural substance that can bind to the target antibody 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 acceptor composition that can accept reactive oxygen species to generate a preset electrical signal; The first structural substance and the second structural substance are not both one of an antigen, a hapten, a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment at the same time; The target antibody 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 antibody 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 antibody analyte.

2. The kit of reagents according to claim 1, wherein, The first structural substance and the second structural substance are antigens, and the antigens can specifically bind to a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment; When the target antibody analyte contacts the first structural substance and the second structural substance, the photosensitive compound is excited by light of a certain wavelength to react with the oxygen acceptor compound to generate an electrical signal.

3. The kit according to claim 1, wherein the first structural substance and the second structural substance are haptens, and the haptens can specifically bind to a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment; When the target antibody analyte contacts the first structural substance and the second structural substance, the photosensitive compound is excited by light of a certain wavelength to react with the oxygen acceptor compound to generate an electrical signal.

4. The kit according to claim 2 or 3, wherein the first structural substance is not directly conjugated with the photosensitive compound, and the kit further contains 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 to generate 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 antibody analyte.

6. The kit according to claim 1, wherein the second structural substance is an antigen, and the first structural substance is 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 immunize the antigen, wherein: When the target antibody analyte contacts the first structural substance and the second structural substance, the target antibody analyte competes with the first structural substance to bind to the second structural substance, and the photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxygen acceptor compound to react with the oxygen acceptor composition of the second structural substance to generate an electrical signal.

7. The kit of reagents according to claim 1, wherein the second structural substance is a hapten, 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 hapten, wherein: When the target antibody analyte contacts the first structural substance and the second structural substance, the target antibody analyte competes with the first structural substance for binding to the second structural substance, and the photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxidized compound that reacts with the oxygen-containing composition of the second structural substance to generate an electrical signal.

8. The kit of reagents according to claim 1, wherein, 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, wherein: When the target antibody analyte contacts the first structural substance and the second structural substance, the target antibody analyte competes with the second structural substance for binding to the first structural substance, and the photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxidized compound that reacts with the oxygen-containing composition of the second structural substance to generate an electrical signal.

9. The kit of reagents according to claim 1, wherein, The first structural substance is a hapten, 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 hapten, wherein: When the target antibody analyte contacts the first structural substance and the second structural substance, the target antibody analyte competes with the second structural substance for binding to the first structural substance, and the photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxidized compound that reacts with the oxygen-containing composition of the second structural substance to generate an electrical signal.

10. The kit of reagents according to claim 1, wherein, 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, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen; When the target antibody analyte contacts the second structural substance and the third structural substance, the target antibody analyte competes with the third structural substance for binding to the second structural substance, wherein the photosensitive compound on the third structural substance is excited by light of a certain wavelength to generate an oxidized compound that reacts with the oxygen-containing composition of the second structural substance to generate an electrical signal.

11. The kit of reagents according to claim 1, wherein, The second structural substance is a 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 being a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, and the third structural substance being capable of receptor-ligand affinity adsorption with the first structural substance, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the hapten; When the target antibody analyte contacts the second structural substance and the third structural substance, the target antibody analyte competitively binds to the second structural substance with the third structural substance, wherein the photosensitive compound on the third structural substance is excited by light of a certain wavelength to generate an oxidized compound that reacts with the oxygen-containing composition of the second structural substance to generate an electrical signal.

12. The kit of reagents according to any one of claims 6 to 9, wherein the intensity of the electrical signal is negatively correlated with the quantity of the target antibody analyte.

13. A method for detecting a target antibody analyte, comprising: using the kit of reagents according to any one of claims 1 to 12 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 antibody analyte.

14. An instrument system for detecting a target antibody 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 12; 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 from the electrode module and process it to determine the presence and / or quantity of the target antibody analyte; Optionally, the liquid reaction module is a container capable of containing liquid, preferably a cuvette, a cuvette array, 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 12.

15. A target antibody analyte detection system, comprising: an electrode that adsorbs or conjugates an immunomolecule; photosensitive microspheres and an oxidized compound reagent, wherein the photosensitive spheres contain a photosensitive compound that generates reactive oxygen species capable of reacting with the oxidized 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.

16. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, the binding ligand is an antigen, the immunomolecule adsorbed or conjugated by the electrode is an antibody, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen; When the target antibody analyte contacts the photosensitive microspheres and the electrode, the target antibody analyte and the antibody 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.

17. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, the binding ligand is a hapten, the immunomolecule adsorbed or conjugated to the electrode is an antibody, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the hapten; When the target antibody analyte contacts the photosensitive microspheres and the electrode, the antibody target antibody analyte and the antibody 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.

18. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, the binding ligand is an antigen, the immunomolecule adsorbed or conjugated to the electrode is an antigen, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen; When the target antibody 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.

19. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, the binding ligand is a hapten, the immunomolecule adsorbed or conjugated to the electrode is an antigen, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the hapten or the antigen; When the target antibody 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.

20. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, the immunomolecule adsorbed or conjugated to the electrode is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, the detection system further comprises an antigen labeled with receptor-ligand affinity adsorption, and the antigen labeled with receptor-ligand affinity adsorption can undergo affinity adsorption with the photosensitive spheres, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen; When the target antibody analyte contacts the photosensitive microspheres and the electrode, the target antibody analyte and the antibody on the electrode competitively bind to the antigen labeled with receptor-ligand affinity adsorption, and the photosensitive compound is excited by light of a certain wavelength to react with the oxidizable compound reagent.

21. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, a ScFv or an antibody fragment, the immunomolecular antigen is adsorbed or coupled to the electrode, the detection system further includes an antigen 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; When the target antibody analyte contacts the electrode and the antigen labeled with receptor-ligand affinity adsorption, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxidized compound reagent.

22. A method for detecting a target antibody analyte, comprising: using the detection system according to any one of claims 15 to 21 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 antibody analyte.

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