A fluorescent infiltration kit and its use method and preparation method

Abstract

The present application discloses a fluorescent percolation kit for detecting a target protein in a sample to be tested, comprising a reagent card, wherein the reagent card comprises a composite layer, a retention layer and a water absorption layer which are stacked in sequence, wherein the composite layer comprises a raw material labeled with a first fluorescent microsphere, the retention layer comprises the raw material, the raw material is used to bind to the target protein, the composite layer does not allow red blood cells to pass through, and the water absorption layer is used to absorb water in the sample to be tested. The fluorescent percolation kit provided by the present application has a simple structure, simple operation, fast detection speed, strong adaptability, small size, accurate results and good repeatability.

Description

Technical Field

[0001] The present application relates to the field of target protein detection, and in particular to a fluorescent filtration kit and a method for using and preparing the same. Background Art

[0002] Fluorescent immunochromatography is a new type of membrane detection technology based on antigen-antibody specific immune reaction. It combines the two advantages of high specificity of immune reaction and high sensitivity of fluorescence signal amplification, and is therefore widely used in the detection of target proteins in human / animal body fluid samples. In existing fluorescent chromatography immunoassays, the sample to be tested and the buffer solution must first be diluted in a certain ratio, and then the diluted sample to be tested is dripped into the sample slot on the fluorescent chromatography reagent card, so that the diluted sample to be tested contacts the chromatography test paper in the reagent card, and the test result is obtained after the chromatography test paper reacts with the sample to be tested for a period of time. Usually, the reagent card is mainly composed of four parts: a sample pad, a marking pad, a nitrocellulose membrane, and absorbent paper. Each part is connected in an overlapping manner and adhered to a polyester board substrate. Since the processing errors when cutting and overlapping these four parts of the material will affect the repeatability and detection accuracy of the reagent card, the fluorescent immunochromatography technology has certain disadvantages; not only that, when using fluorescent immunochromatography technology to chromatograph the samples to be tested, there needs to be enough distance for the samples to flow, which leads to a larger volume of the reagent card, which is not conducive to the miniaturization of the reagent detection instrument. Summary of the invention

[0003] The present application provides a fluorescent filtration kit and a method for using and preparing the kit, aiming to solve the technical problems in the prior art that the reagent card required for the fluorescent chromatography detection technology is large in size, which is not conducive to the miniaturization and automation of the detection instrument, and the chromatography detection technology requires a long detection time, which is not conducive to improving the detection efficiency.

[0004] In order to achieve the above-mentioned purpose, in a first aspect, the present application provides a fluorescent filtration kit for detecting a target protein in a sample to be tested, comprising a reagent card, wherein the reagent card comprises a composite layer, a retaining layer and a water absorbing layer stacked in sequence, the composite layer comprises a raw material labeled with a first fluorescent microsphere, the retaining layer comprises the raw material, the raw material is used to bind to the target protein, the composite layer does not allow red blood cells to pass through, and the water absorbing layer is used to absorb water in the sample to be tested.

[0005] In one embodiment of the present application, the composite layer includes a control protein labeled with a second fluorescent microsphere, and the retaining layer includes a control protein antibody, and the control protein antibody can bind to the control protein.

[0006] In one embodiment of the present application, the fluorescent material of the first fluorescent microsphere is selected from lanthanide metals, and the particle size of the first fluorescent microsphere is selected from one of 100 nm, 200 nm, and 300 nm.

[0007] In one embodiment of the present application, the fluorescent material of the first fluorescent microsphere is selected from trivalent europium, and the particle size of the first fluorescent microsphere is selected from 200 nm.

[0008] In one embodiment of the present application, in the stacking direction, the ratio of the area of ​​the retaining layer to the area of ​​the composite pad is 0.4-0.9.

[0009] In one embodiment of the present application, the reagent card further comprises an adhesive ring, which is an annular structure and is disposed between the retention layer and the water absorption layer. The inner diameter of the adhesive ring is 10%-40% of the diameter of the retention layer.

[0010] In a second aspect, the present application provides a method for using the fluorescent infiltration kit described above, comprising the following steps:

[0011] Dropping the sample to be tested on the composite layer, the target protein in the sample to be tested is combined with the raw material labeled with the first fluorescent microsphere in the composite layer to obtain a first conjugate, so that the first conjugate is filtered into the retention layer;

[0012] The first binding substance is combined with the raw material in the retention layer;

[0013] The composite layer is removed, the fluorescence signal intensity value of the first fluorescent microspheres on the retention layer is detected, and the concentration value of the target protein in the sample to be tested is calculated using a standard curve.

[0014] In a third aspect, the present application provides a method for preparing the fluorescent infiltration kit described above, comprising the following steps:

[0015] Using a sprayer or a spotter to coat the solution containing the raw material on the substrate to obtain the retention layer;

[0016] attaching the retaining layer on the water absorbing layer;

[0017] Using a sprayer to apply the raw material solution containing the first fluorescent microsphere marker on the base material treated with the treatment liquid to obtain the composite layer;

[0018] The composite layer is attached to the retaining layer.

[0019] In one embodiment of the present application, the treatment liquid includes buffer salt, anti-human red blood cell antibody, anti-HAMA antibody and surfactant, the buffer salt is selected from one of phosphate buffer, tris(hydroxymethylaminomethane)-hydrochloric acid buffer, and borate buffer, and the surfactant is selected from one of block copolymers of ethylene oxide and propylene oxide, polysorbate, and polyethylene glycol ether.

[0020] In one embodiment of the present application, the pH value of the buffer solution is 6.0-8.5, and in the treatment solution, the concentration of the anti-human red blood cell antibody is 0.001 mg / ml-2 mg / ml, and the concentration of the anti-HAMA antibody is 0.001 mg / ml-2 mg / ml.

[0021] Compared with the prior art, the fluorescent percolation kit provided by the present application has the characteristics of simple structure, simple operation, fast detection speed, strong adaptability, small volume, accurate results and good repeatability. The fluorescent percolation kit provided by the present application mainly includes a composite layer, a retention layer and a water absorption layer which are stacked in sequence. Due to the stacking arrangement, the structure of the kit is simple, and there is no requirement for the size of the reagent card. Reagent cards and kits of different sizes can be flexibly prepared according to actual needs, so that when the kit is quantitatively detected, there is no requirement for the volume of the detection instrument, which is conducive to the miniaturization of the kit detection instrument; in addition, the stacking arrangement rather than the splicing or overlapping arrangement is more conducive to the flow of the sample to be tested, and there will be no problem that the kit cannot be reused or the detection accuracy is low due to processing errors. Furthermore, the setting of the water absorption layer can absorb the moisture in the sample to be tested, accelerate the percolation of the sample to be tested from the composite layer to the retention layer, thereby shortening the reaction time between the sample to be tested and the reagent card, and improving the detection efficiency of the sample to be tested. Not only that, the composite layer does not allow red blood cells to pass through, and during testing, the composite layer will be removed to expose the retained layer, and only the fluorescent signal on the retained layer will be detected. This makes it possible to directly use whole blood samples for reaction and testing without the need for special pretreatment when testing whole blood samples, which greatly improves the detection efficiency of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 A schematic diagram of a fluorescent infiltration kit provided in one embodiment of the present application;

[0024] Reference numerals

[0025] 1. Sample to be tested; 2. Composite layer; 3. Retention layer; 4. Water absorption layer; 5. Outer shell; 6. Adhesive ring. DETAILED DESCRIPTION

[0026] The present application discloses a fluorescent filtration kit and a method for using and preparing the same. To make the purpose, technical solution and effect of the present application clearer and more specific, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific examples described herein are only used to explain the present application and are not intended to limit the present application.

[0027] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as generally understood by those skilled in the art in the art to which the present invention belongs. It should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with the meanings in the context of the prior art, and will not be interpreted with idealized or overly formal meanings unless specifically defined as herein.

[0028] First, as Figure 1 As shown, one embodiment of the present application provides a fluorescent filtration kit for detecting a target protein in a sample to be tested, including a reagent card, the reagent card including a composite layer, a retaining layer and a water absorption layer stacked in sequence, the composite layer including a raw material labeled with a first fluorescent microsphere, the retaining layer including the raw material, the raw material being used to bind to the target protein, the composite layer not allowing red blood cells to pass through, and the water absorption layer being used to absorb water in the sample to be tested.

[0029] The reagent card is a composite layer, a retention layer and a water-absorbing layer that are stacked in sequence, rather than being spliced ​​or overlapped, that is, the detection principle adopted by the kit provided by the present application is a percolation principle rather than a chromatography principle, thereby avoiding the problem of low reuse rate and low detection accuracy of the reagent card in the fluorescence chromatography technology. In addition, the percolation technology is adopted, compared with the chromatography technology, the reaction time between the sample to be tested and the reagent card is shortened, the detection time of the sample to be tested is greatly shortened, and the detection efficiency of the sample to be tested is improved. Moreover, the fluorescent percolation kit provided by the present application, because each layer in the reagent card adopts a stacking arrangement mode, the size, shape, etc. of the reagent card can be designed according to the actual test needs, that is, the structural design of the kit is more flexible and simple, and the volume of the kit can also be relatively small, so that it is not only convenient to carry, but also conducive to the miniaturization of the kit detection instrument. Not only that, because the composite layer does not allow red blood cells to pass, when performing the whole blood sample detection, there is no need to process the whole blood sample in advance, which greatly simplifies the detection step of the sample to be tested and improves the detection efficiency of the sample to be tested. The water absorption layer can absorb the water in the sample to be tested, thereby accelerating the infiltration of the sample to be tested, shortening the reaction time of the sample to be tested, and further improving the detection efficiency of the sample to be tested.

[0030] Furthermore, the composite layer includes a control protein labeled with a second fluorescent microsphere, and the retaining layer includes a control protein antibody, and the control protein antibody can bind to the control protein.

[0031] After the sample to be tested reacts with the reagent card, the composite layer needs to be removed and the fluorescent signal of the retained layer needs to be detected, so as to calculate the concentration of the target protein in the sample to be tested according to the standard curve. The setting of the control protein and the control protein antibody labeled with the second fluorescent microsphere makes it possible to correct the fluorescent detection signal, thereby reducing the result fluctuation caused by the external environment and operation errors, and greatly improving the accuracy of the detection of the sample to be tested.

[0032] Furthermore, if Figure 1 As shown, the fluorescent filtration kit provided in one embodiment of the present application also includes a shell, the shell is provided with a accommodating space with an opening at one end, the reagent card is arranged in the accommodating space, and the composite layer is arranged near the opening of the shell.

[0033] When the fluorescent percolation kit provided in this embodiment is used for testing a sample, the sample is dripped onto the composite layer from the opening of the housing, reacts with the substance in the composite layer, and then percolates into the retention layer.

[0034] Furthermore, if Figure 1 As shown, in the fluorescent filtration kit provided in one embodiment of the present application, the reagent card also includes an adhesive ring, which is arranged between the retaining layer and the water absorbing layer.

[0035] By setting the adhesive ring, the positions of the retention layer and the water absorption layer can be fixed to prevent the retention layer and the water absorption layer from moving or falling off; and the adhesive ring is ring-shaped, that is, the middle part is empty, so it will not affect the infiltration of the sample to be tested into the water absorption layer, and will not affect the absorption of water in the sample to be tested by the water absorption layer.

[0036] Furthermore, the adhesive ring is an annular structure, the inner diameter of the adhesive ring is 10%-40% of the diameter of the retaining layer, and the outer diameter of the adhesive ring is consistent with the diameter of the retaining layer.

[0037] That is to say, in the embodiment of the present application, the composite layer, the retention layer, and the water-absorbing layer are all circular structures. When the inner diameter of the adhesive ring is 10%-40% of the diameter of the retention layer, it can not only meet the fixation of the positions of the retention layer and the water-absorbing layer, so that the retention layer and the water-absorbing layer will not move or fall off, but also will not affect the infiltration of the sample to be tested from the retention layer to the water-absorbing layer, so that the water-absorbing layer can absorb the moisture in the sample to be tested to a better extent, thereby accelerating the infiltration of the sample to be tested, shortening the reaction time between the sample to be tested and the reagent card, and improving the detection efficiency of the sample to be tested.

[0038] Furthermore, the inner diameter of the adhesive ring is 25% of the diameter of the retaining layer.

[0039] When the inner diameter of the adhesive ring is 25% of the diameter of the retaining layer, the percolation effect of the sample to be tested between the retaining layer and the water absorbing layer is optimal, and the positions of the retaining layer and the water absorbing layer will not move or separate.

[0040] Furthermore, if Figure 1 As shown, in the stacking direction, the ratio of the area of ​​the retained layer to the area of ​​the composite layer is 0.4-0.9.

[0041] That is to say, the area of ​​the composite layer is larger than that of the retaining layer, so that the sample to be tested can be completely gathered in the retaining layer, making it easier to detect the fluorescent signal of the retaining layer.

[0042] In one embodiment of the present application, the fluorescent material of the first fluorescent microspheres is selected from lanthanide metals, and the particle size of the fluorescent microspheres is selected from one of 100 nm, 200 nm, and 300 nm.

[0043] Further, the fluorescent material of the first fluorescent microsphere is selected from one of trivalent lanthanum and trivalent europium. Preferably, the fluorescent material of the first fluorescent microsphere is selected from trivalent europium;

[0044] Furthermore, the particle size of the first fluorescent microsphere is selected from 200 nm.

[0045] In one embodiment of the present application, the first fluorescent microsphere and the second fluorescent microsphere have the same incident light wavelength and different emitted light wavelengths.

[0046] Furthermore, the wavelength of the incident light of the first fluorescent microsphere and the second fluorescent microsphere is 465 nm, the wavelength of the outgoing light of the first fluorescent microsphere is 585 nm, and the wavelength of the outgoing light of the second fluorescent microsphere is 480 nm.

[0047] In a second aspect, an embodiment of the present application provides a method for using the above-mentioned fluorescent infiltration kit, comprising the following steps:

[0048] The sample to be tested is dripped onto the composite layer, and the target protein in the sample to be tested is combined with the raw material labeled with the first fluorescent microsphere in the composite layer to obtain a first conjugate, and the first conjugate is filtered into the retention layer;

[0049] The first binding substance is combined with the raw material in the retention layer;

[0050] The water in the sample to be tested permeates into the water absorbing layer and is absorbed by the water absorbing layer;

[0051] The composite layer is removed, the fluorescence signal intensity value of the first fluorescent microsphere in the retained layer is detected, and the concentration value of the target protein in the sample to be tested is calculated using the standard curve.

[0052] Since the composite layer does not allow red blood cells to pass through, they will not reach the retention layer. During detection, the composite layer is removed to expose the retention layer, and only the retention layer is tested for fluorescent signals. This can prevent the red blood cells and interfering proteins in the sample to be tested from affecting the detection, greatly improving the accuracy of the detection.

[0053] Furthermore, the composite layer also contains a control protein labeled with a second fluorescent microsphere, and the retaining layer also contains a control protein antibody, and the control protein can bind to the control protein antibody.

[0054] Therefore, when the sample to be tested is detected, the first binding substance and the control protein labeled with the second fluorescent microspheres are filtered into the retention layer, and the control protein labeled with the second fluorescent microspheres will bind to the control protein antibody in the retention layer.

[0055] The setting of the control protein and the control protein antibody labeled with the second fluorescent microsphere allows the detection signal of the first fluorescent microsphere to be corrected according to the signal of the second fluorescent microsphere when detecting the sample to be tested, thereby reducing the fluctuation of the detection result of the sample to be tested caused by the external environment and operation errors, and greatly improving the accuracy of the detection result.

[0056] In a third aspect, an embodiment of the present application provides a method for preparing the above-mentioned fluorescent infiltration kit, comprising the following steps:

[0057] preparing a water absorbing layer;

[0058] Preparing the retention layer: selecting a substrate, and coating the solution containing the raw material on the substrate using a sprayer or a spotting machine to obtain the retention layer;

[0059] Preparation of the composite layer: selecting a base material, treating the base material with a treatment liquid, and using a sprayer to apply a raw material solution containing a first fluorescent microsphere marker on the base material treated with the treatment liquid to obtain a composite layer;

[0060] preparing an adhesive ring;

[0061] From bottom to top, the water absorbing layer, the adhesive ring, the retaining layer, and the composite layer are stacked in sequence to obtain a reagent card;

[0062] The reagent card is placed in a housing with a receiving space having an opening at one end, the composite layer is close to the opening of the receiving space, and the water absorption layer is arranged at the bottom of the receiving space away from the opening, thereby obtaining a fluorescent infiltration reagent kit.

[0063] Furthermore, the material used for the water-absorbing layer is selected from one of absorbent paper, absorbent cotton, non-woven fabric, and dust-free paper.

[0064] Furthermore, the substrate is selected from one of nitrocellulose membrane and polyester fiber. Preferably, the substrate is selected from nitrocellulose membrane.

[0065] Furthermore, the adhesive ring is a ring-shaped double-sided adhesive, which is used to fix the water absorbing layer and the retaining layer so that the positions of the water absorbing layer and the retaining layer do not move.

[0066] Furthermore, the base material is selected from one of glass fiber, polyester fiber, and non-woven fabric. Preferably, the base material is selected from glass fiber.

[0067] Furthermore, the treatment liquid includes buffer salt, anti-human red blood cell antibody, anti-HAMA antibody and surfactant, wherein the buffer salt is selected from one of phosphate buffer, tris(hydroxymethylaminomethane)-hydrochloric acid buffer and borate buffer, and the surfactant is selected from one of block copolymers of ethylene oxide and propylene oxide, polysorbate and polyethylene glycol ether.

[0068] Preferably, the buffer salt is selected from phosphate buffer and the surfactant is selected from polyethylene glycol ether.

[0069] Furthermore, the pH value of the buffer solution is 6.0-8.5, preferably, the pH value of the buffer solution is 7.0-7.5.

[0070] Furthermore, in the treatment solution, the concentration of the anti-human red blood cell antibody is 0.001 mg / ml-2 mg / ml, and the concentration of the anti-HAMA antibody is 0.001 mg / ml-2 mg / ml.

[0071] Preferably, in the treatment solution, the concentration of the anti-human red blood cell antibody is 0.1 mg / ml-1 mg / ml, and the concentration of the anti-HAMA antibody is 0.1 mg / ml-1 mg / ml.

[0072] Furthermore, in the stacking direction, the ratio of the area of ​​the retained layer to the area of ​​the composite layer is 0.4-0.9.

[0073] Furthermore, the inner diameter of the adhesive ring is 10%-40% of the diameter of the retaining layer.

[0074] It will be apparent to those skilled in the art that the present application is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present application. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the present application is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present application. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0075] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A fluorescent filtration kit for detecting a target protein in a sample to be tested, characterized in that: The reagent card comprises a composite layer, a retaining layer and a water absorbing layer which are stacked in sequence, the composite layer comprises a raw material marked with a first fluorescent microsphere, the retaining layer comprises the raw material, the raw material is used to bind to the target protein, the composite layer does not allow red blood cells to pass through, and the water absorbing layer is used to absorb water in the sample to be tested; The composite layer includes a control protein labeled with a second fluorescent microsphere, and the retaining layer includes a control protein antibody, and the control protein antibody can bind to the control protein; The preparation method of the fluorescent infiltration kit comprises the following steps: Using a sprayer or a spotter to coat the solution containing the raw material on the substrate to obtain the retention layer; attaching the retaining layer on the water absorbing layer; Using a sprayer to apply the raw material solution containing the first fluorescent microsphere marker on the base material treated with the treatment liquid to obtain the composite layer; attaching the composite layer on the retaining layer; The reagent card further comprises an adhesive ring, which is an annular structure and is arranged between the retention layer and the water absorption layer, and the inner diameter of the adhesive ring is 10%-40% of the diameter of the retention layer; The treatment solution comprises buffer salt, anti-human erythrocyte antibody, anti-HAMA antibody and surfactant.

2. A fluorescent infiltration kit according to claim 1, characterized in that: The fluorescent material of the first fluorescent microsphere is selected from lanthanide metals, and the particle size of the first fluorescent microsphere is selected from one of 100 nm, 200 nm, and 300 nm.

3. A fluorescent infiltration kit according to claim 2, characterized in that: The fluorescent material of the first fluorescent microsphere is selected from trivalent europium, and the particle size of the first fluorescent microsphere is selected from 200 nm.

4. A fluorescent infiltration kit according to claim 1, characterized in that: In the stacking direction, the ratio of the area of ​​the reserved layer to the area of ​​the composite layer is 0.4-0.

9.

5. The fluorescent infiltration kit according to claim 1, characterized in that: The buffer salt is selected from one of phosphate buffer, tris(hydroxymethylaminomethane)-hydrochloric acid buffer and borate buffer, and the surfactant is selected from one of block copolymer of ethylene oxide and propylene oxide, polysorbate and polyethylene glycol ether.

6. The fluorescent infiltration kit according to claim 5, characterized in that: The pH value of the buffer solution is 6.0-8.

5. In the treatment solution, the concentration of the anti-human red blood cell antibody is 0.001 mg / ml-2 mg / ml, and the concentration of the anti-HAMA antibody is 0.001 mg / ml-2 mg / ml.

7. A method for using the fluorescent filtration kit according to any one of claims 1 to 6 for non-diagnostic purposes, characterized in that: The steps include: Dropping the sample to be tested on the composite layer, the target protein in the sample to be tested is combined with the raw material labeled with the first fluorescent microsphere in the composite layer to obtain a first conjugate, so that the first conjugate is filtered into the retention layer; The first binding substance is combined with the raw material in the retention layer; The composite layer is removed, the fluorescence signal intensity value of the first fluorescent microspheres in the retained layer is detected, and the concentration value of the target protein in the sample to be tested is calculated using a standard curve.

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