A POCT detection device and detection method

Abstract

The present invention discloses a POCT detection device and a detection method, wherein the POCT detection device comprises: a base; a magnetic part, arranged on the top of the base; a substrate, arranged above the base and covering the magnetic part. The POCT detection method comprises: S1, preparing detection reagents and the above-mentioned POCT detection device, wherein the detection reagents comprise magnetic microspheres-first monoclonal antibodies and fluorescent microspheres-second monoclonal antibodies; S2, mixing the detection reagents with the sample solution and dripping them onto the position of the substrate corresponding to the magnetic part; S3, measuring the fluorescence intensity on the surface of the substrate to obtain the target content in the sample solution. The POCT detection device of the present invention has the characteristics of simple structure, simple operation and low cost, and the POCT detection method of the present invention has the characteristics of high detection efficiency, high sensitivity and high precision.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnostic reagents, and in particular to a POCT detection device and a detection method. Background Art

[0002] In vitro diagnostic reagents refer to reagents, kits, calibrators, quality control products, etc. that can be used alone or in combination with instruments, appliances, equipment or systems for in vitro testing of human samples (various body fluids, cells, tissue samples, etc.) in the prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation and prediction of genetic diseases.

[0003] The development trend of in vitro diagnostic reagents mainly includes two directions. One is chemiluminescence detection, which is a trace analysis method based on the principle that the concentration of the analyte in the chemical detection system and the chemiluminescence intensity of the system are linearly quantitatively related under certain conditions. It uses instruments to detect the chemiluminescence intensity of the system to determine the content of the analyte. It has the characteristics of high sensitivity, high precision, low detection efficiency and complex operation. The second is POCT detection, which is a new method that analyzes immediately at the sampling site, eliminating the complex processing procedures of the specimen in the laboratory test and quickly obtaining the test results.

[0004] POCT testing is mainly based on immunochromatographic reagent cards and microfluidic chips. The immunochromatographic reagent card is a heterogeneous reaction system with the disadvantages of low detection efficiency, low sensitivity and low precision. The microfluidic chip integrates the basic operation units such as sample preparation, reaction, separation and detection in the biological, chemical and medical analysis processes into a micron-scale chip to automatically complete the entire analysis process. It is a homogeneous reaction system with the characteristics of high detection efficiency, high sensitivity and high precision. However, the preparation process of the microfluidic chip is complicated, and the production cost and production line construction cost are high, which limits its promotion in the market.

[0005] Therefore, researching a low-cost, high-efficiency, high-sensitivity, and high-precision POCT detection device has great clinical and commercial value. Summary of the invention

[0006] In order to solve the above defects in the prior art, one of the objectives of the present invention is to provide a POCT detection device, which has the characteristics of simple structure, simple operation and low cost.

[0007] In order to solve the above-mentioned defects in the prior art, the second object of the present invention is to provide a POCT detection method with the characteristics of high detection efficiency, high sensitivity and high precision.

[0008] The technical solution provided according to one of the purposes of the present invention is as follows:

[0009] A POCT detection device, comprising:

[0010] Pedestal;

[0011] A magnetic member, disposed on the top of the base;

[0012] The substrate is arranged above the base and covers the magnetic part.

[0013] Furthermore, the magnetic member is arranged in the central area of ​​the base.

[0014] Furthermore, the magnetic member is a cylindrical structure, and the circular end surface of the magnetic member is in contact with the bottom surface of the substrate.

[0015] Furthermore, a groove matched with the magnetic component is formed on the top of the base, the magnetic component is arranged in the groove, and the top surface of the magnetic component is not lower than the top surface of the base.

[0016] Furthermore, a slot matching the substrate is provided on the top of the base, and the substrate is arranged in the slot.

[0017] Furthermore, a discontinuous protrusion is provided on the peripheral side of the slot to form a recess at the break of the protrusion, and in the vertical direction, the recess is lower than the top surface of the substrate.

[0018] Further, in the horizontal direction, the edge of the card slot is provided with an inner concave portion sunken toward the central area of ​​the card slot, and the recessed portion is arranged correspondingly to the inner concave portion.

[0019] Furthermore, the substrate is a corrosion-resistant smooth structure.

[0020] The technical solution provided according to the second object of the present invention is as follows:

[0021] A POCT detection method, characterized in that it comprises the following steps:

[0022] S1. Prepare detection reagents and the above-mentioned POCT detection device, wherein the detection reagents include magnetic microspheres-first monoclonal antibodies and fluorescent microspheres-second monoclonal antibodies;

[0023] S2, mixing the detection reagent and the sample solution and then dropping the mixture onto the position of the substrate corresponding to the magnetic element;

[0024] S3. After the liquid on the substrate is dried, the fluorescence intensity of the magnetic microspheres on the substrate is measured to obtain the content of the target substance in the sample solution.

[0025] Beneficial effects:

[0026] 1) The POCT detection device of the present invention comprises three components: a base, a magnetic component and a substrate, and has the characteristics of simple structure, convenient assembly and low cost.

[0027] 2) The POCT detection method of the present invention is implemented by combining the detection reagent with the POCT detection device. The detection reagent is mixed with the sample solution and then dripped onto the POCT detection device. The content of the target in the sample solution is obtained by observing the fluorescence intensity. The detection method of the present invention can quickly and accurately obtain the content of the target in the sample solution, and has the characteristics of high detection efficiency, high sensitivity and high precision.

[0028] The POCT detection principle of the present invention is: the detection reagent, i.e., the magnetic microsphere-first monoclonal antibody and the fluorescent microsphere-second monoclonal antibody, is mixed evenly with the sample solution, and then the mixed solution is dripped onto the POCT detection device in alignment with the position of the magnetic part. When the target is present in the sample solution, the first monoclonal antibody and the second monoclonal antibody sandwich the target, forming a layer structure of magnetic microsphere-first monoclonal antibody-target-second monoclonal antibody-fluorescent microsphere, so that the fluorescent microsphere covers the magnetic microsphere. When the mixed solution of the detection reagent and the sample solution is dripped onto the POCT detection device, due to the magnetic adsorption effect, the magnetic microspheres are adsorbed and aggregated by the magnetic part, and the surface of the magnetic microspheres is fluorescent, that is, the magnetic microspheres show photoluminescence, and the photoluminescence intensity is proportional to the content of the target. Therefore, by measuring the fluorescence intensity of the magnetic microspheres adsorbed by the magnetic part, the content of the target in the sample solution can be known, so as to achieve the purpose of accurate and quantitative detection. When there is no target in the sample solution, there is no coating relationship between the magnetic microspheres and the fluorescent microspheres. When the mixed solution of the detection reagent and the sample solution is dropped onto the POCT detection device, the magnetic microspheres are adsorbed and aggregated by the magnetic part due to the magnetic adsorption effect, and according to the coffee ring effect, the fluorescent microspheres that are not magnetically adsorbed flow away from the magnetic part toward the edge and aggregate at the edge, so that there is no fluorescence on the surface of the magnetic microspheres, and the magnetic microspheres do not exhibit photoluminescence, thereby determining that there is no target in the sample solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is an exploded view of the POCT detection device in the embodiment.

[0030] The meanings of the reference numerals are as follows:

[0031] 1. Base; 11. Groove; 12. Slot; 13. Protrusion; 14. Recess; 15. Inner concave; 2. Magnetic member; 3. Substrate. DETAILED DESCRIPTION

[0032] For better understanding and implementation, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0033] In the description of the present invention, it should be noted that the terms "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" etc. indicating directions or positional relationships are based on the directions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as a limitation on the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] See also Figure 1 The present invention provides a POCT detection device, which includes a base 1, a magnetic member 2 and a substrate 3, wherein the magnetic member 2 is arranged on the top of the base 1, the substrate 3 is arranged above the base 1 and covers the magnetic member 2, and the bottom surface of the substrate 3 is in contact with the top surface of the magnetic member 2. The POCT detection device of the present invention is assembled by the base 1, the magnetic member 2 and the substrate 3, and has the characteristics of simple structure, convenient assembly and low cost.

[0036] The present invention also provides a POCT detection method, comprising the following steps:

[0037] S1. Prepare detection reagents and the above-mentioned POCT detection device, wherein the detection reagents include magnetic microspheres-first monoclonal antibodies and fluorescent microspheres-second monoclonal antibodies;

[0038] S2. Add the detection reagent and the sample solution into the EP tube, and shake the EP tube for 1-2 minutes to mix the detection reagent and the sample solution evenly to form a mixed solution;

[0039] S3, drop the mixed solution onto the substrate 3 at the position aligned with the magnetic element 2, and after the liquid is completely dry, observe whether the position on the substrate 3 corresponding to the magnetic element 2 exhibits fluorescence, and then measure the fluorescence intensity to obtain the content of the target substance in the sample solution.

[0040] The POCT detection method of the present invention is realized by combining the detection reagent with the POCT detection device, the detection reagent is mixed with the sample solution and then dripped onto the POCT detection device, and the content of the target in the sample solution is obtained by measuring the fluorescence intensity of the position corresponding to the magnetic member 2 on the substrate 3. The detection method of the present invention can quickly and accurately obtain the content of the target in the sample solution, and has the characteristics of high detection efficiency, high sensitivity and high precision.

[0041] Compared with the immunochromatographic reagent card in the prior art, the POCT detection device and detection method of the present invention have the advantages of high detection efficiency, high sensitivity, high precision and low cost; compared with the microfluidic chip detection method in the prior art, it has the advantage of low cost and is suitable for large-scale promotion; compared with the chemiluminescence detection method in the prior art, the two have equivalent detection sensitivity and precision, but the present invention has the advantage of high detection efficiency.

[0042] The POCT detection principle of the present invention is: the detection reagent, i.e., the magnetic microsphere-first monoclonal antibody and the fluorescent microsphere-second monoclonal antibody, is mixed evenly with the sample solution, and then the mixed solution is dripped onto the substrate 3 of the POCT detection device in alignment with the position of the magnetic part 2. When the target is present in the sample solution, the first monoclonal antibody and the second monoclonal antibody sandwich the target, forming a layer structure of magnetic microsphere-first monoclonal antibody-target-second monoclonal antibody-fluorescent microsphere, so that the fluorescent microsphere covers the magnetic microsphere. When the mixed solution of the detection reagent and the sample solution is dripped onto the POCT detection device, due to the magnetic adsorption effect, the magnetic microsphere is adsorbed and aggregated by the magnetic part 2, and the surface of the magnetic microsphere is fluorescent, that is, the magnetic microsphere exhibits photoluminescence, and its photoluminescence intensity is proportional to the content of the target. Therefore, by measuring the fluorescence intensity of the magnetic microsphere adsorbed by the magnetic part 2, the content of the target in the sample solution can be known, so as to achieve the purpose of accurate and quantitative detection. When there is no target in the sample solution, there is no coating relationship between the magnetic microspheres and the fluorescent microspheres. When the mixed solution of the detection reagent and the sample solution is dripped onto the POCT detection device, the magnetic microspheres are adsorbed and aggregated by the magnetic member 2 due to the magnetic adsorption effect, and according to the coffee ring effect, other components such as the fluorescent microspheres that are not magnetically adsorbed flow away from the magnetic member 2 toward the edge and aggregate at the edge, so that there is no fluorescence on the surface of the magnetic microspheres, and the magnetic microspheres do not exhibit photoluminescence, thereby determining that there is no target in the sample solution.

[0043] Since the mixed solution of the detection reagent and the sample solution is dripped onto the substrate 3 in alignment with the position of the magnetic part 2, and when there is no target in the sample solution, other components such as fluorescent microspheres will flow toward the edge of the substrate 3 with the magnetic part 2 as the center, in order to prevent other components such as fluorescent microspheres from flowing out of the edge of the substrate 3, the magnetic part 2 in the POCT detection device of the present invention is arranged in the central area of ​​the top of the base 1, and the magnetic part 2 is arranged in the central area of ​​the bottom surface of the substrate 3. In order to better present the coffee ring effect, the magnetic part 2 of the present invention is a cylindrical structure, and the upper circular end surface of the magnetic part 2 is arranged to fit the bottom surface of the substrate 3.

[0044] Specifically, a groove 11 is provided on the top of the base 1, and the size of the groove 11 matches the size of the cylindrical magnetic member 2. The magnetic member 2 is arranged in the groove 11, so that the groove 11 limits the magnetic member 2, and prevents the magnetic member 2 from moving during the detection process and affecting the detection result. The top surface of the magnetic member 2 is not lower than the top surface of the base 1, so that the top surface of the magnetic member 2 can fit with the bottom surface of the substrate 3, so that the magnetic microspheres can be fully adsorbed.

[0045] Similarly, a card slot 12 is provided on the top of the base 1, and the size of the card slot 12 is adapted to the outer peripheral size of the substrate 3. The substrate 3 is arranged in the card slot 12, so that the card slot 12 limits the substrate 3 to prevent the substrate 3 from moving during the detection process and affecting the detection result. Specifically, the card slot 12 is formed by providing a protrusion 13 on the peripheral side of the base 1 so that the area surrounded by the protrusion 13 is concave. The protrusion 13 of the present invention is a discontinuous structure, which includes a plurality of protrusions 13 and is arranged at intervals, so that a recessed portion 14 is formed at the interval between the protrusions 13, and in the vertical height direction, the recessed portion 14 is lower than the top surface of the substrate 3, so that the substrate 3 can be taken out of the card slot 12 through the recessed portion 14, so that the substrate 3 can be easily replaced. During specific production, in the horizontal direction, the recessed portion 14 can be flush with the card slot 12.

[0046] Furthermore, in the horizontal direction, the edge of the card slot 12 is provided with an inner recess 15 that is recessed toward the central area of ​​the card slot 12, the position of the inner recess 15 is arranged corresponding to the position of the recess 14, and the substrate 3 covers the inner recess 15. When the substrate 3 needs to be removed, the operator can lift the substrate 3 upward at the inner recess 15, which makes it easier to remove the substrate 3.

[0047] In the present invention, the substrate 1 is a solid material with a certain hardness such as plastic or metal, the magnetic part 2 is a cylindrical part with good magnetism, and the substrate 3 is a corrosion-resistant smooth part such as glass or plastic to facilitate the formation of the coffee ring effect.

[0048] The POCT detection device of the present invention has consumables of substrate 3 and detection reagents. Compared with immunochromatographic reagent cards and microfluidic chip technology, the cost of the present invention is significantly reduced. In addition, the present invention attaches fluorescent microspheres to the periphery of the magnetic microspheres by attaching magnetic microspheres to the magnetic part 2 in the presence of a target, so as to concentrate the fluorescent light emission, thereby improving the detection sensitivity and accuracy. It should be particularly noted that the detection method of the present invention reads the fluorescence intensity after the liquid on the substrate 3 is completely dried. At this time, the fluorescent microspheres corresponding to the target content will all be attached to the magnetic microspheres, and the detection accuracy will be higher.

[0049] The specific application of the POCT detection device of the present invention is described in detail below in conjunction with a specific embodiment. Preparation of detection reagents: A D-dimer detection reagent, including tube A and tube B, both tube A and tube B are added with a base buffer and an active ingredient, the base buffer in tube A is a 10mM borate buffer with a pH of 8.0, and the active ingredient is a lyophilized powder of a D-dimer monoclonal antibody 1-magnetic microsphere conjugate 2μg (in actual applications, it can be 2-10μg, selected as needed), the base buffer in tube B is a 10mM borate buffer with a pH of 8.0, and the active ingredient is a lyophilized powder of a D-dimer monoclonal antibody 2-time-resolved fluorescent microsphere conjugate 2μg (in actual applications, it can be 2-10μg, selected as needed).

[0050] Detection device: invented POCT detection device.

[0051] Detection process: Take 20 μL of plasma sample and add it to tube A, then add 30 μL of phosphate buffer with a pH of 7.4, shake well and let it stand for 1 minute, then transfer all of it to tube B, shake well and let it stand for 1 minute, assemble the base 1, magnetic part 2 and substrate 3 into a POCT detection device, take 30 μL of the mixed solution and drop it on the position of the substrate 3 corresponding to the magnetic part 2, put the POCT detection device into a constant temperature and humidity incubator and incubate for 10 minutes before taking it out. At this time, all the liquid on the substrate 3 is evaporated, and the fluorescence intensity of the magnetic microsphere position on the substrate 3 is observed by naked eyes or instruments. The fluorescence intensity of the magnetic microsphere is proportional to the D-dimer content in the plasma sample, which is used for quantitative detection of D-dimer in the plasma sample.

[0052] Evaluation of test results: The detection limit of the POCT detection device of the present invention for D-dimer reaches 0.03 mg / L, the upper limit of the linear range is 12 mg / L, and the CV values ​​of the quality control products for detecting high, medium and low concentration levels are all within 8%, which is equivalent to the detection level of microfluidic chips on the market. The detection limit of common immunochromatographic reagent cards on the market is generally 0.1 mg / L, and the upper limit of the linear range is 10 mg / L. The CV values ​​of the quality control products for detecting high, medium and low concentration levels are generally lower than 15%.

[0053] This shows that the detection level of the POCT detection device of the present invention is equivalent to that of the microfluidic chip, but the detection cost is significantly reduced; the detection level of the POCT detection device of the present invention is higher than that of the immunochromatographic reagent card, and the detection cost is slightly lower than that of the immunochromatographic reagent card. Therefore, the POCT detection device and detection method of the present invention have the advantages of high calibration efficiency, high sensitivity, high precision, simple structure and low cost, and are suitable for large-scale promotion.

[0054] The technical means disclosed in the scheme of the present invention are not limited to the technical means disclosed in the above-mentioned implementation mode, but also include technical schemes composed of any combination of the above-mentioned technical features. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also regarded as the protection scope of the present invention.

Claims

1. A POCT detection method, characterized in that: The following steps are involved: S1. Prepare detection reagents and a POCT detection device, wherein the detection reagents include magnetic microspheres-first monoclonal antibodies and fluorescent microspheres-second monoclonal antibodies; Wherein, the POCT detection device comprises: a base; A magnetic member is arranged on the top of the base; the magnetic member is arranged in the central area of ​​the base; a groove matching the magnetic member is opened on the top of the base, the magnetic member is arranged in the groove, and the top surface of the magnetic member is not lower than the top surface of the base; A substrate is arranged above the base and covers the magnetic member; the magnetic member is a cylindrical structure, and the circular end surface of the magnetic member is in contact with the bottom surface of the substrate; the substrate is a corrosion-resistant smooth structure; a slot matching the substrate is provided on the top of the base, and the substrate is arranged in the slot; S2, mixing the detection reagent and the sample solution and then dropping the mixture onto the position of the substrate corresponding to the magnetic element; S3. After the liquid on the substrate is dried, the fluorescence intensity of the magnetic microspheres on the substrate is measured to obtain the content of the target substance in the sample solution.

2. A POCT detection method according to claim 1, characterized in that: A discontinuous protrusion is provided on the peripheral side of the slot to form a recess at the break of the protrusion. In the vertical direction, the recess is lower than the top surface of the substrate.

3. A POCT detection method according to claim 2, characterized in that: In the horizontal direction, an inner concave portion which is sunken toward the central area of ​​the slot is disposed on the edge of the slot, and the sunken portion is disposed correspondingly to the inner concave portion.

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