WO2021012999A1 - 一种试剂卡、检测方法及应用 - Google Patents

一种试剂卡、检测方法及应用 Download PDF

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Publication number
WO2021012999A1
WO2021012999A1 PCT/CN2020/101988 CN2020101988W WO2021012999A1 WO 2021012999 A1 WO2021012999 A1 WO 2021012999A1 CN 2020101988 W CN2020101988 W CN 2020101988W WO 2021012999 A1 WO2021012999 A1 WO 2021012999A1
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WO
WIPO (PCT)
Prior art keywords
hole
sample
reaction
pad
reagent card
Prior art date
Application number
PCT/CN2020/101988
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English (en)
French (fr)
Inventor
黄静峰
Original Assignee
广东盛泽康华生物医药有限公司
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Filing date
Publication date
Application filed by 广东盛泽康华生物医药有限公司 filed Critical 广东盛泽康华生物医药有限公司
Priority to JP2022530340A priority Critical patent/JP7405978B2/ja
Priority to US17/627,614 priority patent/US20220276230A1/en
Publication of WO2021012999A1 publication Critical patent/WO2021012999A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces

Definitions

  • the invention relates to a reagent card, in particular to a reagent card for accurate and simple in vitro detection of a trace sample, a detection method and application.
  • POCT point-of-care testing
  • POCT refers to clinical testing and bedside testing performed next to the patient. It is usually not performed by the clinical laboratory examiner, but immediately analyzed at the sampling site, saving It is a new method to quickly get the test results by removing the complicated processing procedures of specimens in the laboratory.
  • Reagent cards are commonly used tools in point-of-care tests for testing biochemical substances in blood, urine, feces and other body fluids, such as bilirubin and protein, glucose, heme, ketone bodies, and nitrous acid in serum, plasma and urine Biomarkers such as salt and urobilinogen.
  • the reagent card is composed of a card housing (face cover and bottom cover) and a test strip in the card housing.
  • the test strip is fixed in the card slot of the bottom cover, and the face cover and the bottom cover are combined to form a reagent card.
  • the current rapid detection technology generally requires a sample volume of at least 10uL. However, some samples can only get 1uL or less in clinical practice, such as tear samples.
  • the sensitivity of the existing rapid detection technology is generally 0.05 ng/mL (fluorescence immunoassay 0.1 ng/mL, quantum dot 0.05 ng/mL), and the precision is generally 5% to 15%. If the lower limit of detection of the total amount of analyte is calculated: about 0.5pg, it is more difficult to test for small samples.
  • the detection requirements are relatively high; and in actual operation, since the tears, aqueous humor, and ocular surface lotion that can be collected from the eyes are all trace samples, the detection Difficulty; for example, patients with red and swollen eyes and conjunctival congestion, it is difficult for clinicians to determine whether the cause is caused by infection or allergy without the help of detection technology. Accurate and rapid diagnosis of the cause is very important for guiding medication; if it is caused by allergy, antiallergic drugs will be used.
  • antiviral or corresponding anti-amebic infection treatments will be used; if anti-allergic drugs or even hormones are used on the infected eyes due to misdiagnosis, the patient's condition may be more serious , And even cause serious medical accidents.
  • the present invention provides a reagent card.
  • the flow direction and chromatographic speed of the test liquid on the test strip are controlled to achieve the purpose of accurate detection of clinical micro-samples, which is used to solve the problem.
  • the invention provides a reagent card, comprising a face cover, a bottom cover, and a test strip; the face cover and the bottom cover fix the test strip;
  • a card slot structure and a raised platform are arranged inside the bottom cover; a supporting protrusion is arranged in the card slot structure, and the height of the supporting protrusion is less than or equal to the height of the raised platform;
  • the cover is provided with a fixed plate, a reaction window, a sample hole, and a detection liquid hole; the reaction window, a sample hole, and a detection liquid hole are arranged in sequence; the fixed plate is distributed on both sides of the reaction window;
  • test strip After the test strip is inserted into the slot structure of the bottom cover and fixed, the test strip is supported by the raised platform and the supporting protrusion, and the face cover and the bottom cover pass through positioning holes and positioning posts After the clamping, under the squeeze of the cover fixing plate and the detection liquid hole, the middle part of the test strip protrudes upward.
  • the test strip includes a three-layer or four-layer structure, the first layer is a bottom plate, the second layer is a reaction film, and the third layer is an absorbent pad, a bonding pad and a cushion pad; or the first layer is a bottom plate, The second layer is reactive film and cushioning pad, and the third layer is absorbent pad and bonding pad; or the first layer is bottom plate, the second layer is reactive film, the third layer is absorbent pad and bonding pad, and the fourth layer is cushioning pad. ;
  • the reaction membrane is arranged in the middle of the bottom plate, a detection line T and a quality control line C are distributed on the reaction membrane, and the detection line T and the quality control line C correspond to the reaction window;
  • One end of the reaction membrane is provided with a bonding pad, and the other end of the reaction membrane is provided with an absorbent pad, and the bonding pad corresponds to the sample hole;
  • a buffer pad is arranged on or under the bonding pad or the bonding pad is butted with the buffer pad, and the buffer pad corresponds to the detection liquid hole.
  • the number of the slot structure is two, which are distributed on both sides of the convex platform.
  • 1 to 5 supporting protrusions are provided in the slot structure.
  • three supporting protrusions are provided in the card slot structure
  • diversion ports are symmetrically provided on both sides of the slot structure corresponding to the detection liquid hole.
  • the depth of the slot structure is 1.2-1.7 mm
  • the height of the support protrusion is 0.1-0.15 mm
  • the height of the protrusion platform is 0.15-0.25 mm.
  • the number of the fixing plates is 4, which are respectively a first fixing plate for fixing the test strip, a second fixing plate for fixing the test strip, and a second fixing plate for fixing the test strip.
  • the widths of the first fixing plate, the second fixing plate, the third fixing plate and the fourth fixing plate are 3-5mm; the height of the first fixing plate and the second fixing plate are 1.0-1.5mm, and the third fixing plate , The height of the fourth fixing plate is 0.1-0.3mm.
  • the sample loading hole has a funnel-shaped structure
  • the diameter of the front circle of the face cover is 4-6mm
  • the diameter of the inner circle of the face cover is 3-5mm
  • the height of the protrusion is 0.1-0.2mm
  • the hole wall is 30°-45 to the horizontal plane. °Included angle.
  • the detection liquid hole has a funnel-shaped structure
  • the diameter of the front circle of the face cover is 6-8mm
  • the diameter of the inner circle of the face cover is 1-3mm
  • the height of the protrusion is 0.3-0.5mm
  • the hole wall is 30°-45 with the horizontal plane. °Included angle.
  • the reaction window is an elliptical ring structure
  • the front ellipse of the face cover is 150-200mm long and 70-90mm wide
  • the inner circle diameter of the face cover is 130-150mm
  • the height of the protrusion is 0.1-0.2mm.
  • the horizontal plane is at an angle of 30°-45°
  • the contact surface between the bottom of the reaction window and the test strip is a vertical protrusion of 0.01-0.02mm.
  • the face cover includes a hand-held end, and the surface of the hand-held end is a thread-like or convex-point-like convex structure.
  • the face cover and the bottom cover are clamped by positioning holes and positioning posts.
  • this application also provides a detection method using any of the above reagent cards:
  • Step (1) Add sample into the sample hole
  • the sample is added to the binding pad in the loading hole.
  • the binding pad has the antibody coupled with the nanosphere, that is, the antibody nanosphere.
  • the test substance in the sample is immunologically bound to the antibody nanosphere on the binding pad Form antibody nanosphere complexes;
  • Step (2) Add test solution to test solution hole
  • the test solution After adding the test solution to the test solution hole, the test solution starts to chromatograph along the direction of the absorbent pad end of the test strip.
  • the test solution chromatography passes through the loading hole to dissolve the antibody nanospheres on the binding pad that are not bound to the test substance.
  • the chromatography of the test solution on the test strip has undergone "climbing, parallel, and downhill", and the test solution moves on the test strip and is in "parallel”
  • the second immune recognition reaction is carried out in the stage.
  • the antibody nanosphere complex performs the second immune recognition reaction at the detection line T and starts to develop color; antibody nanospheres that do not react with the detection line T Then continue to move forward and combine with the quality control line C to develop color.
  • 0.1-50ul of the sample is added to the sample hole; 0-150ul of the test solution is added to the test solution hole.
  • the concentration of the analyte in the sample is calculated by instrument detection or color card comparison with the signal value or color depth of the T and C lines.
  • the above detection method is used for the detection of trace samples such as tear fluid, wound exudate, tissue fluid, sweat, aqueous humor, ocular surface lotion, etc., and can also be used for routine sample blood (peripheral blood, serum, plasma), Saliva, urine and other conventional samples; it can also be used for other non-clinical samples, such as pets, animal husbandry, agriculture, fish, food, environmental protection, etc.
  • the test strip Since the height of the support protrusion is less than or equal to the height of the protrusion platform, after the test strip is installed in the slot and fixed, the test strip forms a "bridge" structure under the support of the support protrusions in the slot at both ends ;
  • the fixing plate on the face cover, the sample hole, the detection liquid hole and the raised platform are squeezed downward, and the reaction film in the middle part is raised upward, and
  • the horizontal plane forms an "arch bridge" structure.
  • This "arch bridge” structure controls the flow direction and chromatographic speed of the test liquid on the test strip, thereby improving the accuracy and precision of the test results of the reagent card, achieving the purpose of accurately detecting clinically small samples and filling the existing The market for POCT micro-sample testing products is blank.
  • Figure 1 is a schematic diagram of the face cover
  • Figure 2 is a schematic diagram of the bottom cover
  • Figure 3a is a schematic diagram of Example 1 of the test strip
  • Figure 3b is a schematic diagram of Example 2 of the test strip
  • Figure 3c is a schematic diagram of Example 3 of the test strip
  • Figure 4 is a schematic diagram of a reagent card
  • Figure 5a is a schematic front view of the face cover
  • Figure 5b is a schematic diagram of the reverse side of the face cover
  • Figure 5c is a schematic cross-sectional view of the face cover
  • Figure 6a is a schematic front view of the bottom cover
  • Figure 6b is a schematic diagram of the reverse side of the bottom cover
  • Figure 6c is a schematic cross-sectional view of the bottom cover
  • Figure 7a is a schematic diagram of the disassembly of the reagent card
  • Figure 7b is a schematic cross-sectional view of the reagent card
  • Figure 8a is a schematic cross-sectional view of the assembled reagent card
  • Figure 8b is a partial enlarged view of the cross-section of the reagent card after assembly.
  • the embodiment of the present invention provides a reagent card to solve the problems existing in the prior art.
  • the present invention provides a reagent card, including a face cover, a bottom cover, and a test strip; the face cover and the bottom cover are clamped by positioning holes and positioning posts to fix the test strip ;
  • the face cover is provided with a positioning post G01, and the bottom cover is provided with a positioning hole D01.
  • the test strip is fixed by the cooperation of the positioning post G01 and the positioning hole D01; the position of the positioning post G01 is symmetrically distributed in the front, middle and rear three groups, positioning Column G01 has a height of 2.5 ⁇ 3.5mm and a diameter of 1.0 ⁇ 1.5mm.
  • the position of the positioning hole D01 is symmetrically distributed in the front, middle and rear groups, the height of the column hole is 2.5 ⁇ 3.5mm, and the diameter is 1.1 ⁇ 1.6mm. ,Combined with the column on the cover to support and fix the reagent card.
  • the bottom cover is provided with a card slot structure (D02, D06) and a raised platform D04;
  • the card slot structure is provided with a supporting protrusion D03, and the height of the supporting protrusion D03 is less than Equal to the height of the raised platform D04;
  • the face cover is provided with a fixing plate (G02, G03, G04, G06), a reaction window G05, a sample hole A, a detection liquid hole B; the reaction window G05, a sample hole A, The detection liquid holes B are arranged in sequence; the fixing plates (G02, G03, G04, G06) are distributed on both sides of the reaction window G05;
  • test strip After the test strip is inserted into the slot structure (D02, D06) of the bottom cover and fixed, the test strip is supported by the raised platform D04 and the supporting protrusion D03, and the face cover and the bottom After the cover is clamped through the positioning hole and the positioning post, under the squeezing of the cover fixing plate (G02, G03, G04, G06) and the detection liquid hole B, the middle of the test strip protrudes upward.
  • the test strip includes a three-layer or four-layer structure, the first layer is the bottom plate 1, the material is preferably PVC, the second layer is the reaction film 4, and the third layer is the absorbent pad 7, the bonding pad 3. And the cushion 2; or the first layer is the bottom plate 1, the second layer is the reaction film 4, the third layer is the absorbent pad 7 and the bonding pad 3, and the fourth layer is the cushion 2;
  • the reaction film 4 is arranged in the middle of the bottom plate 1, and a detection line T5 and a quality control line C6 are distributed on the reaction film 4, and the detection line T5 and the quality control line C6 correspond to the reaction window G05; There can be two detection lines T5, which are used to distinguish between two diseases.
  • reaction membrane 4 One end of the reaction membrane 4 is provided with a bonding pad 3, the other end of the reaction membrane 4 is provided with an absorbent pad 7, and the bonding pad 3 corresponds to the sample hole A;
  • a cushioning pad 2 is provided on the bonding pad 3 or the bonding pad 3 is butted with the cushioning pad 2, and the cushioning pad 2 corresponds to the detection liquid hole B.
  • the number of the card slot structures is two, respectively, a first card slot structure D02 and a second card slot structure D06.
  • the first card slot structure D02 and the second card slot structure D06 are distributed on both sides of the convex platform D04.
  • Diversion ports D05 are symmetrically provided on both sides of the second slot structure D06 corresponding to the detection liquid hole B; two sets of diversion ports D05 are symmetrically arranged at both ends of the slot to ensure that the detection liquid chromatograms along the test strip , To avoid liquid sticking to the card shell or seeping to the back of the test strip due to the surface tension of the reagent card.
  • the width of the first slot structure D02 and the second slot structure D06 is 3-5mm, the length is 10-18mm, and the depth is 1.2-1.7mm.
  • the height of the supporting protrusion D03 is 0.1-0.15mm and the width is 3-5mm,
  • the height of the raised platform D04 is 0.15-0.25 mm.
  • Supporting protrusions D03 are similar to the working principle of bridge piers. They are distributed at both ends of the card slot, and three are set at the end of the absorbent pad to fix and support the test strip.
  • the number of the fixing plates is 4, which are the first fixing plate G02 for fixing the test strip, the second fixing plate G03 for fixing the test strip, and the second fixing plate G03 for fixing the test strip.
  • the first fixing plate G02, the second fixing plate G03, and the third fixing plate G04 are distributed on the other side of the reaction window G05.
  • the width of the first fixed plate G02, the second fixed plate G03, the third fixed plate G04, and the fourth fixed plate G06 is 3-5mm; the height of the first fixed plate G02 and the second fixed plate G03 is 1.0- 1.5mm, the height of the third fixing plate G04 and the fourth fixing plate G06 is 0.1-0.3mm.
  • the first fixing plate G02 and the second fixing plate G03 are used to fix the test strips after the card housing is assembled, which is different from other manufacturers' test strips using adhesive tape;
  • the third fixing plate G04 is mainly used to fix the absorbent pad on the test strip 7 Combine with the reaction membrane 4 to ensure that the liquid has traction force to continue the chromatographic movement in the late stage of immunochromatography;
  • the fourth fixed plate G06 is mainly used to fix the binding pad 3 on the test strip and the reaction membrane 4 to make the first step reactant and solution smooth Reach the reaction membrane 4 and proceed to the second step of reaction;
  • the sample hole A has a funnel-shaped structure, the diameter of the front circle of the face cover is 4-6mm, the diameter of the inner circle of the face cover is 3-5mm, the height of the protrusion is 0.1-0.2mm, and the hole wall and the horizontal plane are between 30°-45° Angle;
  • the wide-mouth design is conducive to contact with the bonding pad 3 of the test strip when adding a small sample, and it is also convenient for a variety of instruments to collect samples after the sample is fully absorbed by the bonding pad.
  • the detection liquid hole B has a funnel-shaped structure, the diameter of the front circle of the face cover is 6-8mm, the diameter of the inner circle of the face cover is 1-3mm, the height of the protrusion is 0.3-0.5mm, and the hole wall and the horizontal plane are between 30°-45° Angle; funnel-shaped design stores the liquid after adding the detection liquid, forming a hydraulic drive liquid to move to the bonding pad 3, the reaction film 4; the protrusion height 0.3 ⁇ 0.5mm is combined with the bottom cover to press the buffer pad 2 at the slot position, The volume is about 90 ⁇ L, and the liquid is released slowly and directionally under capillary action to avoid excessive liquid flow rate, which may cause insufficient immune response in the first step;
  • the reaction window G05 is an elliptical ring structure, the front ellipse of the face cover is 150-200mm long and 70-90mm wide, the inner circle diameter of the face cover is 130-150mm, the height of the protrusion is 0.1-0.2mm, and the hole wall is in line with the horizontal plane.
  • the angle of 30°-45°, the contact surface between the bottom of the reaction window and the test strip is a vertical protrusion of 0.01-0.02mm; the design of a vertical protrusion of 0.01-0.02mm avoids chromatographic movement on the liquid reaction membrane 4 Affected by the edge effect of the inner protrusion; at the same time, the height of the protrusion 0.1-0.2mm is the same as the height of the sample hole A, and both are lower than the test solution B 0.3-0.5mm, which ensures the air circulation inside the reagent card, so that The liquid chromatography process is not affected by air pressure.
  • the reaction window G05 is the reaction site of the second step of the immune reaction. After the reaction is over, the concentration of the analyte in the sample can be calculated by measuring the signal value of the T and C lines or comparing the color depth of the T and C lines by the instrument;
  • the face cover includes a hand-held end C, and the surface of the hand-held end C is a thread-like or bump-like convex structure.
  • the length of the raised platform D04 is 15.0-0.0mm, the width is 3.0-5.0mm, and the height is 0.15-0.25mm; this raised platform D04 is located in the face-covered reaction window and is the site for the second step of immune binding reaction; due to the support
  • the height of the protrusion D03 is less than or equal to the height of the protrusion platform D04.
  • the test strip After the test strip is inserted into the slot and fixed, the test strip forms a "bridge" structure under the support of the support protrusions D03 in the slot at both ends;
  • the fixing plate G02, G03, G04, G06
  • the sample hole A, the detection liquid hole B and the raised platform D04 are squeezed downward. Press to raise the horizontal surface of the reaction membrane 4 in the middle part of the test strip, and the reaction membrane 4 in the middle part protrudes upward by 0.15-0.25mm, forming an "arch bridge” structure with the horizontal plane (see Figure 8b).
  • this "arch bridge" structure plays an important role in the accuracy and precision of the test results of the reagent card; this design has the following two functions: 1. Control the chromatography speed, and further expand the role of the reaction membrane molecular sieve: In the process of liquid chromatography, the level of the test strip gradually rises, and the liquid chromatography is in a climbing state. At this time, the chromatography speed slows down, prolonging the reaction time of the second step, and the first step is not sufficient. The bound analyte is further identified and bound during the moving process.
  • the reaction membrane molecular sieve is further exerted to separate non-target substances in some samples; the liquid after the second step is fully reacted It starts to go downhill, and under the action of the absorbent pad, the chromatography speed is accelerated. The entire reaction process is completed within 10-15 minutes.
  • the liquid chromatography speed of the reagent card is about 45mm/s; 2.
  • the liquid flow direction is unified: the reagent strip is formed.
  • the structure of “arch bridge” see Figure 8b), the liquid chromatographically moves in the same direction under capillary action, avoiding the liquid spreading around due to the hydraulic pressure when the test liquid is dripped, and the loss of the test object; ensuring a small amount of sample liquid The effective detection concentration.
  • this application also provides a detection method using any of the above reagent cards:
  • Step (1) Add sample into the sample hole
  • the sample is added to the binding pad in the loading hole.
  • the binding pad has the antibody coupled with the nanosphere, that is, the antibody nanosphere.
  • the test substance in the sample is immunologically bound to the antibody nanosphere on the binding pad Form antibody nanosphere complexes;
  • Step (2) Add test solution to test solution hole
  • the test solution After adding the test solution to the test solution hole, the test solution starts to chromatograph along the direction of the absorbent pad end of the test strip.
  • the test solution chromatography passes through the loading hole to dissolve the antibody nanospheres on the binding pad that are not bound to the test substance.
  • the chromatography of the test solution on the test strip has undergone "climbing, parallel, and downhill” (see Figure 8b), and the test solution moves on the test strip , Perform the second immune recognition reaction in the "parallel” stage.
  • the antibody nanosphere complex performs the second immune recognition reaction at the detection line T and starts to develop color; it does not react with the detection line T
  • the antibody nanospheres continue to move forward and combine with quality control line C to develop color.
  • the concentration of the test substance in the sample is calculated by instrument detection or the color card comparison with the signal value or color depth of the T and C lines. Whether the color of the C line is sufficient is to evaluate whether the reaction of the reagent card is completed. An important indicator of the accuracy of the value.
  • the C line is the quality control line, which plays an important role in the interpretation of the test results of the reagent card. The C line needs to be fully colored and reach the established value to judge that the test result is valid; the C line does not show color, the measured value is low, and may exist Interference, the test result is invalid; C-line color rendering is insufficient, and the measured value is low, prompting the user to make a mistake in the operation, and the test result is invalid.
  • the above-mentioned detection method is used for the detection of tear fluid, wound exudate, tissue fluid, sweat, aqueous humor, and ocular surface lotion.
  • this reagent card mainly focuses on the following four aspects to fulfill:
  • the front (face cover) of the reagent card is designed with sample hole A and test solution hole B.
  • sample volume 1 to 5 ⁇ L
  • test solution hole B After a small amount of sample is collected, add the sample to the binding pad in sample hole A, and then place the sample on the binding pad in sample hole A. Drop the test solution into hole B to start the immunochromatographic reaction, the whole process is simple and convenient;
  • the sample hole A is located at the binding pad of the test strip.
  • the binding pad is made of hydrophilic glass cellulose, and the binding pad has a nanoparticle-labeled antibody AB-1.
  • the sample is directly added to the binding pad, hydrophilic
  • the material is conducive to the diffusion of the sample, so that the analyte in the sample is recognized and immunologically combined with the antibody AB-1 labeled with the nanoparticle.
  • the utilization rate of the sample is almost 100%, and the trace amount of liquid is not 0.1-5 ⁇ L. Cause subsequent chromatographic reaction; alternatively, you can add more volume of sample, ⁇ 50 ⁇ L, directly add the sample and perform the test;
  • test substance is combined with the antibody AB-1 labeled with the nanosphere to form the bottom and middle layers of the "sandwich" structure of the double antibody sandwich method, completing the first reaction of immunochromatographic detection ;
  • test liquid After adding the test liquid to the test liquid hole B, the test liquid starts to chromatograph along the direction of the absorbent pad end of the test strip. From the beginning to the completion of the reaction, the liquid on the test strip undergoes chromatography. Climbing, parallel, downhill" (see Figure 8b), the liquid flow direction and flow rate control are all dependent on the special design of the reagent card;
  • the liquid moves on the test strip in the "parallel" stage for the second immune recognition reaction, and forms a complete “sandwich” structure with the detection line T, and begins to develop color.
  • the color depth and the test object are the "sandwich” middle layer
  • the content is positively correlated; the nanoparticles that do not react with the T line continue to move forward and combine with the quality control line C to develop color.
  • the concentration of the analyte in the sample can be calculated by the instrument detection or the color card comparing the signal value or color depth of the T and C lines. Whether the color of the C line is sufficient is to evaluate whether the reaction of the reagent card is completed and the measured value An important indicator of accuracy.
  • the flow direction and chromatographic speed of the test liquid on the test strip are controlled to achieve the purpose of accurate detection of clinical trace samples, filling the market gap of existing POCT trace sample detection products.
  • the reagent card provided in this application can be used for auxiliary diagnosis of dry eye syndrome
  • Step (1) Add sample into the sample hole
  • the binding pad has an alpha lymphotoxin (LTA) antibody coupled to the nanospheres, that is, the antibody nanospheres.
  • LTA alpha lymphotoxin
  • Step (2) Add test solution to test solution hole
  • the test solution After adding the alpha lymphotoxin (LTA) test solution to the test solution hole, the test solution starts to chromatograph along the direction of the absorbent pad end of the test strip, and the test solution chromatography passes through the sample hole position to dissolve the untested substance on the binding pad
  • LTA alpha lymphotoxin
  • step (1) the antibody nanosphere complex performs the second immune recognition reaction at the detection line T and starts to develop color; and The antibody nanospheres that do not react with the detection line T continue to move forward and combine with the quality control line C to develop color.
  • the sensitivity reached 0.15ng/mL; the precision CV reached 5%-10%.
  • the reagent card provided in this application can be used for auxiliary diagnosis of inflammation
  • Step (1) Add sample into the sample hole
  • the binding pad has a matrix metalloproteinase 9 (MMP-9) antibody coupled to nanospheres, that is, antibody nanospheres.
  • MMP-9 matrix metalloproteinase 9
  • the analyte and the antibody nanospheres on the binding pad immunologically combine to form an antibody nanosphere complex;
  • Step (2) Add test solution to test solution hole
  • test solution After adding the matrix metalloproteinase 9 (MMP-9) test solution to the test solution hole, the test solution starts to chromatograph along the direction of the absorbent pad end of the test strip, and the test solution chromatography passes through the sample hole position to dissolve the binding pad.
  • MMP-9 matrix metalloproteinase 9
  • the antibody nanospheres bound to the test substance and the antibody nanosphere complexes that have been bound to the test substance have undergone "climbing, parallel, and downhill” chromatography on the test strip (see Figure 8b) ,
  • the test solution moves on the test strip and performs the second immune recognition reaction in the "parallel" stage.
  • step (1) the antibody nanosphere complex performs the second immune recognition reaction at the test line T, and begins to show The antibody nanospheres that do not react with the detection line T continue to move forward and combine with the quality control line C to develop color.
  • the sensitivity reached 1.0ng/mL; the precision CV reached 5%-10%.
  • the reagent card provided in this application can be used for auxiliary diagnosis of allergic conjunctivitis
  • Step (1) Add sample into the sample hole
  • the binding pad has the total immunoglobulin E (IgE) antibody coupled to the nanospheres, namely the antibody Nanospheres, the test substance in the sample and the antibody nanospheres on the binding pad are immunologically combined to form an antibody nanosphere complex;
  • IgE immunoglobulin E
  • Step (2) Add test solution to test solution hole
  • test solution After adding the total immunoglobulin E (IgE) test solution to the test solution hole, the test solution starts to chromatograph along the direction of the absorbent pad end of the test strip, and the test solution chromatography passes through the sample hole position to dissolve the non-combined pad.
  • the antibody nanospheres bound to the test substance and the antibody nanosphere complexes that have been bound to the test substance have undergone "climbing, parallel, and downhill” chromatography on the test strip (see Figure 8b).
  • the test solution moves on the test strip, and performs the second immune recognition reaction in the "parallel" stage.
  • step (1) the antibody nanosphere complex performs the second immune recognition reaction at the test line T and starts to develop color ;
  • the antibody nanospheres that do not react with the detection line T continue to move forward and combine with the quality control line C to develop color.
  • the sensitivity reached 0.5IU/mL; the precision CV reached 5%-10%.
  • the reagent card provided in this application can be used for the auxiliary diagnosis of conjunctivitis and the differential diagnosis of conjunctivitis caused by allergy or viral infection;
  • Step (1) Add sample into the sample hole
  • the binding pad has the total immunoglobulin E (IgE) antibody and virus antibody coupled to the nanospheres , That is, antibody nanospheres, the test substance in the sample and the antibody nanospheres on the binding pad immunologically combine to form an antibody nanosphere complex;
  • IgE immunoglobulin E
  • Step (2) Add test solution to test solution hole
  • test solution After adding total immunoglobulin E (IgE) and virus detection solution to the test solution hole, the test solution starts to chromatograph along the direction of the absorbent pad end of the test strip, and the test solution chromatography passes through the sample hole position to dissolve the bond pad.
  • the antibody nanospheres bound to the test substance and the antibody nanosphere complexes that have been bound to the test substance have undergone "climbing, parallel, and downhill” chromatography of the test solution on the test strip (see Figure 8b) ), the test solution moves on the test strip, and the second immune recognition reaction is carried out in the "parallel" stage.
  • the antibody nanosphere complex is in two test lines T (respectively the virus test line T and the total
  • the second immune recognition reaction is performed at the IgE test line T), and the color begins to develop.
  • the color development of the virus test line T and the total IgE test line T it can be determined whether the eye disease is caused by a virus or an allergy.
  • the T-reactive antibody nanospheres continue to move forward and combine with the quality control line C to develop color. After many experiments, the sensitivity reached 0.5IU/mL or 0.5ng/mL; the precision CV reached 5%-10%.

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Abstract

一种试剂卡、检测方法及应用，面盖和底盖之间通过定位孔(D01)和定位柱(G01)卡接将试纸条固定；底盖内部设置卡槽结构(D02、D06)、凸起平台(D04)；卡槽结构(D02、D06)内设置有支撑凸起(D03)，支撑凸起(D03)的高度小于等于凸起平台(D04)的高度；面盖设置固定板(G02、G03、G04、G06)、反应窗口(G05)、加样孔(A)、检测液孔(B)；面盖和底盖通过定位孔(D01)和定位柱(G01)卡接后，在面盖固定板(G02、G03、G04、G06)和检测液孔(B)的挤压下，试纸条中部向上凸起，与水平面形成"拱桥"结构。这一"拱桥"结构控制检测液液体在试纸条上的流向及层析速度，进而提高了试剂卡的检测结果准确度和精密度，达到精准检测微量样本的目的。

Description

一种试剂卡、检测方法及应用
本申请要求于2019年7月19日提交中国专利局、申请号为201910657081.5、发明名称为“一种试剂卡、检测方法及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及一种试剂卡，具体说是一种微量样本体外精准简捷检测的试剂卡、检测方法及应用。
背景技术
POCT，即时检验(point-of-care testing)，指在病人旁边进行的临床检测及床边检测(bedside testing)，通常不一定是临床检验师来进行，而是在采样现场即刻进行分析，省去标本在实验室检验时的复杂处理程序，快速得到检验结果的一类新方法。
试剂卡是即时检验中常用的工具，用于检验血液、尿、粪便和其他体液中的生化物质，例如血清、血浆和尿中的胆红素和蛋白质、葡萄糖、血红素、酮体、亚硝酸盐、尿胆素原等生物标记物。
试剂卡由卡壳(面盖及底盖)与卡壳内的试纸条组成，试纸条固定于底盖的卡槽内，将面盖与底盖结合后组装成试剂卡。
目前的快速检测技术，对样本体积要求一般至少10uL。可是临床上有些样本仅可获得1uL甚至更少，例如泪液样本。
现有的快速检测技术，对于样本量体积极为微量的情况下如2μL，如果对样本进行额外步骤的操作处理，例如稀释，这会不可避免地降低检测灵敏度、精密度和准确度，增加检测操作的复杂性、对工具的要求较高和产生差错的机率较大。
现有的快速检测技术其灵敏度一般在0.05ng/mL(荧光免疫检测0.1ng/mL，量子点0.05ng/mL)，精密度一般在5％～15％。如果以待测物的总量检测下限计算：约为0.5pg，对于微量样本来说测试较为困难。
对于眼类疾病来说，由于眼睛器官的敏感和脆弱，其对检测要求比较 高；而在实际操作中，由于眼睛可采的样本泪液、房水、眼表润洗液均属微量样本，检测难度大；例如患者眼睛红肿、结膜充血，临床医生不借助检测技术很难判断病因，是感染还是过敏引起，准确快速诊断病因对指导用药具有十分重要作用；如果是过敏引起会使用抗过敏药，如果是感染，例如病毒感染或者阿米巴感染会使用抗病毒或相应的抗阿米巴感染治疗；如果因为误诊对感染的眼睛使用了抗过敏药甚至激素的话，有可能会导致患者病情更加严重，甚至造成严重的医疗事故。
发明内容
有鉴于此，本发明提供了一种试剂卡，通过卡壳结构的特殊设计，控制检测液液体在试纸条上的流向及层析速度，达到精准检测临床上微量样本的目的，用于解决现有技术中存在的问题。
本发明提供了一种试剂卡，包括面盖、底盖、试纸条；所述面盖和所述底盖将试纸条固定；
所述底盖内部设置卡槽结构、凸起平台；所述卡槽结构内设置有支撑凸起，所述支撑凸起的高度小于等于所述凸起平台的高度；
所述面盖设置固定板、反应窗口、加样孔、检测液孔；所述反应窗口、加样孔、检测液孔依次设置；所述固定板分布在所述反应窗口两侧；
所述试纸条装入所述底盖的卡槽结构固定后，所述试纸条被所述凸起平台和支撑凸起支撑，所述面盖和所述底盖通过定位孔和定位柱卡接后，在所述面盖固定板和检测液孔的挤压下，所述试纸条中部向上凸起。
优选地，所述试纸条包括三层或四层结构，第一层为底板，第二层为反应膜，第三层为吸收垫、结合垫和缓冲垫；或者第一层为底板，第二层为反应膜和缓冲垫，第三层为吸收垫、和结合垫；或者第一层为底板，第二层为反应膜，第三层为吸收垫和结合垫，第四层为缓冲垫；
所述反应膜设置在所述底板中部，所述反应膜上分布有检测线T和质控线C，所述检测线T和质控线C与所述反应窗口对应；
所述反应膜的一端设置结合垫，所述反应膜的另外一端设置吸收垫，所述结合垫与所述加样孔对应；
所述结合垫上或结合垫下设置缓冲垫或者所述结合垫与所述缓冲垫对接，所述缓冲垫与所述检测液孔对应。
优选地，所述卡槽结构数量为两个，分布在所述凸起平台两侧。
优选地，所述卡槽结构内设置1-5个支撑凸起。
优选地，所述卡槽结构内设置3个支撑凸起
优选地，与所述检测液孔对应的所述卡槽结构的两边对称设置有导流口。
优选地，所述卡槽结构的深度为1.2-1.7mm，所述支撑凸起的高度为0.1-0.15mm，所述凸起平台高度为0.15-0.25mm。
优选地，所述固定板的数量为4个，分别为用于固定所述试纸条的第一固定板、用于固定所述试纸条的第二固定板、用于固定所述试纸条上所述吸收垫和反应膜结合的第三固定板、用于固定所述试纸条上所述反应膜和结合垫结合的第四固定板；所述第四固定板设置在所述加样孔和所述反应窗口之间，所述第一固定板、第二固定板、第三固定板分布在所述反应窗口另一侧。
优选地，第一固定板、第二固定板、第三固定板、第四固定板的宽度为3-5mm；第一固定板、第二固定板的高度为1.0-1.5mm，第三固定板、第四固定板的高度为0.1-0.3mm。
优选地，所述加样孔为漏斗状结构，面盖正面圆直径4-6mm，面盖内部圆直径为3-5mm，凸起高度为0.1-0.2mm，孔壁与水平面呈30°-45°夹角。
优选地，所述检测液孔为漏斗状结构，面盖正面圆直径6-8mm，面盖内部圆直径为1-3mm，凸起高度为0.3～0.5mm，孔壁与水平面呈30°-45°夹角。
优选地，所述反应窗口为椭圆形环结构，面盖正面椭圆长150-200mm，宽度为70-90mm，面盖内部圆直径为130-150mm，凸起高度为0.1-0.2mm，孔壁与水平面呈30°-45°夹角，反应窗内底部与试纸条接触面为0.01-0.02mm的垂直凸起。
优选地，所述面盖包括手持端，所述手持端的表面为螺纹状或凸点状凸起结构。
优选地，所述面盖和所述底盖之间通过定位孔和定位柱卡接。
所述反应膜上分布的检测线T为一条、两条或多条。
此外，本申请还提供了一种使用上述任一试剂卡实现的检测方法：
步骤(1)加样孔内加样
样本采集后，将样本加入所述加样孔内的结合垫上，结合垫上有纳米微球偶联的抗体，即抗体纳米微球，样本中的待测物与结合垫上的抗体纳米微球免疫结合形成抗体纳米微球复合物；
步骤(2)检测液孔内加入检测液
在检测液孔中加入检测液后，检测液开始沿着试纸条的吸水垫端的方向开始层析，检测液层析经过加样孔位置溶解结合垫上未与待测物结合的抗体纳米微球及已与待测物结合的抗体纳米微球复合物，检测液在试纸条上的层析经历了“爬坡、平行、下坡”，检测液在试纸条上移动，在“平行”阶段进行第二次免疫识别反应，步骤(1)中抗体纳米微球复合物在检测线T处进行第二次免疫识别反应，并且开始显色；而不与检测线T反应的抗体纳米微球则继续向前运动，与质控线C结合显色。
优选地，加样孔内加样为0.1-50ul；检测液孔内加入检测液0-150ul。
优选地，所述试剂卡反应完成后通过仪器检测或者色卡对比T、C线的信号值或色深来计算样本中待测物的浓度。
优选地，上述检测方法用于对泪液、伤口渗出液、组织液、汗液、房水、眼表润洗液等微量样本的检测，也可用于对于常规样本血液(末梢血、血清、血浆)、唾液、尿液等常规样本的检测；也可用于对于其它非临床样本的检测，例如宠物、畜牧业、农业、鱼类、食品、环保检测等。
由于所述支撑凸起的高度小于等于所述凸起平台的高度，试纸条装入卡槽固定后，试纸条在两端卡槽内的支撑凸起的支撑下形成“桥”的结构；在面盖和底盖通过定位孔和定位柱卡接过程中，面盖上固定板、加样孔、检测液孔与凸起平台配合向下挤压，中间部位反应膜向上凸起，与水平面形成“拱桥”结构。这一“拱桥”结构控制检测液液体在试纸条上的流向及层析速度，进而提高了试剂卡的检测结果准确度和精密度，达到精准检测临床上微量样本的目的，填补了现有POCT微量样本检测产品的市场空白。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为面盖示意图；
图2为底盖示意图；
图3a为试纸条实施例1示意图；
图3b为试纸条实施例2示意图；
图3c为试纸条实施例3示意图；
图4为试剂卡示意图；
图5a为面盖正面示意图；
图5b为面盖反面示意图；
图5c为面盖截面示意图；
图6a为底盖正面示意图；
图6b为底盖反面示意图；
图6c为底盖截面示意图；
图7a为试剂卡拆解示意图；
图7b为试剂卡截面示意图；
图8a为试剂卡组装后的截面示意图；
图8b为试剂卡组装后的截面局部放大图。
图中：1.底板，2.缓冲垫，3.结合垫，4.反应膜，5.检测线T，6.质控线C，7.吸收垫，G01.定位柱，G02.第一固定板，G03.第二固定板，G04.第三固定板，G05.反应窗口，G06.第四固定板，A.加样孔，B.检测液孔，C.手持端，D01.定位孔，D02.第一卡槽结构，D03.支撑凸起，D04.凸起平台，D05.导流口，D06.第二卡槽结构。
具体实施方式
本发明实施例提供了一种试剂卡，用于解决现有技术中存在的问题。
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
为了更详细说明本发明，下面结合附图对本发明提供的一种试剂卡，进行具体地描述。
参见图1-4，本发明提供了一种试剂卡，包括面盖、底盖、试纸条；所述面盖和所述底盖之间通过定位孔和定位柱卡接将试纸条固定；面盖上设有定位柱G01，底盖上设置有定位孔D01，通过定位柱G01和定位孔D01的配合将试纸条固定；定位柱G01位置前、中、后三组对称分布，定位柱G01高度2.5～3.5mm，直径1.0～1.5mm，主要支撑和固定试剂卡的作用；定位孔D01位置前、中、后三组对称分布，柱孔高度2.5～3.5mm，直径1.1～1.6mm，与面盖的柱子结合支撑和固定试剂卡的作用。
如图6A-6C所示，所述底盖内部设置卡槽结构(D02、D06)、凸起平台D04；所述卡槽结构内设置有支撑凸起D03，所述支撑凸起D03的高度小于等于所述凸起平台D04的高度；
如图5A-5C所示，所述面盖设置固定板(G02、G03、G04、G06)、反应窗口G05、加样孔A、检测液孔B；所述反应窗口G05、加样孔A、检测液孔B依次设置；所述固定板(G02、G03、G04、G06)分布在所述反应窗口G05两侧；
所述试纸条装入所述底盖的卡槽结构(D02、D06)固定后，所述试纸条被所述凸起平台D04和支撑凸起D03支撑，所述面盖和所述底盖通过定位孔和定位柱卡接后，在所述面盖固定板(G02、G03、G04、G06)和检测液孔B的挤压下，所述试纸条中部向上凸起。
如图3所示，所述试纸条包括三层或四层结构，第一层为底板1，材料优选为PVC，第二层为反应膜4，第三层为吸收垫7、结合垫3和缓冲垫2；或者第一层为底板1，第二层为反应膜4，第三层为吸收垫7和结合 垫3，第四层为缓冲垫2；
所述反应膜4设置在所述底板1中部，所述反应膜4上分布有检测线T5和质控线C6，所述检测线T5和质控线C6与所述反应窗口G05对应；所述检测线T5可以为两条，用于两种疾病的区分。
所述反应膜4的一端设置结合垫3，所述反应膜4的另外一端设置吸收垫7，所述结合垫3与所述加样孔A对应；
所述结合垫3上设置缓冲垫2或者所述结合垫3与所述缓冲垫2对接，所述缓冲垫2与所述检测液孔B对应。
所述卡槽结构数量为两个分别为第一卡槽结构D02和第二卡槽结构D06，第一卡槽结构D02和第二卡槽结构D06分布在所述凸起平台D04两侧。
所述卡槽结构内设置1-5个支撑凸起；优选地，所述卡槽结构内设置3个支撑凸起，如图6A-6C所示，第一卡槽结构D02和第二卡槽结构D06内分别设置有三个支撑凸起D03。
与所述检测液孔B对应的所述第二卡槽结构D06的两边对称设置有导流口D05；两组导流口D05于卡槽两端对称设置，确保检测液沿试纸条层析，避免因试剂卡表面张力导致液体粘在卡壳上或者渗到试纸条背面。
第一卡槽结构D02和第二卡槽结构D06的宽度3-5mm，长度10-18mm，深度为1.2-1.7mm，所述支撑凸起D03的高度为0.1-0.15mm，宽度3-5mm，所述凸起平台D04高度为0.15-0.25mm。支撑凸起D03类似于桥墩的工作原理，在卡槽内的两端均有分布，在吸收垫端设置了三个，起固定和支持试纸条的作用。
所述固定板的数量为4个，分别为用于固定所述试纸条的第一固定板G02、用于固定所述试纸条的第二固定板G03、用于固定所述试纸条上所述吸收垫和反应膜结合的第三固定板G04、用于固定所述试纸条上所述反应膜和结合垫结合的第四固定板G06；所述第四固定板G06设置在所述加样孔和所述反应窗口之间，所述第一固定板G02、第二固定板G03、第三固定板G04分布在所述反应窗口G05另一侧。
所述第一固定板G02、第二固定板G03、第三固定板G04、第四固定 板G06的宽度为3-5mm；所述第一固定板G02、第二固定板G03的高度为1.0-1.5mm，第三固定板G04、第四固定板G06的高度为0.1-0.3mm。
第一固定板G02、第二固定板G03用于固定卡壳组装后的试纸条，区别于其他厂家的试纸条采用胶布固定的方式；第三固定板G04主要作用固定试纸条上吸收垫7与反应膜4结合，确保免疫层析后期液体有牵引力继续层析移动；第四固定板G06主要作用固定试纸条上结合垫3与反应膜4结合，使第一步反应物和溶液顺利到达反应膜4，进行第二步的反应；
所述加样孔A为漏斗状结构，面盖正面圆直径4-6mm，面盖内部圆直径为3-5mm，凸起高度为0.1-0.2mm，孔壁与水平面呈30°-45°夹角；广口设计有利于微量样本加样时与试纸条的结合垫3接触，也方便多种器具采集样本后的加样，充分的被结合垫吸收。
所述检测液孔B为漏斗状结构，面盖正面圆直径6-8mm，面盖内部圆直径为1-3mm，凸起高度为0.3-0.5mm，孔壁与水平面呈30°-45°夹角；漏斗形设计在加检测液后存储液体、形成液压驱动液体向结合垫3、反应膜4方向运动；凸起高度0.3～0.5mm与底盖结合后压紧卡槽部位的缓冲垫2，容量约90μL，在毛细作用下定向的缓慢释放液体，避免液体流速过快，导致第一步免疫反应不充分；
所述反应窗口G05为椭圆形环结构，面盖正面椭圆长150-200mm，宽度为70-90mm，面盖内部圆直径为130-150mm，凸起高度为0.1-0.2mm，孔壁与水平面呈30°-45°夹角，反应窗内底部与试纸条接触面为0.01-0.02mm的垂直凸起；0.01-0.02mm的垂直凸起的设计避免了在液体反应膜4上层析运动时受到内凸起边缘效应的影响；同时，凸起的高度0.1-0.2mm与加样孔A的高度相同，均低于检测液B0.3-0.5mm，保证了试剂卡内部的空气流通，使得液体层析过程中不受气压的影响。反应窗口G05为免疫反应第二步反应的部位，反应结束后，可以通过仪器检测T、C线的信号值或者色卡对比T、C线色深来计算样本中待测物的浓度；
所述面盖包括手持端C，所述手持端C的表面为螺纹状或凸点状凸起结构。
凸起平台D04的长度15.0-0.0mm，宽度3.0-5.0mm，高度0.15-0.25mm； 这一凸起平台D04位于面盖反应窗口，是进行第二步免疫结合反应的部位；由于所述支撑凸起D03的高度小于等于所述凸起平台D04的高度，试纸条装入卡槽固定后，试纸条在两端卡槽内的支撑凸起D03的支撑下形成“桥”的结构；在面盖和底盖通过定位孔和定位柱卡接过程中，面盖上固定板(G02、G03、G04、G06)、加样孔A、检测液孔B与凸起平台D04配合向下挤压，使试纸条中间部位反应膜4水平面抬高，中间部位反应膜4向上凸起0.15-0.25mm，与水平面形成“拱桥”结构(参见图8b)。这一“拱桥”结构的形成对于试剂卡的检测结果准确度和精密度有着重要的作用；这一设计具有以下两方面的作用：一、控制层析速度，使反应膜分子筛的作用进一步扩大：液体在层析的过程中，试纸条水平面逐渐上升，液体层析时处于一种爬坡状态，此时层析速度减慢，延长了第二步反应的时间，在第一步未被充分结合的待测物在移动的过程中被进一步识别结合，在减慢层析速度的情况下，进一步发挥反应膜分子筛的作用，将一些样本中的非目标物分离；第二步充分反应后液体开始下坡状态，并且在吸收垫的作用下，层析速度加快，整个反应过程于10～15min内完成，试剂卡液体层析速度约为45mm/s；二、液体流向统一：试剂条形成“拱桥”的结构(参见图8b)，液体在毛细作用下向同一方向层析运动，避免在滴加检测液时由于液压导致液体向四周扩散，使待测物流失的情况；保证了微量样品液的有效检测浓度。
此外，本申请还提供了一种使用上述任一试剂卡实现的检测方法：
步骤(1)加样孔内加样
样本采集后，将样本加入所述加样孔内的结合垫上，结合垫上有纳米微球偶联的抗体，即抗体纳米微球，样本中的待测物与结合垫上的抗体纳米微球免疫结合形成抗体纳米微球复合物；
步骤(2)检测液孔内加入检测液
在检测液孔中加入检测液后，检测液开始沿着试纸条的吸水垫端的方向开始层析，检测液层析经过加样孔位置溶解结合垫上未与待测物结合的抗体纳米微球及已与待测物结合的抗体纳米微球复合物，检测液在试纸条上的层析经历了“爬坡、平行、下坡”(参见图8b)，检测液在试纸条上移 动，在“平行”阶段进行第二次免疫识别反应，步骤(1)中抗体纳米微球复合物在检测线T处进行第二次免疫识别反应，并且开始显色；而不与检测线T反应的抗体纳米微球则继续向前运动，与质控线C结合显色。
加样孔内加样为0.1-50ul；检测液孔内加入检测液0-150ul。
所述试剂卡反应完成后通过仪器检测或者色卡对比T、C线的信号值或色深来计算样本中待测物的浓度，C线显色是否充分是评价本试剂卡反应是否完成，测值是否准确的重要指标。C线为质控线，对试剂卡测试结果判读起重要的作用，C线需充分显色并达到既定的数值，才能判断检测结果是有效的；C线不显色，测值低，可能存在干扰，检测结果无效；C线显色不充分，测值偏低，提示使用者可能操作错误，检测结果无效。
上述检测方法用于对泪液、伤口渗出液、组织液、汗液、房水、眼表润洗液的检测。
对于微量样本的检测，提高样本的利用率，充分的让待测物被相应的抗体识别并发生免疫结合的反应是确保检测结果准确可靠的关键，在本试剂卡的设计中主要从以下四方面来实现：
(1)加样
试剂卡正面(面盖)的设计有加样孔A和检测液孔B，当样本量1～5μL时，微量的样本采集后，将样本加入加样孔A内的结合垫上，再在检测液孔B滴加检测液，开始免疫层析的反应，整个过程操作简捷；
加样孔A处于试纸条的结合垫处，结合垫的材质为亲水性的玻璃纤维素，在结合垫上有纳米颗粒标记的抗体AB-1，将样本直接加于结合垫上，亲水性的材质有利于样本的扩散，从而使样本中的待测物与纳米颗粒标记的抗体AB-1识别并发生免疫结合，样本的利用率几乎达到了100％，而且微量的液体0.1～5μL并不引起后续的层析反应；备选也可以加更多体积的样本，≥50μL，直接加样后进行检测；
(2)第一步免疫反应
样本加入加样孔A后，待测物便与纳米微球标记的抗体AB-1结合，形成双抗体夹心法“三明治”结构的底层和中间层，完成免疫层析法检测的第一步反应；
(3)免疫层析过程中液体流动方向及流速的控制
在检测液孔B中加入检测液后，检测液液体开始沿着试纸条吸水垫端的方向开始层析，从开始至整个反应完成的过程中，液体在试纸条上的层析经历了“爬坡、平行、下坡”(参见图8b)，液体流动方向及流速的控制均依赖于试剂卡特殊的设计来完成；
(4)第二步免疫反应
液体在试纸条上移动在“平行”阶段进行第二次免疫识别反应，与检测线T形成完整的“三明治”结构，并且开始显色，颜色的深浅与待测物即“三明治”中间层的含量成正相关；而不与T线反应的纳米颗粒则继续向前运动，与质控线C结合显色。试剂卡反应完成后可以通过仪器检测或者色卡对比T、C线的信号值或色深来计算样本中待测物的浓度，C线显色是否充分是评价本试剂卡反应是否完成，测值是否准确的重要指标。
通过卡壳结构的特殊设计，控制检测液液体在试纸条上的的流向及层析速度，达到精准检测临床上微量样本的目的，填补了现有POCT微量样本检测产品的市场空白。
检测方法实施例1
本申请提供的试剂卡可用于干眼症的辅助诊断；
步骤(1)加样孔内加样
泪液样本采集后，将泪液样本1μL加入所述加样孔内的结合垫上，结合垫上有纳米微球偶联的α淋巴毒素(LTA)抗体，即抗体纳米微球，样本中的待测物与结合垫上的抗体纳米微球免疫结合形成抗体纳米微球复合物；
步骤(2)检测液孔内加入检测液
在检测液孔中加入α淋巴毒素(LTA)检测液后，检测液开始沿着试纸条的吸水垫端的方向开始层析，检测液层析经过加样孔位置溶解结合垫上未与待测物结合的抗体纳米微球及已与待测物结合的抗体纳米微球复合物，检测液在试纸条上的层析经历了“爬坡、平行、下坡”(参见图8b)，检测液在试纸条上移动，在“平行”阶段进行第二次免疫识别反应，步骤(1) 中抗体纳米微球复合物在检测线T处进行第二次免疫识别反应，并且开始显色；而不与检测线T反应的抗体纳米微球则继续向前运动，与质控线C结合显色。经过多次试验，灵敏度达到0.15ng/mL；精密度CV达到5％～10％。
检测方法实施例2
本申请提供的试剂卡可用于炎症的辅助诊断；
步骤(1)加样孔内加样
房水样本采集后，将房水样本0.5μL加入所述加样孔内的结合垫上，结合垫上有纳米微球偶联的基质金属蛋白酶9(MMP-9)抗体，即抗体纳米微球，样本中的待测物与结合垫上的抗体纳米微球免疫结合形成抗体纳米微球复合物；
步骤(2)检测液孔内加入检测液
在检测液孔中加入基质金属蛋白酶9(MMP-9)检测液后，检测液开始沿着试纸条的吸水垫端的方向开始层析，检测液层析经过加样孔位置溶解结合垫上未与待测物结合的抗体纳米微球及已与待测物结合的抗体纳米微球复合物，检测液在试纸条上的层析经历了“爬坡、平行、下坡”(参见图8b)，检测液在试纸条上移动，在“平行”阶段进行第二次免疫识别反应，步骤(1)中抗体纳米微球复合物在检测线T处进行第二次免疫识别反应，并且开始显色；而不与检测线T反应的抗体纳米微球则继续向前运动，与质控线C结合显色。经过多次试验，灵敏度达到1.0ng/mL；精密度CV达到5％～10％。
检测方法实施例3
本申请提供的试剂卡可用于过敏性结膜炎的辅助诊断；
步骤(1)加样孔内加样
眼表润洗液样本采集后，将眼表润洗液样本2.2μL加入所述加样孔内的结合垫上，结合垫上有纳米微球偶联的总免疫球蛋白E(IgE)抗体，即抗体纳米微球，样本中的待测物与结合垫上的抗体纳米微球免疫结合形成抗体纳米微球复合物；
步骤(2)检测液孔内加入检测液
在检测液孔中加入总免疫球蛋白E(IgE)检测液后，检测液开始沿着试纸条的吸水垫端的方向开始层析，检测液层析经过加样孔位置溶解结合垫上未与待测物结合的抗体纳米微球及已与待测物结合的抗体纳米微球复合物，检测液在试纸条上的层析经历了“爬坡、平行、下坡”(参见图8b)，检测液在试纸条上移动，在“平行”阶段进行第二次免疫识别反应，步骤(1)中抗体纳米微球复合物在检测线T处进行第二次免疫识别反应，并且开始显色；而不与检测线T反应的抗体纳米微球则继续向前运动，与质控线C结合显色。经过多次试验，灵敏度达到0.5IU/mL；精密度CV达到5％～10％。
检测方法实施例4
本申请提供的试剂卡可用于结膜炎的辅助诊断，对过敏或病毒感染引起的结膜炎鉴别诊断；
步骤(1)加样孔内加样
眼表润洗液样本采集后，将眼表润洗液样本2.2μL加入所述加样孔内的结合垫上，结合垫上有纳米微球偶联的总免疫球蛋白E(IgE)抗体和病毒抗体，即抗体纳米微球，样本中的待测物与结合垫上的抗体纳米微球免疫结合形成抗体纳米微球复合物；
步骤(2)检测液孔内加入检测液
在检测液孔中加入总免疫球蛋白E(IgE)和病毒检测液后，检测液开始沿着试纸条的吸水垫端的方向开始层析，检测液层析经过加样孔位置溶解结合垫上未与待测物结合的抗体纳米微球及已与待测物结合的抗体纳米微球复合物，检测液在试纸条上的层析经历了“爬坡、平行、下坡”(参见图8b)，检测液在试纸条上移动，在“平行”阶段进行第二次免疫识别反应，步骤(1)中抗体纳米微球复合物在两条检测线T(分别为病毒检测线T和总IgE检测线T)处进行第二次免疫识别反应，并且开始显色，根据病毒检测线T和总IgE检测线T的显色情况可以判断眼疾具体为病毒感染还是过敏引起；而不与检测线T反应的抗体纳米微球则继续向前运动，与质控线C结合显色。经过多次试验，灵敏度达到0.5IU/mL或0.5ng/mL；精密 度CV达到5％～10％。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (19)

  1. 一种试剂卡，包括面盖、底盖、试纸条；所述面盖和所述底盖将试纸条固定；其特征在于：
    所述底盖内部设置卡槽结构、凸起平台；所述卡槽结构内设置有支撑凸起，所述支撑凸起的高度小于等于所述凸起平台的高度；
    所述面盖设置固定板、反应窗口、加样孔、检测液孔；所述反应窗口、加样孔、检测液孔依次设置；所述固定板分布在所述反应窗口两侧；
    所述试纸条装入所述底盖的卡槽结构固定后，所述试纸条被所述凸起平台和支撑凸起支撑，所述面盖和所述底盖通过定位孔和定位柱卡接后，在所述面盖固定板和检测液孔的挤压下，所述试纸条中部向上凸起。
  2. 根据权利要求1所述的一种试剂卡，其特征在于，所述试纸条包括三层或四层结构，第一层为底板，第二层为反应膜，第三层为吸收垫、结合垫和缓冲垫；或者第一层为底板，第二层为反应膜和缓冲垫，第三层为吸收垫、和结合垫；或者第一层为底板，第二层为反应膜，第三层为吸收垫和结合垫，第四层为缓冲垫；
    所述反应膜设置在所述底板中部，所述反应膜上分布有检测线T和质控线C，所述检测线T和质控线C与所述反应窗口对应；
    所述反应膜的一端设置结合垫，所述反应膜的另外一端设置吸收垫，所述结合垫与所述加样孔对应；
    所述结合垫上或结合垫下设置缓冲垫或者所述结合垫与所述缓冲垫对接，所述缓冲垫与所述检测液孔对应。
  3. 根据权利要求2所述的一种试剂卡，其特征在于，所述卡槽结构数量为两个，分布在所述凸起平台两侧。
  4. 根据权利要求3所述的一种试剂卡，其特征在于，所述卡槽结构内设置1-5个支撑凸起。
  5. 根据权利要求4所述的一种试剂卡，其特征在于，所述卡槽结构内设置3个支撑凸起。
  6. 根据权利要求5所述的一种试剂卡，其特征在于，与所述检测液孔对应的所述卡槽结构的两边对称设置有导流口。
  7. 根据权利要求6所述的一种试剂卡，其特征在于，所述卡槽结构的深度为1.2-1.7mm，所述支撑凸起的高度为0.1-0.15mm，所述凸起平台高度为0.15-0.25mm。
  8. 根据权利要求7所述的一种试剂卡，其特征在于，所述固定板的数量为4个，分别为用于固定所述试纸条的第一固定板、用于固定所述试纸条的第二固定板、用于固定所述试纸条上所述吸收垫和反应膜结合的第三固定板、用于固定所述试纸条上所述反应膜和结合垫结合的第四固定板；所述第四固定板设置在所述加样孔和所述反应窗口之间，所述第一固定板、第二固定板、第三固定板分布在所述反应窗口另一侧。
  9. 根据权利要求8所述的一种试剂卡，其特征在于，第一固定板、第二固定板、第三固定板、第四固定板的宽度为3-5mm；第一固定板、第二固定板的高度为1.0-1.5mm，第三固定板、第四固定板的高度为0.1-0.3mm。
  10. 根据权利要求9所述的一种试剂卡，其特征在于，所述加样孔为漏斗状结构，面盖正面圆直径4-6mm，面盖内部圆直径为3-5mm，凸起高度为0.1-0.2mm，孔壁与水平面呈30°-45°夹角。
  11. 根据权利要求10所述的一种试剂卡，其特征在于，所述检测液孔为漏斗状结构，面盖正面圆直径6-8mm，面盖内部圆直径为1-3mm，凸起高度为0.3～0.5mm，孔壁与水平面呈30°-45°夹角。
  12. 根据权利要求11所述的一种试剂卡，其特征在于，所述反应窗口为椭圆形环结构，面盖正面椭圆长150-200mm，宽度为70-90mm，面盖内部圆直径为130-150mm，凸起高度为0.1-0.2mm，孔壁与水平面呈30°-45°夹角，反应窗内底部与试纸条接触面为0.01-0.02mm的垂直凸起。
  13. 根据权利要求12所述的一种试剂卡，其特征在于，所述面盖包括手持端，所述手持端的表面为螺纹状或凸点状凸起结构。
  14. 根据权利要求13所述的一种试剂卡，其特征在于，所述面盖和所述底盖之间通过定位孔和定位柱通过定位孔和定位柱卡接。
  15. 根据权利要求12所述的一种试剂卡，其特征在于，所述反应膜上分布的检测线T为一条或两条或多条。
  16. 使用权利要求1-15任一试剂卡实现的检测方法，其特征在于：
    步骤(1)加样孔内加样
    样本采集后，将样本加入所述加样孔内的结合垫上，结合垫上有纳米微球偶联的抗体，即抗体纳米微球，样本中的待测物与结合垫上的抗体纳米微球免疫结合形成抗体纳米微球复合物；
    步骤(2)检测液孔内加入检测液
    在检测液孔中加入检测液后，检测液开始沿着试纸条的吸水垫端的方向开始层析，检测液层析经过加样孔位置溶解结合垫上未与待测物结合的抗体纳米微球及已与待测物结合的抗体纳米微球复合物，检测液在试纸条上的层析经历了“爬坡、平行、下坡”，检测液在试纸条上移动，在“平行”阶段进行第二次免疫识别反应，步骤(1)中抗体纳米微球复合物在检测线T处进行第二次免疫识别反应，并且开始显色；而不与检测线T反应的抗体纳米微球则继续向前运动，与质控线C结合显色。
  17. 如权利要求16所述的检测方法，其特征在于：加样孔内加样为0.1-50ul；检测液孔内加入检测液0-150ul。
  18. 如权利要求17所述的检测方法，其特征在于：所述试剂卡反应完成后通过仪器检测或者色卡对比T、C线的信号值或色深来计算样本中待测物的浓度。
  19. 使用权利要求15-18任一检测方法用于对泪液、伤口渗出液、组织液、汗液、房水、眼表润洗液微量样本的检测。
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