Polymeric particle detection reagent, detection particle and detection sample preparation method

Abstract

The polymer particle detection reagent, the detection particles and the detection sample preparation method are used for target object detection, the detection particles in a sample form the detection sample, a to-be-detected object in the detection sample and the detection particles are combined and aggregated to form aggregated microparticles, the detection sample is subjected to image shooting, a detection sample image is obtained, and through image analysis, a detection result is obtained. The content of a target object in a detection sample is obtained based on the number or area information of the agglomerated microparticles, and a detection reagent B is included; the detection reagent B comprises detection particles; the surface of the detection particle comprises any one of antigen, antibody, protein or enzyme; the detection reagent B comprises a buffering agent, and the concentration of the buffering agent is 10-100 mmoL.

Description

Technical Field

[0001] This application belongs to the technical field of in vitro diagnostic immunology, and particularly relates to a polymer particle detection reagent, a detection particle, and a method for preparing a detection sample. Background Art

[0002] In vitro diagnostic immunology is a method for diagnosing diseases by detecting specific immune markers in body fluids. There are multiple different technical paths. The mature technologies include enzyme-linked immunosorbent assay, radioimmunoassay, immunoturbidimetry, immunofluorescence assay, and chemiluminescent immunoassay.

[0003] Chinese Patent Application No. "CN201410197209.1", "A Method for Detecting Biological Macromolecules or Microorganisms", in this patent, a photo of the aggregate is obtained, and the concentration of biological macromolecules or microorganisms in the sample to be detected is determined by quantifying the gray value of the photo.

[0004] This type of technology is used for target detection. Detection particles in the sample form a detection sample. The analyte in the detection sample binds and aggregates with the detection particles to form aggregated particulate bodies. An image of the detection sample is taken to obtain a detection sample image. Through image analysis, the content of the target in the detection sample is obtained based on the number or area information of the aggregated particulate bodies.

[0005] This type of technology is not yet mature. The diameter of the detection particles is in the micron level. How to prepare, store, and transport these particles requires in-depth research.

[0006] The applicant has conducted a large number of experiments and found that the detection particles are very easy to become ineffective. How to maintain the activity of the detection particles is the key to the industrial application of this type of technology.

[0007] The activation principle of EDC and NHS is to make carboxylic acids more reactive through chemical reactions, so that they can undergo coupling reactions with groups such as amino groups.

[0008] EDC, that is, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, is a commonly used carboxylic acid activation reagent. During the activation process, EDC reacts with carboxylic acids to form an unstable intermediate product - O-acylurea. This intermediate product has high reactivity and can undergo nucleophilic addition reactions with amino groups to form stable amide bonds. Through this process, carboxylic acids with relatively low reactivity become capable of efficient coupling with amino groups.

[0009] NHS, namely N-hydroxysuccinimide, is also a commonly used carboxylic acid activation reagent. When NHS reacts with carboxylic acid, an activated ester, N-hydroxysuccinimide ester, is formed. This activated ester has high reactivity and can undergo nucleophilic substitution reaction with amino groups to form amide bonds and N-hydroxy succinic acid. The key to the activation principle of NHS lies in its ability to enhance the reactivity of carboxylic acid by forming activated esters, thus promoting the coupling reaction with amino groups.

[0010] Blocking solution: The proteins in the blocking solution can bind to the blank positions on the surface and also bind to the membrane in the ways of mechanical filling (accumulation) and adsorption covering. Since there is "filling" and "covering" of protein binding sites to avoid non-specific binding of the primary antibody, there is the term "blocking". The blocking solution should block all unbound sites without replacing the target protein on the surface, not binding to the epitope of the target protein, and not having cross-reaction with antibodies or detection reagents. BSA is the most commonly used blocking solution, with a single component and suitable for most cases. Summary of the Invention

[0011] In this application, the inventor proposes to add detection microparticles into a liquid for storage and transportation. The liquid includes a buffer to prevent the detection microparticles from binding to form aggregated microparticles before storage, transportation or use, resulting in inoperability. The inventor also proposes to use a stabilizer to maintain the activity of antigens, antibodies, proteins or enzymes on the surface of the detection microparticles and extend the available time of the detection microparticles.

[0012] A polymeric microparticle detection reagent for detecting a target substance. In a sample, the detection microparticles form a detection sample. The analyte in the detection sample binds and aggregates with the detection microparticles to form aggregated microparticles. An image of the detection sample is taken to obtain a detection sample image. Through image analysis, the content of the target substance in the detection sample is obtained based on the number or area information of the aggregated microparticles. It is characterized in that it includes detection reagent B; in detection reagent B, there are detection microparticles; on the surface of the detection microparticles, there is any one of an antigen, an antibody, a protein or an enzyme; in detection reagent B, there is a buffer, and the concentration of the buffer is 10 mmol - 100 mmol.

[0013] It may be that the buffer includes one or more of tris, Hepes, PBS, MES. It may be that a stabilizer is further included. It may be that the stabilizer includes one or more of sucrose, trehalose, mannitol, BSA, glycine, casein, preservatives.

[0014] It may be that the mass concentrations of the optional components of the stabilizer are as follows: the sucrose concentration is 0.5%-10%, the trehalose concentration is 0.5%-5%, the mannitol concentration is 0.5%-3%, the BSA concentration is 0.1%-5%, the glycine concentration is 0.1%-5%, the casein concentration is 0.1%-1%, and the preservative concentration is 0.01%-0.5%.

[0015] It may be that the sucrose concentration is 1.0%-10%, the trehalose concentration is 0.5%-5%, the mannitol concentration is 0.5%-3%, the BSA concentration is 0.1%-1%, the glycine concentration is 0.1%-1%, the casein concentration is 0.1%-1%, and the preservative concentration is 0.1%-0.5%.

[0016] It may be that the detection reagent B is used for detecting transparent and impurity-free samples, and the samples include any one of serum, body fluid, urine without visible formed elements or fecal filtrate. It may be that the detection reagent B is used for any one of serum, body fluid, urine or fecal diluent.

[0017] It may be that the detection reagent B is stored or transported at low temperature, and the low temperature range is 2-8°C. It may also include detection reagent A, and detection reagent A includes a staining agent; detection reagent A and the detection reagent B are separately packaged and are mixed with the sample successively during use.

[0018] It may be that during use, first mix detection reagent A with the sample, and then take the mixed sample and mix it with detection reagent B again.

[0019] It may be that first mix detection reagent A and detection reagent B, and then take the mixed reagent and mix it with the sample again

[0020] It may be that the staining agent includes any one or more of methylene blue, new methylene blue, brilliant cresyl blue, toluidine blue, hematoxylin, neutral red, crystal violet, methyl green.

[0021] It may be that the sample includes any one of blood, serum, body fluid, urine or fecal diluent.

[0022] It may be that the surface of the detection particle includes an antigen or an antibody, and the particle and the particle conjugate are antigen-antibody binding.

[0023] It may be that the surface of the detection particle includes a protein or an enzyme, and the particle and the particle conjugate are affinity binding.

[0024] It may be that the detection particles are polymer particles, polymer particles or magnetic bead particles.

[0025] It may be that the surface of the detection particle includes two antigens or antibodies, and a fluorescent group or a quenching group is modified on the antigen or antibody. When the antigen or antibody is attracted and aggregated by the same antigen or antibody, the fluorescent group is quenched and no longer emits fluorescence. The more aggregated, the less fluorescence.

[0026] It may be that the surface of the detection particle includes two antigens or antibodies, and the two antigens or antibodies are modified with fluorescent groups. When the two antigens or antibodies are attracted and aggregated by the same antigen or antibody, the fluorescent groups emit fluorescence. The more aggregated, the more fluorescence. It may be that the diameter of the detection particle is greater than 0.1 micrometer.

[0027] A method for preparing a detection particle, the detection particle is used for detecting a target. The detection particle is added to a sample to form a detection sample. The analyte in the detection sample binds and aggregates with the detection particle to form an aggregated particulate body. An image of the detection sample is taken to obtain a detection sample image. Through image analysis, the content of the target in the detection sample is obtained based on the number or area information of the aggregated particulate body. The steps for preparing the detection particle include: Step A30: Coupling, taking microspheres and adding a coupling solution, mixing evenly; Step A40: Blocking, adding a blocking solution, mixing evenly, removing the supernatant to obtain a microsphere precipitate; Step A50: Final washing, adding a coupling solution, mixing evenly, removing the supernatant to obtain a microsphere precipitate; Step A51: Preservation, adding a preservation solution to the microsphere precipitate, filtering with a filter, and a syringe filter can be used for filtering to obtain the detection particle.

[0028] It may be that before Step A30, there is: Step A20: Activation, taking latex microspheres and adding a coupling solution, mixing evenly, removing the supernatant to obtain a microsphere precipitate.

[0029] It may be that the coupling solution includes NHS and EDC; the concentration of NHS is 1 - 100 mg / mL NHS, and the concentration of EDC is 1 - 100 mg / mL EDC.

[0030] It may be that the coupling solution includes NHS and EDC; the concentration of NHS is 10 mg / mL NHS, and the concentration of EDC is 10 mg / mL EDC.

[0031] It may be that the preservation solution includes a buffer and a stabilizer; the buffer includes any one or more of Tris, HEPES, and PBS; the buffer concentration is 10 - 200 mmol / L; the stabilizer includes one or more of sucrose, trehalose, mannitol, BSA, glycine, casein, and preservatives.

[0032] A method for preparing a detection sample, which is used for detecting a target substance. A reagent is added to a sample to form a detection sample. The substance to be detected in the detection sample binds and aggregates with detection particles to form aggregated particulate bodies. An image of the detection sample is taken to obtain a detection sample image. Through image analysis, the content of the target substance in the detection sample is obtained based on the number or area information of the aggregated particulate bodies. Detection reagent A is added to the sample; after mixing evenly, an intermediate sample is obtained; detection reagent B is added to the intermediate sample; after mixing evenly, a detection sample is obtained; the detection reagent A includes a staining agent, and the detection reagent B includes detection particles; the surface of the detection particles includes any one of an antigen, an antibody, a protein, or an enzyme.

[0033] It may be that the detection reagent B is stored or transported at a low temperature, and the low temperature range is 2 - 8°C.

[0034] It may be that the staining agent includes any one or more of methylene blue, new methylene blue, brilliant cresyl blue, toluidine blue, hematoxylin, neutral red, crystal violet, and methyl green.

[0035] It may be that the sample includes any one of blood, serum, body fluid, urine, or fecal diluent.

[0036] The technical effects include: adding detection particles to a liquid, and the detection particles are dispersed and suspended in the liquid, reducing the probability of natural aggregation.

[0037] The technical effects include: storing and transporting in a liquid; the liquid includes a buffer to further increase the storage time and extend the service life; it can prevent the detection particles from aggregating and forming aggregated particulate bodies before storage, transportation, or use, resulting in unusability.

[0038] The technical effects include: using a stabilizer to maintain the activity of the antigen, antibody, protein, or enzyme on the surface of the detection particles and extend the available time of the detection particles.

[0039] The technical effects include: in scenarios such as serum where staining is not required, only using the detection reagent B can form visible aggregated particulate bodies with antigens, antibodies, or other target substances in the serum.

[0040] The technical effects include: in scenarios such as other body fluids like urine or feces after centrifugation or filtration layering, only using the detection reagent B can also form visible aggregated particulate bodies with antigens, antibodies, or other target substances in the serum.

[0041] The technical effects include: if stored at a low temperature, the active maintenance time of the detection particles can be extended to more than 12 months.

[0042] The technical effects include: adopting this solution, the storage time of the detection particles is extended to be equivalent to the storage period of conventional reagents, enabling this type of technology to be industrially applied.

[0043] The technical effects include: By staining cells with detection reagent A, the detection ability of cells can be further improved, and at the same time, morphological detection and immunological detection of cells can be achieved.

[0044] The technical effects include: The staining agent and detection microparticles are separately prepared into two reagents. Although the use process is complex, the active maintenance time of the detection microparticles is effectively extended, enabling industrialization to be realized.

[0045] The technical effects include: Coupling and then blocking are adopted. After blocking, a preservation solution is added to maintain the activity of the detection microparticles and prevent natural aggregation of the detection microparticles. After filtration, the diameter range of the microparticles is made consistent, which is beneficial for subsequent pattern recognition processing.

[0046] The impossible becomes engineering-feasible. The preservation solution includes a buffer solution and a stabilizer. The detection microparticles are dispersed and suspended in the preservation solution, reducing the probability of natural aggregation.

[0047] The technical effects include: It is stored and transported in a liquid, and the liquid includes a buffer agent, further improving the storage time and extending the service life.

[0048] The technical effects include: Using a stabilizer to maintain the activity of antigens, antibodies, proteins or enzymes on the surface of the detection microparticles, extending the available time of the detection microparticles.

[0049] The technical effects include: First, the sample can be stained and diluted with detection reagent A; the volume of the diluted sample increases, and detection reagent B is added to enable the detection microparticles in detection reagent B to be effectively dispersed and prevent ineffective aggregation. Description of the Drawings

[0050] Figure 1 It is a schematic diagram of the steps of a method for detecting a target substance based on microparticle aggregation;

[0051] Figure 2 It is a schematic diagram of a substance to be detected that is invisible in the sample distribution;

[0052] Figure 3 It is a schematic diagram of the structures of traditional monoclonal antibodies, heavy-chain antibodies, and nanobodies;

[0053] Figure 4 It is a schematic diagram of an antibody or antigen coated on microparticles such as latex or magnetic beads;

[0054] Figure 5 It is a schematic diagram of the coated microparticles adsorbing and aggregating with each other;

[0055] Figure 6 It is a micrograph showing that the detection microparticles are relatively uniform without the target substance;

[0056] Figure 7It is a microscopic magnification image containing the target substance, where the detected particles show aggregation;

[0057] Figure 8 These are the particles with aggregation recognized by AI;

[0058] Figure 9 It is a schematic diagram of the detection area corresponding to one image when taking an array image;

[0059] Figure 10 It is a top view schematic diagram of a detection chip;

[0060] Figure 11 It is a cross-sectional schematic diagram of the detection cavity and the sample injection channel inside a detection chip;

[0061] Figure 12 It is a cross-sectional schematic diagram of the detection cavity, the sample injection channel, and the exhaust channel inside a detection chip;

[0062] Figure 13 It is a calculation method for detecting the content of the target substance based on the particle aggregation image;

[0063] Figure 14 It is a schematic diagram for obtaining the number or area or volume of aggregated particulate matter through AI image recognition;

[0064] Figure 15 It is a schematic diagram for obtaining the number or area or volume of aggregated particulate matter by binarizing the test image area;

[0065] Figure 16 It is a schematic diagram for obtaining an image by binarizing the test image area;

[0066] Figure 17 It is a schematic diagram of the method for preparing detected particles. Detailed implementation manners

[0067] The following further details the content of this application in conjunction with each attached drawing. It should be noted that the following is an illustration of the preferred embodiments of the present invention and does not constitute any limitation to the present invention. The illustration of the preferred embodiments of the present invention is only for the illustration of the general principle of the present invention. The numbers such as "first", "second", "A", and "B" involved in the present invention are only for the convenience of illustration and do not represent the sequence relationship in time or space. The combinations of letters and numbers such as "TA", "TB", and "H" involved in the present invention are only for the convenience of illustration, and the specific meanings are determined by the specific words they represent.

[0068] An aggregate particle detection reagent for detecting a target substance. In the sample, the detected particles form a detection sample. The substance to be detected in the detection sample binds and aggregates with the detected particles to form aggregated particulate bodies. An image of the detection sample is taken to obtain a detection sample image. Through image analysis, the content of the target substance in the detection sample is obtained based on the number or area information of the aggregated particulate bodies. It is characterized in that it includes detection reagent B; in detection reagent B, it includes detected particles; on the surface of the detected particles, there is any one of antigen, antibody, protein or enzyme; in detection reagent B, it includes a buffer, and the buffer concentration is 10 mmol - 100 mmol.

[0069] The detected particles are added to a liquid, and the detected particles are dispersed and suspended in the liquid, reducing the probability of natural aggregation. It is stored and transported in the liquid, and the liquid includes a buffer, further improving the storage time and extending the service life. It can prevent the detected particles from aggregating and forming aggregated particulate bodies before storage, transportation or use, resulting in unusability.

[0070] It can be that the buffer includes one or several of tris, Hepes, PBS, MES.

[0071] It can be that a stabilizer is further included.

[0072] It can be that the stabilizer includes one or more of sucrose, trehalose, mannitol, BSA, glycine, casein, preservative.

[0073] The stabilizer is used to maintain the activity of the antigen, antibody, protein or enzyme on the surface of the detected particles and extend the available time of the detected particles.

[0074] It can be that the mass concentration of the optional components of the stabilizer is: the sucrose concentration is 1.0% - 10%, the trehalose concentration is 0.5% - 5%, the mannitol concentration is 0.5% - 3%, the BSA concentration is 0.1% - 1%, the glycine concentration is 0.1% - 1%, the casein concentration is 0.1% - 1%, and the preservative concentration is 0.1% - 0.5%.

[0075] It can be that detection reagent B is used for detecting a sample that is transparent and free of impurities, and the sample includes any one of serum, body fluid, urine without visible formed elements or fecal filtrate.

[0076] It can be that detection reagent B is used for any one of serum, body fluid, urine or fecal diluent.

[0077] In scenarios such as serum where staining is not required, only detection reagent B can form visible aggregated particulate bodies with antigens, antibodies or other target substances in the serum. Simple image algorithms can be used for image processing to obtain the characteristics of the aggregated particulate bodies.

[0078] In scenarios where other body fluids such as urine or feces are centrifuged or filtered and layered, it is also possible to use only detection reagent B to form visual aggregated microparticles with antigen-antibody or other target substances in serum.

[0079] It can be that the detection reagent B is stored or transported at low temperature, and the low temperature range is 2 - 8°C.

[0080] After long-term repeated experiments, even with a liquid protective agent, that is, when the detection microparticles are in a liquid, at room temperature, the active maintenance time of the detection microparticles is very short and cannot be well applied to industrialization. If stored at low temperature, the active maintenance time of the detection microparticles can be extended to more than 12 months.

[0081] After the hospital purchases the reagent, if it is not used in time, it can only be discarded after the reagent expires, resulting in a large amount of waste and increasing the detection and use costs. With this solution, the storage time of the detection microparticles is extended to be equivalent to the storage period of conventional reagents, enabling this type of technology to be industrially applied.

[0082] It can be that it further includes detection reagent A, and detection reagent A includes a staining agent; detection reagent A and the detection reagent B are separately packaged and are mixed with the sample successively during use.

[0083] In the prior art, immunological detection is carried out through the gray scale or aggregation size of binary graphics, and it is impossible to distinguish cells and aggregated microparticles.

[0084] The applicant proposes immunological detection based on pattern recognition, so it has the ability to distinguish cells and aggregated microparticles. Staining the cells with detection reagent A can further improve the detection ability of cells and simultaneously realize morphological detection and immunological detection of cells.

[0085] Preparing the staining agent and the detection microparticles into two reagents respectively, although the use process is complex, effectively extends the active maintenance time of the detection microparticles, enabling industrialization to be achieved.

[0086] It can be that during use, first mix detection reagent A with the sample, and then take the mixed sample and mix it with detection reagent B again.

[0087] It can be that the staining agent includes any one or more of methylene blue, new methylene blue, brilliant cresyl blue, toluidine blue, hematoxylin, neutral red, crystal violet, methyl green.

[0088] It can be that the sample includes any one of blood, serum, body fluid, urine or feces diluent.

[0089] It can be that the surface of the detection microparticles includes an antigen or an antibody, and the binding of the microparticles to the microparticle conjugate is an antigen-antibody binding.

[0090] It may be that the surface of the detection particle includes a protein or an enzyme, and the binding between the particle and the particle conjugate is an affinity binding.

[0091] It may be that the detection particles are polymer particles, polymer particles or magnetic bead particles.

[0092] It may be that the surface of the detection particle includes two antigens or antibodies, and a fluorescent group or a quenching group is modified on the antigen or antibody. When the antigen or antibody is attracted and aggregated by the same antigen or antibody, the fluorescent group is quenched and no longer emits fluorescence. The more aggregated, the less fluorescence.

[0093] It may be that the surface of the detection particle includes two antigens or antibodies, and the two antigens or antibodies are modified with fluorescent groups. When the two antigens or antibodies are attracted and aggregated by the same antigen or antibody, the fluorescent groups emit fluorescence. The more aggregated, the more fluorescence.

[0094] It may be that the diameter of the detection particle is greater than 0.1 micrometer.

[0095] Such as Figure 17 A method for preparing detection particles, the detection particles are used for detecting a target. The detection particles are added to a sample to form a detection sample. The analyte in the detection sample binds and aggregates with the detection particles to form an aggregated particulate body. An image of the detection sample is taken to obtain a detection sample image. Through image analysis, the content of the target in the detection sample is obtained based on the number or area information of the aggregated particulate bodies. The steps for preparing the detection particles include: Step A30: Coupling. Take microspheres and add a coupling solution, mix well; Step A40: Blocking. Add a blocking solution, mix well, remove the supernatant to obtain a microsphere precipitate; Step A50: Final washing. Add a coupling solution, mix well, remove the supernatant to obtain a microsphere precipitate; Step A51: Preservation. Add a preservation solution to the microsphere precipitate and filter it with a syringe filter to obtain the detection particles.

[0096] By using coupling plus blocking, and adding a preservation solution after blocking, the activity of the detection particles is maintained while preventing the natural aggregation of the detection particles. After filtration, the particle diameter range is consistent, which is beneficial for subsequent pattern recognition processing.

[0097] It may be that before Step A30, there is: Step A20: Activation. Take latex microspheres and add a coupling solution, mix well, remove the supernatant to obtain a microsphere precipitate.

[0098] It may be that the coupling solution includes NHS and EDC; the concentration of NHS is 1 - 100 mg / mL NHS, and the concentration of EDC is 1 - 100 mg / mL EDC.

[0099] It may be that the preservation solution includes a buffer and a stabilizer; the buffer includes any one or more of Tris, HEPES, and PBS; the buffer concentration is 10-200 mmol / L; the stabilizer includes one or more of sucrose, trehalose, mannitol, BSA, glycine, casein, and preservatives.

[0100] The preservation solution includes a buffer and a stabilizer, which can detect the dispersion and suspension of microparticles in the preservation solution and reduce the probability of natural aggregation. When placed in a liquid for storage and transportation, the liquid includes a buffer agent, which can further extend the storage time and prolong the service life. It can prevent the detection microparticles from aggregating and forming aggregated microparticles before storage, transportation, or use, resulting in unusability.

[0101] Use a stabilizer to maintain the activity of antigens, antibodies, proteins, or enzymes on the surface of the detection microparticles and extend the usable time of the detection microparticles.

[0102] A method for preparing a detection sample for detecting a target substance. A reagent is added to a sample to form a detection sample. The analyte in the detection sample binds and aggregates with the detection microparticles to form aggregated microparticles. An image of the detection sample is taken, and a detection sample image is obtained. Through image analysis, the content of the target substance in the detection sample is obtained based on the number or area information of the aggregated microparticles. A detection reagent A is added to the sample; it is mixed evenly to obtain an intermediate sample; the intermediate sample is added with a detection reagent B; it is mixed evenly to obtain a detection sample; the detection reagent A includes a staining agent, and the detection reagent B includes detection microparticles; the surface of the detection microparticles includes any one of antigens, antibodies, proteins, or enzymes.

[0103] First, the detection reagent A can be used to stain and dilute the sample; the volume of the diluted sample increases, and the detection reagent B is added so that the detection microparticles in the detection reagent B can be effectively dispersed to prevent ineffective aggregation.

[0104] It may be that the detection reagent B is stored or transported at a low temperature, and the low temperature range is 2-8 °C. It may be that the staining agent includes any one or more of methylene blue, new methylene blue, brilliant cresyl blue, toluidine blue, hematoxylin, neutral red, crystal violet, and methyl green. It may be that the sample includes any one of blood, serum, body fluid, urine, or fecal diluent.

[0105] Such as Figure 1 , a detection method for detecting a target substance based on particle aggregation, including adding detection microparticles to a sample; the target substance in the sample binds to the detection microparticles to form particle conjugates; the particle conjugates aggregate to form aggregated microparticles; an image of the sample is taken under a microscope; the image is analyzed to obtain the number, area, or volume of the aggregated microparticles, and based on the binding degree of the target substance and the detection microparticles, the number or content of the target substance is obtained.

[0106] Such asFigure 2 For target substances in a sample, such as proteins, small molecules, amino acids, sugars, enzymes, etc., substances that cannot be observed or are difficult to observe under an optical microscope, they cannot be directly observed and detected using an ordinary optical microscope.

[0107] For example Figure 3 In an antigen-antibody detection system, through the reaction of specific antibodies with the substance to be detected, but the antibodies and antigens cannot be directly observed and detected under an ordinary optical microscope. Traditional monoclonal antibodies, heavy chain antibodies, and nanobodies cannot be directly observed under a traditional microscope.

[0108] For example Figure 4 When antibodies or antigens are coated on microparticles such as latex or magnetic beads, the diameter of the microparticles is greater than 0.1 μm, and the microparticles can be directly observed and detected under an ordinary optical microscope.

[0109] For example Figure 5 When the coated microparticles adsorb or aggregate with each other to form large agglomerated microparticle bodies, by observing the number, area, or volume of the agglomerated microparticle bodies under a microscope, the quantity or content information of the target substance can be obtained.

[0110] For example Figure 6 For the actually photographed detection microparticles, when there is no target substance in the sample, the detection microparticles are relatively evenly distributed, and only a very small number of microparticles aggregate into two small clusters. For large particle size detection microparticles (such as 1000 nm magnetic bead detection microparticles), when there is no target substance in the sample or the content is extremely low, the detection microparticles have good dispersibility.

[0111] For example Figure 7 For the actually photographed detection microparticles, when the sample contains the target substance, some microparticles aggregate into clusters. As the concentration of the corresponding target substance increases, the individually dispersed detection microparticles aggregate to produce aggregated detection microparticles, and the aggregated detection microparticles are clearly distinguishable after aggregation.

[0112] For example Figure 8 During detection, detection microparticles with a fixed concentration are added, the number of aggregated detection microparticles is identified, and the antigen concentration is quantified. By identifying and counting the microparticles that have aggregated into clusters, and based on the binding degree between the microparticles and the target substance, the quantity or content of the target substance can be calculated. The binding degree between the microparticles and the target substance is calculated based on the particle size, the amount of coated antibody, and the degree of aggregation.

[0113] The above detection method; the microparticles can be polymer microparticles or magnetic bead microparticles. The polymer microparticles can be polystyrene microspheres, i.e., latex microparticles. Polystyrene microspheres, i.e., latex microparticles, adsorb proteins. Through adsorption, the invisible microparticles can expand to visible microparticles, and by detecting the number and size of the visible microparticles, the amount of protein can be detected.

[0114] The surface of polystyrene can be modified in various ways, such as hydrophilic modification. The surface modification groups of polymer microparticles mainly include functional groups such as polysaccharides, acrylamides, polyvinyl alcohols, polyamines, etc. After modification, polystyrene can selectively adsorb different target substances. Therefore, the present invention can not only be applied to the detection of antibodies and antigens, but also to the detection of various target substances or non-target substances.

[0115] The above detection method; detecting microparticles adsorbed or conjugated with antibodies or antigens. The detection of antibodies and antigens is a very important detection item. The modification methods or coating methods of magnetic beads and microspheres in various existing technologies can all be applied to the modification and coating of microparticles in this application. The modified or coated microparticles can selectively aggregate when encountering the target to be detected.

[0116] The above detection method; the sample includes body fluids or excreta; body fluids include serum, plasma, whole blood, saliva, local body fluid effusions, and excreta include urine, feces; feces are diluted feces.

[0117] The above detection method; obtaining the sample volume according to the sample area and sample height corresponding to the captured image, and calculating the unit volume content of the aggregated microparticles according to the number of aggregated microparticles and the sample volume.

[0118] Such as Figure 9 , during the microscopic imaging process, the field of view is relatively small, and the volume corresponding to each imaging is very small. By selecting the sample area and sample height corresponding to the area of the image, the volume calculated for each imaging can be obtained. By increasing the number of captured images, a larger sample volume can be obtained, improving the accuracy of detection.

[0119] The above detection method; calculating the unit volume content of the target analyte according to the binding degree between the target analyte and the detection microparticles and the unit volume content of the aggregated microparticles.

[0120] Such as Figure 9 , by identifying the aggregated microparticles in the sample volume, the content or quantity per unit volume corresponding to the sample volume can be calculated, and the detection result can be converted into a measurement index in the existing technology, such as virus content, specific protein unit volume content, etc.

[0121] The above detection method; irradiating the aggregated microparticles with excitation light, and the aggregated microparticles are excited by the excitation light to emit fluorescence, obtaining the number, area, volume or fluorescence intensity of the aggregated microparticles emitting fluorescence.

[0122] Such as Figure 10, a detection chip for detecting antibodies and antigens, comprising a sample injection channel 1010, a detection cavity 1020, and a sample injection port 1011; the detection cavity is used to accommodate a sample; one end of the sample injection channel is communicated with the detection cavity; the other end of the sample injection channel is communicated with the sample injection port, and the sample injection port is communicated with the external atmosphere; the upper and lower parts of the detection cavity include transparent windows, and external illumination light can enter the detection cavity through the transparent windows; when placed horizontally, the sample injection port is higher than the top of the detection cavity; the sample includes detection particles, and the detection particles adsorb or conjugate with antibodies or antigens; the target analyte in the sample binds to the detection particles to form particle conjugates; the particle conjugates aggregate to form aggregated particulate bodies; through the transparent windows, an image of the sample can be taken; the number, area, or volume of the aggregated particulate bodies is obtained.

[0123] Such as Figure 11 , a cross-sectional schematic diagram of the detection cavity and the sample injection channel of the detection chip, the sample is accommodated inside the detection cavity 1120, the sample injection channel 1110 is communicated with the sample injection port 1111, the sample injection port 1111 is higher than the upper surface 1121 of the detection cavity, and the sample will not overflow from the sample injection port 1111. At the same time, due to the internal pressure of the liquid, air will be automatically discharged.

[0124] The upper and lower surfaces of the detection cavity are transparent windows, which can introduce light to obtain an image of the internal aggregated particulate bodies.

[0125] The above detection chip; the detection particles are polymer particles or magnetic bead particles; such as Figure 12 , it may further include an exhaust channel 1210 and an exhaust port 1211; one end of the exhaust channel is communicated with the detection cavity; the other end of the exhaust channel is communicated with the exhaust port, and the exhaust port is communicated with the external atmosphere; when placed horizontally, the exhaust port is higher than the top of the detection cavity.

[0126] The above detection chip; the polymer particles include polystyrene microspheres, i.e., latex particles.

[0127] According to the area of the sample corresponding to the captured image and the height of the detection cavity, the sample volume is obtained. The height H of the detection cavity is used to calculate the sample volume, and the sample volume = the area of the sample corresponding to the image × the height H of the detection cavity.

[0128] The above detection chip; according to the binding degree of the target analyte and the particles, and the unit volume content of the aggregated particulate bodies, the unit volume content of the target analyte is calculated. The unit volume content of the aggregated particulate bodies is calculated according to the number of the aggregated particulate bodies and the sample volume. The number of the aggregated particulate bodies is obtained from the image; the unit volume content of the aggregated particulate bodies = the number of the aggregated particulate bodies ÷ the sample volume; the unit volume content of the target analyte = the unit volume content of the aggregated particulate bodies × the binding degree of the target analyte and the particles.

[0129] Such as Figure 9, during the process of taking pictures, multiple pictures are taken to increase the volume of the sample. Among the taken images, all or part are selected, such as Figure 9 , the area of the selected image corresponds to an area inside the chip that is 0.3 mm long, 0.2 mm wide, and 0.4 mm high. The height is determined by the height of the internal cavity of the chip.

[0130] Furthermore, according to the binding degree between the target analyte and the microparticles, and the unit volume content of the aggregated microparticles, the unit volume content of the target analyte is calculated and obtained.

[0131] Within a certain concentration range, the binding degree between the target analyte and the microparticles is relatively fixed. Given the unit volume content of the aggregated microparticles, multiplying it by the binding degree gives the unit volume content of the target analyte. The binding degree of the microparticles is obtained through calibration experiments.

[0132] The sample can be serum, and the target analyte can be various viruses, bacteria, antibodies caused by various pathogens, and the microparticles are coated with the corresponding antigens.

[0133] A reagent for antibody-antigen detection includes detection microparticles, and the detection microparticles adsorb or couple with antibodies or antigens; during the detection process, the reagent is mixed with the sample; the target analyte in the sample binds to the detection microparticles to form a microparticle conjugate; the microparticle conjugates aggregate to form aggregated microparticles; an image of the aggregated microparticles is obtained, and by analyzing the number of aggregated microparticles in the image, the amount of antibodies or antigens in the sample is obtained.

[0134] The detection microparticles are added to various detection liquids, and the concentration of the detection microparticles can be pre-prepared so that the detection microparticles are dispersed in the detection liquid. During the test, it is added according to the volume ratio, which is convenient for the detection process. There are detection microparticles in the reagent, which can serve as the target for focusing of the optical microscopy system to assist in focusing. The detection microparticles can be polymer microparticles or magnetic bead microparticles. The polymer microparticles can be polystyrene microspheres, i.e., latex microparticles. The diameter of the detection microparticles can be greater than 0.1 μm. The diameter of the detection microparticles can be 0.3 - 3 μm. If the diameter is too small, such as less than 0.1 μm, it cannot be seen under a conventional microscope with a magnification of 40 times. If it is greater than 3 μm, the antigen-antibody binding force cannot cause it to aggregate, and it is difficult to form aggregates and thus impossible to perform detection.

[0135] Such as Figure 13, A calculation method for detecting the content of a target substance based on particle aggregation images, including selecting one or more microscopic test pictures as calculation selection pictures; selecting all or part of them as test image areas in the calculation selection pictures; obtaining the test sample volume according to the test image area and the sample height; obtaining the number, area or volume of aggregated particulate bodies in the test image area; obtaining the number or content of the target substance according to the number, area or volume information of the aggregated particulate bodies; and obtaining the content or number per unit volume of the target substance by dividing the number or content of the target substance by the test sample volume.

[0136] The method for calculating the sample volume can be to obtain the test image area by multiplying the pixel area of the image sensor by the number of pixel points in the test image area; obtain the test sample area by dividing the test image area by the microscope magnification; and multiply the test sample area by the test sample height to obtain the test sample volume.

[0137] Such as Figure 14 , To obtain the number, area or volume of aggregated particulate bodies in the test image area, it can be to obtain the number, area or volume of aggregated particulate bodies through AI image recognition.

[0138] Such as Figure 15 , To obtain the number, area or volume of aggregated particulate bodies in the test image area, it can be to binarize the test image area, calculate the area of the binarized image, and obtain the area of the large mass.

[0139] Such as Figure 16 , Photos of the target substance at 3 concentrations taken with the detection device of "Shenzhen Anlv Medical Technology Co., Ltd.", Picture A corresponds to a very low content of the target analyte in the sample, Picture B contains a small amount of the target analyte, and Picture C contains a high concentration of the target analyte. The pictures are binarized to obtain pictures A1, B1, and C1, and it can be clearly seen that the area of the aggregated particulate bodies changes with the content of the target analyte.

[0140] By AI image recognition of the number, area or volume of non-aggregated particulate bodies in the test image area, the more the number, area or volume of non-aggregated particulate bodies recognized, the less the content of the target substance.

[0141] The target substance can be a substance with antigenic activity, and the substance with antigenic activity includes any one of protein, nucleic acid, and polysaccharide.

[0142] The target substance can also be a substance with cell structure, and the substance with cell structure includes any one of platelets, blood parasites, and red blood cells.

[0143] Although the present invention has been illustrated and described with reference to preferred embodiments and several alternatives, the invention is not limited to the specific descriptions in this specification. Other additional alternatives or equivalent components may also be used to practice the present invention.

Claims

1. A polymer particle detection reagent for detecting a target. In the sample, the detected particles form a detection sample. The analyte in the detection sample binds and aggregates with the detected particles to form an aggregated particulate body. An image of the detection sample is taken to obtain a detection sample image. Through image analysis, the content of the target in the detection sample is obtained based on the number or area information of the aggregated particulate bodies. It is characterized in that, Comprising detection reagent B; In the detection reagent B, detection microparticles are included; on the surface of the detection microparticles, any one of antigen, antibody, protein or enzyme is included; In the detection reagent B, a buffer is included, and the concentration of the buffer is 10 mmol - 100 mmol.

2. The polymeric microparticle detection reagent according to claim 1, wherein, The buffer includes one or more of tris, Hepes, PBS, MES.

3. The detection reagent for aggregated microparticles according to claim 1, wherein It further includes a stabilizer.

4. The polymeric microparticle detection reagent according to claim 3, wherein, The stabilizer includes one or more of sucrose, trehalose, mannitol, BSA, glycine, casein, preservative.

5. The aggregation particle detection reagent according to claim 4, characterized in that, The mass concentration of the optional components of the stabilizer is: the sucrose concentration is 0.5% - 10%, the trehalose concentration is 0.5% - 5%, the mannitol concentration is 0.5% - 3%, the BSA concentration is 0.1% - 5%, the glycine concentration is 0.1% - 5%, the casein concentration is 0.1% - 1%, and the preservative concentration is 0.01% - 0.5%.

6. The aggregation particle detection reagent according to any one of claims 1 to 5, characterized in that, The detection reagent B is used for detecting a transparent and impurity-free sample, and the sample includes any one of serum, body fluid, urine without visible formed components or fecal filtrate.

7. The aggregate particle detection reagent according to any one of claims 1 to 5, characterized in that The detection reagent B is used for any one of serum, body fluid, urine or fecal diluent.

8. The aggregate particle detection reagent according to any one of claims 1 to 5, characterized in that, The detection reagent B is stored or transported at low temperature, and the low temperature range is 2 - 8°C.

9. The aggregation particle detection reagent according to any one of claims 1 to 5, characterized in that, It further includes detection reagent A, and the detection reagent A includes a staining agent; the detection reagent A and the detection reagent B are packaged separately and are mixed with the sample successively during use.

10. The polymeric microparticle detection reagent according to claim 9, wherein, It includes any one of the following features: TG10: During use, first mix the detection reagent A with the sample, and then take the mixed sample and mix it with the detection reagent B again; TG20: First mix the detection reagent A and the detection reagent B, and then take the mixed reagent and mix it with the sample again.

11. The aggregated particle detection reagent according to claim 9, wherein The staining agent includes any one or more of methylene blue, new methylene blue, brilliant cresyl blue, toluidine blue, hematoxylin, neutral red, crystal violet, methyl green.

12. The aggregation particle detection reagent according to claim 9, wherein The sample includes any one of blood, serum, body fluid, urine or fecal diluent.

13. The aggregation particle detection reagent according to claim 1, characterized in that, It includes any one or more of the following features; Feature TA10: On the surface of the detection microparticles, antigen or antibody is included, and the binding between the microparticles and the microparticle conjugate is antigen-antibody binding; Feature TA20: On the surface of the detection microparticles, protein or enzyme is included, and the binding between the microparticles and the microparticle conjugate is affinity binding; Feature TA30: The detection microparticles are polymer microparticles, polymer microparticles or magnetic bead microparticles; Feature TA40: On the surface of the detection microparticles, two antigens or antibodies are included, and fluorescent groups or quenching groups are modified on the antigen or antibody. When the antigen or antibody is attracted and aggregated by the same antigen or antibody, the fluorescent group is quenched and no longer emits fluorescence. The more the aggregation, the less the fluorescence; Feature TA41: On the surface of the detection microparticles, two antigens or antibodies are included, and the two antigens or antibodies are modified with fluorescent groups. When the two antigens or antibodies are attracted and aggregated by the same antigen or antibody, the fluorescent group excites fluorescence. The more the aggregation, the more the fluorescence; Feature TA50: The diameter of the detected particles is greater than 0.1 micrometer.

14. A method for preparing detection particles, the detection particles being used for detecting a target. A detection sample is formed by adding the detection particles into a sample. The analyte to be detected in the detection sample binds and aggregates with the detection particles to form agglomerated particulate bodies. An image of the detection sample is taken to obtain a detection sample image. Through image analysis, the content of the target in the detection sample is obtained based on the number or area information of the agglomerated particulate bodies, and is characterized in that, The steps for preparing the detected particles include: Step A30: Coupling. Take microspheres, add coupling solution, and mix evenly. Step A40: Blocking. Add blocking solution, mix evenly, remove the supernatant, and obtain microsphere precipitate. Step A50: Final washing. Add coupling solution, mix evenly, remove the supernatant, and obtain microsphere precipitate. Step A51: Preservation. Add preservation solution to the microsphere precipitate, filter with a filter, and obtain the detected particles.

15. The method for preparing the detection particles according to claim 14, wherein, Before step A30, it includes: Step A20: Activation. Take latex microspheres, add coupling solution, mix evenly, remove the supernatant, and obtain microsphere precipitate.

16. The method for preparing the detection particles according to claim 14, wherein, The coupling solution includes NHS and EDC; the concentration of NHS is 1 - 100 mg / mL NHS, and the concentration of EDC is 1 - 100 mg / mL EDC.

17. The method for preparing the detection particles according to claim 14, wherein, The preservation solution includes a buffer and a stabilizer; the buffer includes any one or more of Tris, HEPES, and PBS; the buffer concentration is 10 - 200 mmol / L; the stabilizer includes any one or more of sucrose, trehalose, mannitol, BSA, glycine, casein, and preservatives.

18. A method for preparing a test sample for detecting a target. In the sample, a reagent is added to form a test sample. The analyte in the test sample binds and aggregates with the detected particles to form aggregated particulate bodies. An image of the test sample is taken to obtain a test sample image. Through image analysis, the content of the target in the test sample is obtained based on the number or area information of the aggregated particulate bodies. It is characterized in that Add test reagent A to the sample; mix evenly to obtain an intermediate sample; Add test reagent B to the intermediate sample; mix evenly to obtain a test sample; The test reagent A includes a staining agent, and the test reagent B includes detected particles; the surface of the detected particles includes any one of an antigen, an antibody, a protein, or an enzyme.

19. The method for preparing a test sample according to claim 18, wherein, The test reagent B is stored or transported at a low temperature, and the low temperature range is 2 - 8 °C.

20. The method for preparing a detection sample according to claim 18, wherein, The staining agent includes any one or more of methylene blue, new methylene blue, brilliant cresyl blue, toluidine blue, hematoxylin, neutral red, crystal violet, and methyl green.

21. The method for preparing a detection sample according to claim 18, wherein, The sample includes any one of blood, serum, body fluid, urine, or fecal diluent.

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