Test device

Abstract

A test device, comprising a base plate, which is provided with a sample application zone and a result display zone, wherein the sample application zone is provided with a sample pad and a reagent layer; the result display zone comprises a test pad, which comprises a free segment not fixedly connected to the base plate and a fixed segment fixedly connected to the base plate, the free segment being close to the sample application zone; the free segment of the test pad is in linear contact with the lower surface of the reagent layer closest to the base plate; and the base plate is provided with a spacer that is located below the test pad so that the free segment of the test pad is not fixedly connected to the base plate. The test device has small difference in test performance, and the test results have good consistency and high accuracy.

Description

Detection device Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, specifically relating to detection devices and their applications. Background Technology

[0002] Clinically, disease diagnosis is performed by detecting the content or presence of a certain component (analyte) in samples such as blood, urine, and saliva.

[0003] Dry testing devices are a commonly used clinical diagnostic method. These portable devices or test cards are for single use only, preventing cross-contamination. They are also simple to operate and provide rapid testing. Results can be visually assessed or tested on demand using a portable analyzer, allowing doctors and patients to obtain quick and accurate results and take timely measures to prevent and reduce disease risks.

[0004] Common dry detection devices include test strip structures such as lateral flow detection devices, vertical flow detection devices, or detection devices that combine both lateral and vertical flow. Detection principles can employ chemical, immunological, electrochemical, or combined analytical methods. Depending on the detection purpose, they can be divided into quantitative and qualitative tests. Analytes in samples can include respiratory pathogens, digestive pathogens, metabolic hormones, chronic disease-related substances such as blood glucose, blood lipids, urea, uric acid, and creatinine, tumor markers, specific biochemical proteins such as C-reactive protein and homocysteine, allergens, myocardial markers such as cardiac enzymes, troponin, and myoglobin, and drug abuse substances such as morphine.

[0005] Lateral flow detection devices generally include a base plate, on which a sample application pad, a marking pad, a detection pad, and an absorbent pad are stacked sequentially from upstream to downstream. The liquid sample, dropped onto the sample application pad, flows sequentially through these pads. In current lateral flow detection devices, the detection pad is flatly adhered to the base plate, serving only to transfer the liquid. After detection, the intensity of the color in the test area on the detection pad is linearly correlated with the concentration of the analyte in the sample, thus determining the level of the corresponding analyte in the sample.

[0006] As shown in Figures 11 and 22, existing lateral flow chromatography detection devices generally include a base plate 105. On the base plate 105, a sample pad 101, a marker pad 102, a detection pad 103, and an absorbent pad 104 are sequentially stacked from upstream to downstream. Liquid samples dropped onto the sample pad 101 flow sequentially through the sample pad 101, marker pad 102, detection pad 103, and absorbent pad 104. In current lateral flow chromatography detection devices, the entire detection pad 103 is flatly adhered to the base plate 105. After detection, the intensity of the detection line color on the detection pad is linearly correlated with the concentration of the analyte in the sample, thus determining the level of the analyte in the sample.

[0007] Vertical flow detection devices generally include a base plate, on which a sample pad, a separation pad, and a detection pad are arranged sequentially from top to bottom. An observation hole is provided at the detection pad location on the base plate. Dry detection devices can also have various test strip formats with different detection pads spaced apart on the base plate, such as in urine multi-sample testing devices. Testing can be performed directly using the dry detection device, or the device can be assembled into a housing for testing as needed.

[0008] US Patent 4876067 employs a dry chemical detection device combining lateral and vertical flow. During detection, the sample is added to a plasma separation layer, flows through a reagent pretreatment layer to the detection pad, and the detection pad transfers the processed sample to the detection area. After sample addition, the analyzer presses down on a transparent cover film, bringing the reagent layer into contact with the detection pad, thereby generating a detectable color signal. The detection pad of the device is flatly adhered to a base plate, with the separation layer and reagent pretreatment layer located above one end of the detection pad, and the reagent layer and cover film located on the opposite side of the separation layer and reagent pretreatment layer, and not in contact with the detection pad before sample addition. US Patent 5302346 has a similar structure, with a mesh protective layer above the separation layer of the detection device.

[0009] When existing detection devices measure the content of analytes in blood samples, such as the content of alanine aminotransferase (ALT) in blood samples, the detection performance of different detection devices varies greatly, and the consistency is poor. Summary of the Invention

[0010] This invention overcomes the shortcomings of existing technologies by improving the structure of the detection device and enhancing its performance.

[0011] The first aspect of the present invention provides a detection device, including a base plate, a sample application area and a result display area on the base plate, the sample application area having a sample pad and a reagent layer, the result display area having a detection pad, the detection pad having a free segment not fixedly connected to the base plate and a fixed segment fixedly connected to the base plate, the free segment being close to the sample application area, the free segment of the detection pad being in linear contact with the lower surface of the reagent layer closest to the base plate, the base plate having a shim element located below the detection pad so that the free segment of the detection pad is not fixedly connected to the base plate.

[0012] Furthermore, the reagent layer is a labeling pad, and the detection device includes a sample pad, a labeling pad, a detection pad, and an absorbent pad that are attached to the base plate and sequentially overlapped from upstream to downstream.

[0013] Furthermore, the contact between the free segment of the detection pad and the lower surface of the marking pad is a linear contact.

[0014] Furthermore, the end of the marker pad closest to the sample pad is attached to the base plate, while the end of the marker pad closest to the detection pad is tilted upwards.

[0015] Furthermore, the reagent layer of the sample application area includes an enzyme layer, and the substrate also includes a colorimetric area. The sample application area and the colorimetric area do not directly contact each other. The detection pad located on the substrate transfers the components of the sample application area to the colorimetric area. There is a first gap between the enzyme layer and the substrate. The colorimetric area includes a colorimetric layer. There is a second gap between the colorimetric layer and the substrate. The detection pad includes a free segment and a fixed segment. At least a portion of the free segment is inserted into the first gap between the enzyme layer and the substrate and contacts the lower surface portion of the enzyme layer.

[0016] Furthermore, the sample application area includes, from top to bottom, a cover layer, a sample absorption layer, a substrate layer, a whole blood separation layer, and an enzyme layer. The outer edges of the cover layer, sample absorption layer, substrate layer, whole blood separation layer, and enzyme layer are fixed to the base plate from top to bottom by a first adhesive element.

[0017] Furthermore, the end of the developing layer furthest from the sample application area is connected to the substrate via a second adhesive element.

[0018] Furthermore, the materials used for the jacking elements are selected from polystyrene, polyvinyl chloride, and polyethylene terephthalate.

[0019] Furthermore, the height of the shim element is between 0.1mm and 1mm. Preferably, the height of the shim element is between 0.1mm and 0.5mm. More preferably, the height of the shim element is between 0.1mm and 0.4mm.

[0020] Furthermore, the shim element is located below the free section of the detection pad or in the fixed section of the detection pad.

[0021] A second aspect of the present invention provides a detection device combining vertical and lateral flow. The device includes a base plate, on which a sample application area and a colorimetric area are respectively disposed. The sample application area and the colorimetric area do not directly contact each other. A detection pad located on the base plate transfers the components from the sample application area to the colorimetric area. The sample application area includes an enzyme layer, and a first gap exists between the enzyme layer and the base plate. The colorimetric area includes a colorimetric layer, and a second gap exists between the colorimetric layer and the base plate. The detection pad includes a free section and a fixed section. A shim element is disposed below the detection pad to prevent the free section from adhering to the base plate. The fixed section of the detection pad is fixedly connected to the base plate. At least a portion of the free section is inserted into the first gap between the enzyme layer and the base plate and contacts the lower surface portion of the enzyme layer. The fixed section of the detection pad can be directly or indirectly fixedly connected to the base plate.

[0022] Furthermore, the shim element is located below the free section of the detection pad.

[0023] Furthermore, the free segment of the detection pad curves upward toward the enzyme layer side.

[0024] Furthermore, the shim element is located below the detection pad fixing section.

[0025] Furthermore, the sample application area also includes a cover layer, a sample absorption layer, a substrate layer, and a whole blood separation layer. The outer edges of the cover layer, sample absorption layer, substrate layer, whole blood separation layer, and enzyme layer are fixed to the base plate from top to bottom by a first adhesive element.

[0026] Furthermore, the end of the developing layer furthest from the sample application area is connected to the substrate via a second adhesive element.

[0027] Furthermore, the height of the shim element is between 0.1mm and 1mm. Preferably, the height of the shim element is between 0.1mm and 0.5mm. More preferably, the height of the shim element is between 0.1mm and 0.4mm.

[0028] Furthermore, the detection device is used to detect alanine aminotransferase (ALT).

[0029] Furthermore, the enzyme layer contains pyruvate oxidase and peroxidase.

[0030] Furthermore, the substrate layer contains L-alanine.

[0031] Furthermore, the colorimetric layer contains α-ketoglutaric acid and an indicator on the side facing the substrate.

[0032] A third aspect of the present invention provides a chromatography test strip, comprising a sample pad, a marking pad, a detection pad, and an absorbent pad that are pasted onto a base plate and sequentially overlapped from upstream to downstream. The detection pad includes a free section that is not fixedly connected to the base plate and a fixed section that is fixedly connected to the base plate. The free section is located on the side closer to the marking pad.

[0033] Furthermore, the free segment of the test pad curves upward and contacts the marking pad.

[0034] Furthermore, a shim element is provided below the free section of the detection pad, and the shim element is located on the base plate.

[0035] Furthermore, the contact between the free segment of the detection pad and the lower surface of the marking pad is a linear contact.

[0036] Furthermore, the end of the marker pad closest to the sample pad is attached to the base plate, while the end of the marker pad closest to the detection pad is tilted upwards.

[0037] Furthermore, the materials used for the jacking elements are selected from polystyrene, polyvinyl chloride, and polyethylene terephthalate.

[0038] Furthermore, the height of the shim element is between 0.1mm and 1mm. Preferably, the height of the shim element is between 0.1mm and 0.5mm. More preferably, the height of the shim element is between 0.1mm and 0.4mm.

[0039] In this invention, the contact area between the free section of the detection pad and the reagent layer closest to the base plate is reduced, for example, a linear contact is formed between the two. This effectively controls sample flow and diffusion, promoting more uniform diffusion of the sample and reactants on the detection pad (NC membrane), resulting in smaller differences in detection performance, better consistency of detection results, and improved accuracy of test results. Attached Figure Description

[0040] Figure 1 is a structural diagram of the front of the detection device of the control group in Example 4.

[0041] Figure 2 is a perspective view of the detection device in Figure 1.

[0042] Figure 3 is a structural diagram of the front of the detection device in Example 1.

[0043] Figure 4 is a perspective view of the detection device in Figure 3.

[0044] Figure 5 is a structural diagram of the front of the detection device in Example 2.

[0045] Figure 6 is a three-dimensional view of the detection device in Figure 5.

[0046] Figure 7 is a schematic diagram showing the detection device inserted into the analyzer, with the analyzer's optical sampling element not pressed onto the colorimetric layer.

[0047] Figure 8 is a schematic diagram showing the detection device inserted into the analyzer, with the analyzer's optical sampling element pressed against the colorimetric layer.

[0048] Figure 9 is a linear graph of the whole blood test results in Example 6.

[0049] Figure 10 is a linear graph of the plasma detection results in Example 6.

[0050] Figure 11 is a schematic diagram of the structure of a chromatography detection device in the prior art.

[0051] Figure 12 is a three-dimensional schematic diagram of the chromatography detection device in Figure 11.

[0052] Figure 13 is a structural diagram of the chromatography detection device of the present invention.

[0053] Figure 14 is a three-dimensional schematic diagram of the chromatography detection device in Figure 13.

[0054] Figure 15 is an exploded view of Figure 14.

[0055] Figure 16 is an enlarged view of point A in Figure 13.

[0056] Figure 17 is an enlarged view of section B in Figure 14.

[0057] Figure 18 is an exploded view of the test plate equipped with a chromatography detection device.

[0058] Figure 19 is a schematic diagram of sample addition and testing on the test plate. Detailed Implementation

[0059] The present invention will now be described in detail with reference to the accompanying drawings.

[0060] The detection device shown in Figures 3 to 6 includes a sample loading area 1, a color development area 2, and a detection pad 3 that transports the liquid sample from the sample loading area 1 to the color development area 2 (result display area). The detection pad 3 is fixed on the base plate 4.

[0061] The sample application area 1 has a reagent layer, which can be a single layer or multiple layers stacked together. A gap exists between the reagent layer and the substrate in the sample application area. The color development area 2 has a color development layer, with reagents applied to the side of the color development layer facing the substrate 4. A gap exists between the color development layer and the substrate.

[0062] The test pad 3 includes a free section 31 and a fixed section 32. A shim element 12 is provided below the test pad to prevent the free section 31 from being adhered to the base plate. The fixed section 32 of the test pad is fixedly connected to the base plate. At least a portion of the free section 31 is inserted into the gap between the reagent layer in the sample application area and the base plate, and contacts a portion of the lower surface of the bottommost reagent layer in the sample application area.

[0063] When the detection device is not in testing mode, the chromogenic layer 11 maintains a certain distance from the detection pad 3, and the chromogenic layer 11 does not contact the detection pad 3. When the detection device is in testing mode, the sample added to the sample application area and the reagent in the sample application area form a mixture that flows into the detection pad 3. The chromogenic layer 11 is pressed down and comes into contact with the detection pad 3. The reagent on the chromogenic layer comes into contact with the mixture on the detection pad 3 and reacts to obtain the test result.

[0064] Example 1: Alanine aminotransferase (ALT) detection device

[0065] Alanine aminotransferase (ALT) catalyzes the amino transfer between alanine and α-ketoglutarate to form pyruvate and glutamate. It is an important metabolic enzyme and is the most commonly used indicator of liver damage.

[0066] The reaction principle of the alanine aminotransferase (ALT) detection device is as follows:

[0067] As shown in Figures 3 and 4, the ALT detection device of the present invention includes a base plate 4, on which a sample application area 1 and a color development area 2 are respectively arranged. The sample application area 1 and the color development area 2 do not directly contact each other. A detection pad 3, which is adhered to the base plate 4, transfers the components of the sample application area 1 to the color development area 2. The reagents and samples in the sample application area 1 are transported to the color development area 2 via the detection pad 3. The reaction results of the sample and reagents are presented in the color development area 2, and the analyzer reads the color change information of the color development area to give the detection result.

[0068] As shown in Figures 3 and 4, the sample application area 1 includes a cover layer 5, a sample absorption layer 6, a substrate layer 7, a whole blood separation layer 8, and an enzyme layer 9 stacked together from top to bottom. The outer edges of the cover layer 5, sample absorption layer 6, substrate layer 7, whole blood separation layer 8, and enzyme layer 9 in the same direction are fixed to the base plate 4 by a first adhesive element 10. A first gap 14 exists between the bottommost enzyme layer 9 and the base plate 4. To ensure the space of the first gap 14 between the enzyme layer 9 and the base plate 4, the enzyme layer material has a certain rigidity, so that there is a certain distance between the enzyme layer and the base plate without external mechanical pressure; or the enzyme layer 9 slopes upward from the first adhesive element 10, so that there is a certain distance between the enzyme layer and the base plate without external mechanical pressure.

[0069] The topmost covering layer 5 has a mesh structure to assist the diffusion of the blood sample dropped onto it in the sample application area. The material of the covering layer is selected from, but is not limited to, polyester, nylon, polypropylene, etc. In this embodiment, polyester (SAATI, model 03-35K-400) is used.

[0070] The sample absorption layer 6 is located below the cover layer 5 and is used to absorb and store blood samples, allowing for slow release of the blood samples. In this embodiment, the material of the sample absorption layer 6 is selected from, but is not limited to, glass fiber, polyester, etc. In this embodiment, glass fiber (Hangzhou Jinyi Biotechnology Co., Ltd., model T8964) is selected.

[0071] Substrate layer 7 is located below sample absorption layer 6 and contains a substrate required for the alanine aminotransferase (ALT) detection reaction. In this embodiment, substrate layer 7 is treated with reagent 2, which includes 5% w / w L-alanine, buffer solution, and non-reactant, wherein the buffer solution and non-reactant are 50 mmol K-PBS at pH 8.5 and erythrocyte antibody, respectively. The material of the substrate layer is selected from, but is not limited to, filter paper, glass fiber, etc. In this embodiment, filter paper (Hangzhou Special Paper Co., Ltd., model A, 0.1 mm thick) is used.

[0072] The whole blood separation layer 8 (also known as a blood filtration membrane) is located below the substrate layer 7. The whole blood separation layer 8 is used to filter red blood cells and separate serum / plasma. The separated serum / plasma continues to flow downwards to the enzyme layer 9. The material of the whole blood separation layer 8 can be selected from, but is not limited to, glass fiber, cotton fiber, and mixtures of glass fiber and cotton fiber. In this embodiment, glass fiber, cotton fiber, mixtures of glass fiber and cotton fiber, polyethersulfone, etc., are used. In this embodiment, a mixture of glass fiber and cotton fiber (Glassray Technology (Shanghai) Co., Ltd., model 8121-6621) is used.

[0073] The bottom enzyme layer 9 contains the enzymes required for the alanine aminotransferase (ALT) detection reaction. In this embodiment, enzyme layer 9 is treated with reagent 3, which includes pyruvate oxidase 125 U / ml, thiamine pyrophosphate 0.4% w / w, flavin adenine dinucleotide 0.01% w / w, peroxidase 500 U / ml, buffer (100 mmol K-PBS, pH 8.5), and 92.8% w / w of non-reactants (the non-reactants include sugars (2% trehalose), inorganic salts (0.1% MgCl2), and 1.5% surfactant S-24). The material of the enzyme layer can be selected from, but is not limited to, filter paper, polyethersulfone, etc. In this embodiment, filter paper (Hangzhou Special Paper Co., Ltd., model A, 0.1 mm thick) is used.

[0074] The developing area 2 includes a developing layer 11. One end of the developing layer 11, away from the sample application area, is connected to the base plate 4 via a second adhesive element 15. The second adhesive element 15 has a certain height, creating a second gap 16 between the developing layer 11 and the base plate 4. The developing layer 11 is made of a material with a certain rigidity, preventing it from bending downwards and contacting the detection pad 3 without external mechanical pressure. A certain distance is maintained between the developing layer 11 and the detection pad 3. The material of the developing layer 11 is selected from, but is not limited to, transparent or translucent plastic sheets (Tekra Corporation, model Makrofol DE 6-2).

[0075] In this embodiment, the side of the color developing layer 11 facing the substrate 4 is coated with a reaction reagent (reagent 4). The reaction reagent includes a reaction substrate and a color developing agent. Specifically, it includes a reaction substrate of 1% w / w α-ketoglutarate and a color developing agent of 0.8% w / w 4-aminoantipyrine.

[0076] The detection pad 3 includes a free segment 31 and a fixed segment 32. The free segment 31 is not adhered to the base plate 4 and does not contact the base plate 4 without external mechanical pressure. The fixed segment 32 is adhered to the base plate 4. At least a portion of the free segment 31 is inserted into the first gap 14 between the enzyme layer 9 and the base plate 4 and is located below the enzyme layer 9. The raised leading edge 33 of the free segment 31 can contact the lower surface of the enzyme layer 9.

[0077] A shim element 12 is also fixedly disposed on the base plate. In one embodiment, the shim element 12 is located below the free segment 31 of the detection pad and within the first gap 14 between the enzyme layer 9 and the base plate 4. In a further embodiment, the shim element 12 is also located within the first gap 14 between the enzyme layer 9 and the base plate 4. The shim element 12 is adhered to the base plate, and the shim element 12 has a certain height so that the free segment 31 of the detection pad 3 overlapping on it is tilted upward. In this embodiment, the shim element 12 is located at the front end of the free segment 31 of the detection pad.

[0078] The detection pad fixing section 32, or most of the fixing section 32, is located at the second gap 16 between the color developing layer 11 and the base plate 4. Without external mechanical pressure, the color developing layer 11 will not bend downward and come into contact with the detection pad fixing section 32. There is a certain distance between the color developing layer 11 and the detection pad 3.

[0079] The material of the detection pad 3 is selected from, but not limited to, NC film, etc. In this embodiment, the detection pad uses NC film (Global Life Sciences Technologies (Shanghai) Co., LTD, model 80HP). The detection pad is treated with reagent 5, which includes S-24 at a concentration of 0.2% (by mass). The material of the elevation element 12 is selected from, but not limited to, polyethylene terephthalate (PET) and other materials that are not easily deformed. In this embodiment, PET (DuPont, model Melinex ST328) is used.

[0080] The preparation of the alanine aminotransferase (ALT) detection device includes the following steps:

[0081] Reagents required for configuring the detection device.

[0082] Reagent 2 (the reagent on the substrate layer) includes 5% w / w L-alanine, 50 mmol K-PBS pH 8.5, and red blood cell antibody.

[0083] Reagent 3 (the reagent on the enzyme layer) includes pyruvate oxidase 125 U / ml, thiamine pyrophosphate 0.4% w / w, flavin adenine dinucleotide 0.01% w / w, peroxidase 500 U / ml, 100 mmol K-PBS pH 8.5, and non-reactants 92.8% w / w, wherein the non-reactants include 2% trehalose, 0.1% MgCl2, and 1.5% surfactant S-24.

[0084] Reagent 4 (the reaction reagent on the chromogenic layer) includes the reaction substrate α-ketoglutaric acid 1% w / w and the indicator 0.8% w / w 4-aminoantipyrine.

[0085] Reagent 5 (the reagent on the test pad) contains S-24 at a concentration of 0.2% by mass.

[0086] Preparation of test pad 3: Immerse the NC membrane in reagent 5, remove the filter paper after immersion, remove excess reagent from the surface of the filter paper, and then dry it in an oven at 55℃.

[0087] Preparation of substrate layer 7: Immerse filter paper in reagent 2, remove the filter paper after immersion, remove excess reagent from the surface of the filter paper, and then dry it in an oven at 55°C.

[0088] Preparation of enzyme layer 9: Soak filter paper in reagent 3, remove the filter paper after soaking, remove excess reagent from the surface of the filter paper, and then dry it in an oven at 55℃.

[0089] Preparation of color development layer 11: Spray the solution of reagent 4 evenly onto the frosted plastic sheet and let it air dry naturally.

[0090] Assembly of the detection device: A shim element 12 is attached to a preset position on the base plate 4. Then, the detection pad 3 is attached to the preset position on the base plate 4, with the free section 31 of the detection pad overlapping the shim element 12. The fixed section 32 of the detection pad is attached to the base plate 4. A cover layer 5, a sample absorption layer 6, a substrate layer 7, a whole blood separation layer 8, and an enzyme layer 9 are stacked from top to bottom at the preset position on the base plate 4. Each layer is fixed to the base plate 4 with a first adhesive element 10 on its outer side in the same direction. The raised free section 31 of the detection pad 3 is inserted below the enzyme layer at the first gap 14 between the enzyme layer 9 and the base plate 4. The raised front edge 33 of the free section 31 contacts part of the lower surface of the enzyme layer 9. The free section 31 of the detection pad 3 overlaps the shim element 12 and needs to contact the lower part of the enzyme layer 9, thus creating a semi-finished large card of the detection device. Then, a roller cutter is used to cut the semi-finished large card of the detection device into single detection devices with a certain width as shown in Figure 4. In this embodiment, the width of a single detection device is 6 mm.

[0091] The lengths and widths of the cover layer 5, sample absorption layer 6, substrate layer 7, whole blood separation layer 8, and enzyme layer 9 are 5 mm and 6 mm, respectively. The length and width of the chromogenic layer 11 are 25 mm and 6 mm, respectively. The length and width of the base plate 4 are 80 mm and 6 mm, respectively. The length and width of the detection pad 3 are 17 mm and 6 mm, respectively. The front end of the free segment 31 of the detection pad 3 overlaps the lower surface of the enzyme layer 9 by approximately 1-4 mm. In this embodiment, the front end of the free segment 31 of the detection pad 3 overlaps the lower surface of the enzyme layer 9 by approximately 2-3 mm. The length direction refers to the longitudinal direction of the detection device from the sample application area 1 to the chromogenic area 2, and the width refers to the direction perpendicular to the longitudinal direction of the detection device.

[0092] Example 2

[0093] Figures 5 and 6 show another design of the ALT detection device according to the present invention. The difference between the detection device shown in Figures 5 and 6 and the detection device of Example 1 (shown in Figures 3 and 4) is that the elevation element 12 of the detection device in this embodiment is not located below the enzyme layer. In a further embodiment, the elevation element 12 is not located within the sample application area. In some embodiments, the elevation element is located within the chromogenic zone; in other embodiments, the elevation element is located at the fixed segment 32 within the chromogenic zone.

[0094] The detection device shown in Figures 5 and 6 includes a base plate 4, on which a sample application area 1 and a color development area 2 are respectively arranged. The sample application area 1 and the color development area 2 do not directly contact each other. A detection pad 3, which is adhered to the base plate 4, transfers the components of the sample application area 1 to the color development area 2. The sample application area 1 includes a cover layer 5, a sample absorption layer 6, a substrate layer 7, a whole blood separation layer 8, and an enzyme layer 9 stacked together from top to bottom. Each layer is fixed to the base plate 4 by a first adhesive element 10. The color development area 2 includes a color development layer 11, the end of which is connected to the base plate 4 away from the sample application area by a second adhesive element 15.

[0095] The detection pad 3 includes a free segment 31 and a fixed segment 32. The free segment 31 is not adhered to the base plate 4, meaning that the free segment 31 does not contact the base plate 4. At least a portion of the free segment 31 of the detection pad 3 is inserted into the first gap 14 between the enzyme layer 9 and the base plate 4, and the leading edge 33 of the free segment 31 contacts a portion of the lower surface of the enzyme layer 9. In this embodiment, the leading edge of the free segment 31 of the detection pad 3 overlaps the lower surface of the enzyme layer 9 by approximately 2mm-3mm.

[0096] A shim element 12 is provided between the fixed section 32 and the base plate 4. The shim element 12 is located in the color development area 2, and the leading edge of the shim element does not extend into the sample application area 1. The shim element 12 is located below the fixed section 32 of the test pad 3. The fixed section 32 is attached to the shim element, and the shim element 12 is attached to the base plate 4.

[0097] Example 3

[0098] Figures 7 and 8 illustrate the detection process of alanine aminotransferase (ALT) in a sample using an analyzer and detection device. The analyzer includes an optical sampling element 13, which acts as a pressure block, pressing down on the display layer 11 and bending it downwards to contact the detection pad 3. The detection device shown in Figure 4 or 6 is placed into the analyzer, and the sample to be tested is added to the cover layer 5 of the sample application area of ​​the detection device. The sample permeates through the cover layer 5, the absorption layer 6, the substrate layer 7, and the whole blood separation layer 8 to the enzyme layer 9. The sample and reagents reaching the enzyme layer are transferred to the detection pad 3. As the optical sampling element 13 of the analyzer moves downwards and presses against the chromogenic layer 11, the sampling element 13 moves further downwards, causing the chromogenic layer 11 to bend downwards under the mechanical pressure of the sampling element 13 and contact the detection pad 3. The reagents on the chromogenic layer 11 are dissolved by the sample on the detection pad 3 and come into contact with the sample and other reagents flowing onto the detection pad 3, triggering a color reaction. The optical system of the analyzer reads this color change and obtains the detection result.

[0099] Example 4: Performance Comparison of Detection Devices

[0100] Experimental Group 1: The detection device of Example 1 was used, wherein the height of the shim element 12 was selected from 0.2 mm, 0.3 mm and 0.4 mm, and is labeled as Experimental Group 1-1, Experimental Group 1-2 and Experimental Group 1-3 in Table 1, respectively. The detection pad 3 has a membrane layer of about 2 mm overlapping the enzyme layer 9.

[0101] Experimental Group 2: The detection device of Example 2 was used, wherein the height of the shim element was selected from 0.1 mm, 0.2 mm and 0.3 mm, and is marked as Experimental Group 2-1, Experimental Group 2-2 and Experimental Group 2-3 in Table 2, respectively. The detection pad 3 has a membrane layer of about 2 mm overlapping the enzyme layer 9.

[0102] Control group: The detection device of the control group is shown in Figures 1 and 2. The detection device of the control group is basically the same as that of Example 1 or Example 2. The difference is that the detection device of the control group does not have the shim element 12. The detection pad 3 is pasted on the base plate 4. The front end of the detection pad 3 is fully covered by the enzyme layer 9. The rear end of the detection pad is located between the color development layer and the base plate.

[0103] The plasma samples were tested using the same detection devices as the experimental group 1 and the control group. The ALT concentrations in the plasma samples were 40 U / L and 160 U / L, respectively. The experimental results are shown in Table 1.

[0104] The plasma samples were tested using the same detection devices as the experimental group 2 and the control group. The ALT concentrations in the plasma samples were 40 U / L and 160 U / L, respectively. The experimental results are shown in Table 2.

[0105] Table 1. Experimental results of experimental group 1

[0106] Table 2. Experimental results of experimental group 2

[0107] The coefficient of variation (CV), also known as the "coefficient of dispersion," is the ratio of the standard deviation of the original data to the mean of the original data. The CV value objectively compares the degree of dispersion between two sets of data. A small CV value indicates that the detection performance of different detection devices varies little and the consistency is good.

[0108] The test results show that the CV values ​​of both experimental groups 1 and 2 are better than those of the control group without the shim. The CV value of experimental group 1, which used a 0.4mm height shim, was relatively better.

[0109] Example 5: Precision and Accuracy Experiment of the Detection Device

[0110] Experimental Group 3: The detection device used in Experimental Group 1 of Example 4 with a thickness of 0.4 mm for the shim element was selected.

[0111] Control group: The detection device used in the control group of Example 4 was selected.

[0112] The ALT levels in whole blood samples were measured using the same detection devices as those used in experimental group 3 and the control group. The ALT concentrations in the whole blood samples were 10 U / L, 40 U / L, 80 U / L, and 160 U / L, respectively. The experimental results are shown in Table 3.

[0113] The ALT levels in plasma samples were measured using the same detection devices as those used in experimental group 3 and the control group. The ALT concentrations in the plasma samples were 10 U / L, 40 U / L, 80 U / L, and 160 U / L, respectively. The experimental results are shown in Table 4.

[0114] Table 3. Results of Whole Blood Test

[0115] Table 4. Plasma Test Results

[0116] The reference value for a biochemical analyzer refers to the concentration of alanine aminotransferase (ALT) in a whole blood sample tested by a biochemical analyzer, and is used as a standard to judge the accuracy of the test results. In this example, the biochemical analyzer is a HITACHI model 7100.

[0117] The deviation percentage is the percentage of the deviation compared to the reference value of the biochemical analyzer.

[0118] The criteria for judging whether a testing device is qualified are:

[0119] Precision: CV < 8.0%.

[0120] Accuracy: ALT < 40 U / L (< 0.68 μkat / L), deviation within ± 6 U / L (± 0.10 μkat / L); ≥ 40 U / L, ≤ 500 U / L (≥ 0.68 μkat / L, ≤ 8.50 μkat / L), deviation within ± 15%.

[0121] The test results show that the detection precision and accuracy of the detection device of the present invention are better than those of the control group.

[0122] Example 6 Linear Experiment

[0123] The ALT content in whole blood samples was determined using the detection device from Experimental Group 1 of Example 4, with a riser element thickness of 0.4 mm, and linear analysis was performed. The experimental results are shown in Table 5 and Figure 9.

[0124] The ALT content in plasma samples was determined using the detection device from Experimental Group 1 of Example 4, with a riser element thickness of 0.4 mm, and linear analysis was performed. The experimental results are shown in Table 6 and Figure 10.

[0125] Table 5. Results of linear experiments using whole blood as samples

[0126] Table 6. Results of linear experiments using plasma as a sample.

[0127] As can be seen from the test results of the linear experiment in Figures 9 and 10, the detection device of the present invention has good linearity and sensitivity.

[0128] As shown in Figures 13 to 17, the chromatography detection apparatus 100 includes a sample pad 101, a marking pad 102, a detection pad 103, and an absorbent pad 104, which are sequentially overlapped from upstream to downstream. The sample pad 101, marking pad 102, detection pad 103, and absorbent pad 104 overlap and adhere to a base plate 105. The detection pad 103 includes a detection line 171 and a control line 172.

[0129] The detection pad 103 includes a free section 131 not fixedly connected to the base plate and a fixed section 132 fixedly connected to the base plate 105. A shim element 106 is provided below the detection pad 103, preventing the free section 131 from being directly fixedly adhered to the base plate 105 and causing the free section 131 of the detection pad 103 to curve upwards. The front edge 133 of the curved detection pad contacts the lower surface of the marking pad 102. Compared to the chromatography detection apparatus shown in Figures 11 and 12, the contact between the detection pad 103 and the lower surface of the marking pad 102 changes from a large-area contact to a smaller-area linear contact. The end of the marking pad 102 near the sample pad is adhered to the base plate 105, and the end of the marking pad 102 near the detection pad 103 curves upwards, overlapping the front edge 133 of the free section of the detection pad. The shim element 106 is fixedly adhered to the base plate 105 and located below the free section 131 of the detection pad. The material of the shim is selected from, but is not limited to, polystyrene, polyvinyl chloride, and polyethylene terephthalate. The shape of the shim is not limited, as long as it can raise the free section and does not protrude beyond the entire detection device strip, that is, it can meet a certain height requirement. The shape is such as: cuboid, cube, cylinder, irregular shape, etc., and the height is between 0.1mm and 0.4mm.

[0130] As shown in Figure 18, the detection plate 200 includes a base plate 122 and a top cover 121. The chromatography detection device 100 shown in Figure 13 is installed between the base plate and the top cover. The top cover 121 has a sample application hole 123 corresponding to the sample pad, and a detection result observation hole 124 corresponding to the detection pad 13 of the chromatography detection device.

[0131] Example 7: Preparation and Detection Procedures of the Glycated Hemoglobin Chromatography Detection Apparatus

[0132] The detection of glycated hemoglobin (HbA1c) is of clinical significance for the timely diagnosis of thrombotic diseases and the monitoring of the efficacy of thrombolytic therapy. For example, elevated HbA1c levels can be observed in diseases such as deep vein thrombosis, pulmonary embolism, disseminated intravascular coagulation, and severe hepatitis, as well as after thrombolytic therapy.

[0133] The glycated hemoglobin chromatography detection device described in this embodiment adopts the structure of the chromatography detection device shown in Figures 13 to 17. The chromatography detection device 100 includes a sample pad 101, a labeling pad 102, a detection pad 103, and an absorbent pad 104, which are attached to a base plate 105 and overlapped sequentially from upstream to downstream. A lifting element 106 is provided below the end of the detection pad 103 near the labeling pad 102. The lifting element 106 causes the detection pad near the labeling pad to tilt upwards and not adhere to the base plate, resulting in a small contact area between the detection pad 103 and the labeling pad 102.

[0134] Detection principle:

[0135] The glycated hemoglobin chromatography detection device uses the principle of a double-antibody sandwich reaction and fluorescence immunoassay to quantitatively detect the concentration of glycated hemoglobin in human blood samples. When the sample is added to the sample pad of the chromatography detection device, the glycated hemoglobin in the sample reacts with the mouse anti-human glycated hemoglobin monoclonal antibody labeled with fluorescent particles in the labeling pad to form a complex. The complex migrates upward on the detection pad (NC membrane) through capillary action, reacts with the mouse anti-human hemoglobin monoclonal antibody pre-coated on the detection line of the detection pad, and forms the final complex deposited on the detection line (T line). The glycated hemoglobin content in the sample is proportional to the fluorescence signal intensity.

[0136] Goat anti-rabbit IgG antibody is pretreated on a labeling pad to bind with fluorescent particles. Rabbit IgG is pre-coated on the control line of the test pad. After the sample flows to the test pad, the goat anti-rabbit IgG antibody binds to the rabbit IgG, and the fluorescent particles are deposited on the control line (C line).

[0137] The dry fluorescence immunoassay analyzer uses a scanning chromatography detection device to obtain the corresponding fluorescence signal. The fluorescence signal is analyzed and processed to quantify the content of glycated hemoglobin.

[0138] Sample pad preparation method:

[0139] The material of the sample pad is selected from, but not limited to, glass fiber, polyester film, etc. In this embodiment, glass fiber (Ahlstrom, 8964) is used.

[0140] Method for preparing the marking pad:

[0141] (1) Add glycated hemoglobin antibody rapidly to a time-resolved fluorescent microsphere dispersion with a particle size of 190-210 nm and a concentration of 0.2-0.4 wr% at a ratio of 0.2-0.5 mg / mL, and stir rapidly with a stirrer. After reacting for 12-24 hours, add blocking solution (1% PEG, 0.1% NaN3) rapidly at a ratio of 0.1 mL / mL, react for 4-4.5 hours, centrifuge at 10000-20000 rpm for 15-30 minutes, discard the supernatant to obtain the precipitate, add reconstitution solution (25 mM Tris buffer, 1% BSA, 0.1% NaN3) to the precipitate at a ratio of 1:1, and sonicate to reconstitute to prepare mouse anti-human glycated hemoglobin antibody-time-resolved fluorescent microsphere complex.

[0142] (2) Biotin was rapidly added to a dispersion of time-resolved fluorescent microspheres with a particle size of 190-210 nm and a concentration of 0.2-0.4 wr% at a ratio of 0.2-0.5 mg / mL, and the mixture was stirred rapidly with a stirrer. After reacting for 12-24 hours, blocking solution (1% BSA, 0.1% NaN3) was rapidly added at a ratio of 0.1 mL / mL. After reacting for 4-4.5 hours, the mixture was centrifuged at 10000-20000 rpm for 15-30 minutes. The supernatant was discarded to obtain the precipitate. The preservation solution (25 mM Tris buffer, 1% BSA, 0.1% NaN3) was added to the precipitate at a ratio of 1:1, and the mixture was reconstituted by sonication to prepare the rabbit IgG-time-resolved fluorescent microsphere complex.

[0143] (3) Based on the titer, the above-mentioned mouse anti-human glycated hemoglobin antibody-time-resolved fluorescent microsphere complex and rabbit IgG-time-resolved fluorescent microsphere complex were mixed and diluted with diluent, then evenly spread on the labeling pad, and dried at 37°C for 12-24 hours. The material of the labeling pad is selected from, but not limited to, glass fiber, polyester film, etc. In this embodiment, glass fiber (Ahlstrom, 8964) was selected. The diluent is 25mM Tris buffer, which contains 1% BSA, 20% sucrose, and 0.1% NaN3.

[0144] Method for preparing the test pad:

[0145] The test pad has a test line (T line) and a control line (C line). The test line is coated with anti-human hemoglobin antibody, and the control line is coated with goat anti-rabbit antibody.

[0146] (1) The coating method for the detection line is as follows: the anti-human hemoglobin antibody is diluted to 2.0 mg / mL with buffer (0.01 M Tris buffer, 0.5% sucrose, 0.1% NaN3) and then coated, and then dried at 37℃ for 12-24 hours.

[0147] (2) The coating method for the quality control line is as follows: dilute the goat anti-rabbit antibody with buffer (0.01M PBS buffer, 10% sucrose, 0.1% NaN3) to 1.0.0mL and then coat it, and then dry it at 37℃ for 12-24 hours.

[0148] The material of the detection pad is selected from, but not limited to, nitrocellulose membrane (Sartorius, CN110, CN110 CN95 CN140), etc. In this embodiment, nitrocellulose membrane (Sartorius, CN110) is used.

[0149] Material of the shim element:

[0150] The material of the shim is selected from, but is not limited to, polystyrene, polyvinyl chloride, and polyethylene terephthalate. In this embodiment, polystyrene is used. The shape of the shim is not limited, as long as it can raise the free section without protruding too much from the overall detection device; that is, it only needs to meet a certain height requirement. Shapes include, for example, cuboids, cubes, cylinders, irregular shapes, etc., with a height between 0.1mm and 0.4mm. In this embodiment, a cuboid shim is selected, with a width W between 1mm and 5mm, and a height H between 0.1mm and 0.4mm. Furthermore, the length of the shim is the same as the width of the detection pad and the marking pad.

[0151] Assembly method of the test reagent:

[0152] A shim element 106 is attached to a predetermined position on the base plate 105. Then, the detection pad 103 is attached to a predetermined position on the base plate 104, wherein the free section 131 of the detection pad 103 near the marking pad overlaps the shim element 106 and is tilted upwards, while the other parts of the detection pad 103 are directly attached to the base plate 104. Then, the marking pad, sample pad, and absorbent pad are sequentially overlapped and adhered to the base plate according to the structure of the chromatography detection device.

[0153] Diluent:

[0154] 0.01M MOPS buffer containing 0.2% Tween 20 and 0.1% NaN3.

[0155] The detection process for glycated hemoglobin using the detection reagent described in this invention is as follows:

[0156] (1) Before testing, the sample, diluent, chromatography detection device and dry fluorescence immunoassay analyzer (analyzer model iFIA-100) should be restored to 15℃~30℃.

[0157] (2) Place the chromatography detection apparatus on a flat and clean surface. The chromatography detection apparatus should be used as soon as possible after opening the bag.

[0158] (3) Take 10 μL of sample and add it to a centrifuge tube containing 1000 μL of diluent. After capping the tube, shake it by hand 10 times or mix it with a mixer for 1 minute. Take 100 μL of the mixture and add it vertically to the sample well of the detection plate equipped with the chromatography detection device, as shown in Figure 19.

[0159] (4) Click the “Test” button on the screen of the dry fluorescence immunoassay analyzer to enter the test interface.

[0160] (5) Wait 5 minutes to read the test results.

[0161] Interpretation of test results:

[0162] The dry fluorescence immunoassay analyzer scans the detection line and control line of the chromatography detection device to obtain the corresponding fluorescence signal. The fluorescence signal is analyzed and processed, and the processed signal is substituted into the calibration curve to calculate and quantify the glycated hemoglobin content. If the result is less than 2.0%, it is reported as "<2.0%"; if the result is greater than 14%, it is reported as ">14.0%".

[0163] Example 8 compares the performance of chromatographic detection devices with different structures

[0164] Experimental group: The chromatography detection device of Example 7 was used, as shown in Figures 13 to 16. The width W of the shim was 1 mm, 2 mm, 3 mm and 5 mm, and the height H of the shim was 0.12 mm, 0.24 mm and 0.36 mm. The combination of the width and height of the shim is shown in Table 7.

[0165] Control group: The chromatography detection device shown in Figures 11 and 12 was used. The chromatography detection device of the control group was basically the same as that of Example 7. The difference was that the chromatography detection device of the control group did not have a shim element, and the entire detection pad was flatly pasted on the base plate.

[0166] Whole blood samples were analyzed using chromatographic detection devices for the experimental and control groups. The samples used were low-concentration whole blood samples with an HbA1C content of 6.3% and high-concentration whole blood samples with an HbA1C content of 10.2%. The experimental results are shown in Table 7.

[0167] Table 7

[0168] The coefficient of variation (CV), also known as the "coefficient of dispersion," is the ratio of the standard deviation of the original data to the mean of the original data. The CV value objectively compares the degree of dispersion between two sets of data. A small CV value indicates that the detection performance of the chromatographic detection device varies less and has good consistency.

[0169] The test results show that the CV value of the chromatography detection device with the elevation element (experimental group) is better than that of the control group without the elevation element. Among them, the chromatography detection device with an elevation element width of 2mm and a height of 0.24mm in the experimental group has a relatively better CV value.

Claims

1. A detection device, comprising a base plate, a sample application area and a result display area on the base plate, the sample application area comprising a sample pad and a reagent layer, and the result display area comprising a detection pad, characterized in that, The test pad includes a free section that is not fixedly connected to the base plate and a fixed section that is fixedly connected to the base plate. The free section is close to the sample application area. The free section of the test pad is in linear contact with the lower surface of the reagent layer closest to the base plate. The base plate is provided with a shim element located below the test pad so that the free section of the test pad is not fixedly connected to the base plate.

2. The detection device according to claim 1, characterized in that, The reagent layer is a labeling pad, and the detection device includes a sample pad, a labeling pad, a detection pad, and an absorbent pad that are attached to the base plate and overlapped sequentially from upstream to downstream.

3. The detection device according to claim 2, characterized in that, The contact between the free segment of the test pad and the lower surface of the marker pad is linear.

4. The detection device according to claim 2, characterized in that, The end of the marker pad closest to the sample pad is attached to the base plate, while the end of the marker pad closest to the test pad is raised upwards.

5. The detection device according to claim 1, characterized in that, The reagent layer in the sample application area includes an enzyme layer, and the substrate also includes a colorimetric area. The sample application area and the colorimetric area do not directly contact each other. The detection pad located on the substrate transfers the components of the sample application area to the colorimetric area. There is a first gap between the enzyme layer and the substrate. The colorimetric area includes a colorimetric layer. There is a second gap between the colorimetric layer and the substrate. The detection pad includes a free segment and a fixed segment. At least a portion of the free segment is inserted into the first gap between the enzyme layer and the substrate and contacts the lower surface portion of the enzyme layer.

6. The detection device according to claim 5, characterized in that, The sample application area includes, from top to bottom, a cover layer, a sample absorption layer, a substrate layer, a whole blood separation layer, and an enzyme layer. The outer edges of the cover layer, sample absorption layer, substrate layer, whole blood separation layer, and enzyme layer are fixed to the base plate from top to bottom by a first adhesive element.

7. The detection device according to claim 5, characterized in that, The end of the developing layer away from the sample application area is connected to the base plate via a second adhesive element.

8. The detection device according to claim 1, characterized in that, The materials used for the jacking elements are selected from polystyrene, polyvinyl chloride, and polyethylene terephthalate.

9. The detection device according to claim 1, characterized in that, The height of the shim is between 0.1mm and 1mm.

10. The detection device according to claim 1, characterized in that, The shim is located below the free section of the test pad or in the fixed section of the test pad.

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