CN107367609B - Lateral flow test piece and detection method - Google Patents

Lateral flow test piece and detection method Download PDF

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CN107367609B
CN107367609B CN201610311026.7A CN201610311026A CN107367609B CN 107367609 B CN107367609 B CN 107367609B CN 201610311026 A CN201610311026 A CN 201610311026A CN 107367609 B CN107367609 B CN 107367609B
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CN107367609A (en
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李欢
黄国栋
廖玲妮
宁晨曦
王陈成
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Shanghai I Reader Biological Technology Co ltd
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

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Abstract

The invention provides a lateral flow test strip and a detection method, the test strip comprises a sample pad, a combination area, a first quality control area, a detection area, a second quality control area and an absorption pad,the first quality control area is a narrowing area with a radial width d1The detection area is a narrow area with a radial width d2The second quality control region is an amplification region with a radial width d3The radial width of the bonding area is d0The radial width of the absorption pad is d4And, furthermore,
Figure DDA0000987471250000011
and is

Description

Lateral flow test piece and detection method
Technical Field
The invention belongs to the field of detection, and particularly relates to a lateral flow test strip, a detection method and application thereof.
Background
The immunochromatography detects a biomarker in a body fluid by using a body fluid component of a human or an animal as a measurement target, and reads and quantifies the depth of a reaction line or a spot color tone displayed by the biomarker. The reflection photometry method of the test paper is applied to the measurement of urine and blood samples. At present, the test paper, the measuring instrument, the daily urine test paper and the like are widely applied to resident health diagnosis and hospital screening. The test items include sugar, protein, bilirubin, hemoglobin, urobilin, etc. And the test strip is provided with a detection line and a quality control line, wherein the detection line is used for quantitatively indicating the concentration of the analyte, and the control line is used for quality control. At present, related products in the market have low sensitivity, and the concentration content of most biomarkers is low, so that the conventional detection method is difficult to detect. Therefore, there is an urgent need in the art to develop methods for improving detection sensitivity.
Disclosure of Invention
The invention aims to provide a lateral flow test strip, a detection method and application thereof.
In a first aspect of the present invention, there is provided a lateral flow test strip comprising: a sample pad, a binding region, a first quality control region, a detection region, a second quality control region, and an absorption pad,
wherein the binding zone comprises detection binding agents for detecting the n analytes, each detection binding agent being for a respective one of the n analytes, the analytes forming a "detection binding agent-analyte" complex with the detection binding agents, the detection binding agents being labeled with a detectable label;
the detection area is provided with m detection lines, wherein m is a positive integer and is more than or equal to n, and each detection line is used for capturing a detection binding agent-analyte complex formed by the binding of the analyte and the detection binding agent thereof;
the first quality control area is provided with a first quality control line, and the first quality control line is used for capturing a complex formed by the combination of the first quality control substance and the corresponding detection binding agent;
the second quality control area contains a second quality control line, and the second quality control line is used for capturing a complex formed by the second quality control substance and the corresponding detection binding agent;
wherein the first quality control area is a narrowing area with a radial width d1And the narrowing region is gradually narrowed;
the detection zone is a narrow zone having a radial width d2；
The second quality control region is an amplification region with a radial width d3；
The radial width of the bonding region is d0；
The radial width of the absorption pad is d4；
And, the radial width satisfies:
Figure BDA0000987471230000021
and is
Figure BDA0000987471230000022
In another preferred embodiment, the
Figure BDA0000987471230000023
Are respectively d0、d1、d2、d3、d4Average value of (a).
In another preferred example, the average is a weighted average.
In another preferred embodiment, d is2In the range of 1-15mm (preferably 2-10mm, more preferably 3-8 mm).
In another preferred embodiment, d is0In the range of 3-20mm (preferably 4-15mm, more preferably 5-10 mm).
In another preferred embodiment, the narrow zone
Figure BDA0000987471230000024
And said amplification region
Figure BDA0000987471230000025
The width ratio of (a) is in the range of 0.1 to 0.7 (preferably 0.2 to 0.6, more preferably 0.3 to 0.5).
In another preferred embodiment, the narrow zone
Figure BDA0000987471230000026
And said narrowing region
Figure BDA0000987471230000027
The width ratio of (a) is in the range of 0.1 to 0.7 (preferably 0.2 to 0.6, more preferably 0.3 to 0.5).
In another preferred embodiment, the
Figure BDA0000987471230000028
In another preferred embodiment, the
Figure BDA0000987471230000029
In another preferred embodiment, the number of the first control lines is 1 to 10, preferably 1 to 7, and more preferably 1 to 5.
In another preferred embodiment, the number of the second control lines is 1 to 10, preferably 1 to 7, and more preferably 1 to 5.
In another preferred embodiment, the detection binding agent is selected from the group consisting of: an antibody, an antigen, a receptor, a ligand, or a combination thereof.
In another preferred embodiment, the detection binding agent is a specific antibody.
In another preferred embodiment, the lateral flow test strip is an immunoreaction-based test strip.
In another preferred embodiment, the amplification region is gradually enlarged.
In another preferred embodiment, the lateral flow test strip is in the lateral flow direction, d1Gradually narrowing and d3Gradually enlarging.
In another preferred embodiment, the type of the narrowing, and enlarging regions is selected from the group consisting of: linear, arc, parabolic, spindle, multi-curved, or combinations thereof.
In another preferred embodiment, the narrowing region and the enlarging region are arc-shaped.
In another preferred embodiment, the arc line has the following characteristics:
(a) for the narrowing region, the slope of the tangent to the arc gradually decreases along the direction of sample flow; and/or
(b) For the amplification zone, the slope of the tangent to the arc increases gradually along the direction of sample flow.
In another preferred embodiment, the narrow region is curved, linear or a combination thereof.
In another preferred example, the narrow region is an arc line type, and the arc line includes: a first constriction region in which the slope of the tangent to the arc gradually decreases and a second constriction region in which the slope of the tangent to the arc gradually increases in the direction along which the sample flows.
In another preferred embodiment, the narrowing, narrowing and enlarging regions are of an arc-shaped line, and the arc-shaped line is a single body and has a continuous smooth arc line (profile).
In another preferred embodiment, the first control substance and the second control substance are the same or different.
In another preferred embodiment, the first and second quality control materials are selected from the group consisting of: an anti-rabbit polyclonal antibody, an anti-sheep polyclonal antibody, an anti-donkey polyclonal antibody, an anti-rabbit monoclonal antibody, an anti-sheep monoclonal antibody, an anti-donkey monoclonal antibody, or a combination thereof.
In another preferred embodiment, the detectable label is selected from the group consisting of: colloidal gold, gold nanorods, silver nanorods, fluorescent microspheres, quantum dots, magnetic microparticles, or a combination thereof.
In another preferred embodiment, the number n of analytes is 1 to 10 (preferably 2 to 8, more preferably 3 to 8).
In another preferred embodiment, the sample pad region is a proximal end of the test strip, the absorption zone is a distal end of the test strip, the first control line is located at the proximal end, the second control line is located at the distal end, the first control line is located at a distance of 1-10mm, preferably 2-8mm, more preferably 3-6mm from the first detection line, and the second control line is located at a distance of 1-10mm, preferably 2-8mm, more preferably 3-6mm from the m-th detection line.
In another preferred embodiment, the distance between the m inspection lines is 0.5-10mm, preferably 1-8mm, more preferably 2-6 mm.
In another preferred embodiment, the number of detection binding agents is 1-10, preferably 2-8, more preferably 3-8.
In another preferred embodiment, the bonding region is a colloidal gold pad.
In another preferred embodiment, the analyte is selected from the group consisting of procalcitonin, C-reactive protein, troponin, myosin, myoglobin, creatine kinase MB isozyme, B-type natriuretic peptide, D-dimer, N-terminal brain natriuretic peptide precursor, cardiac fatty acid binding protein, microalbumin, neutrophil gelatinase-associated lipocalin, β 2-microglobulin, amyloid, cystatin C, or a combination thereof.
In another preferred embodiment, the test strip has a sensitivity that is increased by 10% to 50% (preferably 20 to 40%) over an uncut test strip.
In a second aspect of the invention, there is provided a method for non-diagnostically detecting n analytes in a sample, n being from 1 to 10, comprising the steps of:
(1) providing a sample to be tested and a test strip according to the first aspect of the invention;
(2) dropwise adding the sample to be detected on the sample pad of the test piece, so that the sample to be detected flows towards the direction of the absorption pad and flows through the combination area, the first quality control area, the detection area and the second quality control area;
(3) respectively reading the optical intensity P of the first quality control line in the first quality control region1And the optical intensity P of the second quality control line in the second quality control region2(ii) a And the optical intensity of the detection line in the detection zone corresponding to the i-th analyte, denoted as PiWherein i is 1 to n; and
(4) the optical intensity Pi and the optical intensity P are measured1And P2And comparing to obtain the qualitative and/or quantitative detection result of the ith analyte respectively.
In another preferred embodiment, the optical intensity includes: the intensity of the color signal.
In another preferred embodiment, the optical intensity is a quadratic function of the concentration of the complex formed on the detection line.
In another preferred example, in the step (4), the concentration of the quality control substance is calculated according to the following formula:
C1＝(P1-b)/a
C2＝(P2-b)/a
in the formula, a and b are respectively a constant.
In another preferred embodiment, a and b are the slope and intercept, respectively, of a "concentration-signal intensity" standard curve (fitted straight line).
In another preferred example, in the step (4), the concentration of each analyte is calculated according to the following formula:
Ci＝(C1+C2)(Pi-Pblank)/(P1+P2-2Pblank)，
in the formula (I), the compound is shown in the specification,
Ciis as followsThe concentration of the analyte at the i detection zones,
C1and C2In order to control the concentration of the substance,
Pithe signal intensity at the i-th detection zone of the detection zone,
Pblankis the signal strength of the optical background,
P1and P2The signal intensity of the quality control region.
In another preferred embodiment, the signal intensity includes optical intensity.
In a third aspect of the present invention, there is provided a detection kit comprising:
(a) a lateral flow test strip according to the first aspect of the invention; and
(b) optionally a first container, and a first test reagent in the first container for mixing with a sample to be tested.
In another preferred embodiment, the kit comprises instructions for use of the detection method.
In a fourth aspect of the invention, there is provided the use of a lateral flow test strip according to the first aspect of the invention for the preparation of a kit for detecting the concentration of an analyte in a biological sample.
In a fifth aspect of the present invention, there is provided a lateral flow test strip, wherein the lateral flow test strip is provided with a housing, a sample port, and a chromatographic window, the lateral flow test strip of the first aspect of the present invention is provided inside the lateral flow test strip, and the first quality control region, the detection region, and the second quality control region of the lateral flow test strip are positioned to correspond to the chromatographic window.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
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FIG. 1 shows a test strip housing.
FIG. 2 shows a test strip of the present invention, which is, in order from left to right: sample pad, conjugate pad, detection line T, quality control line C, absorption pad, wherein chromatography membrane region is cut through the arc.
Fig. 3 shows a test strip picture of the present invention (right) and a conventional test strip (left).
Figure 4 shows a rectangular cut test piece.
Detailed Description
The inventors of the present invention have conducted extensive and intensive studies to find out that, when a specific cleavage is performed on a detection line position of a conventional test strip, the local concentration of an analyte at the T detection line position can be significantly increased, thereby increasing the binding efficiency of a binder-analyte complex and improving the detection sensitivity. The present invention has been completed based on this finding.
Description of the terms
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
Immunochromatography
The immunochromatography detects a biomarker in a body fluid by using a body fluid component of a human or an animal as a measurement object, and reads and quantifies the depth of a reaction line or a spot color tone shown by the biomarker.
The reflection photometry method of the test paper is applied to the measurement of urine and blood samples. At present, the test paper, the measuring instrument, the daily urine test paper and the like are widely applied to resident health diagnosis and hospital screening. The test items include sugar, protein, bilirubin, hemoglobin, urobilin, etc. And a T detection line and a C control line are arranged on the test piece, wherein the T detection line is used for quantitatively indicating the concentration of the analyte, and the C control line is used for quality control.
Immunochromatography techniques
The Immunochromatography (Immunochromatography) is a rapid detection technology established in the 80 th and the 90 th of the 20 th century. The immunochromatography technology does not need to separate the combined marker from the free marker, so the operation is simple and quick, and the immunochromatography technology is very suitable for field detection.
Immunochromatographic test strip (or test paper strip)
In the present invention, a lateral flow test strip is provided, the test strip comprising: a sample pad, a binding zone, a detection zone, a quality control zone, and an absorbent pad.
In the present invention, the terms "lateral flow strip", "test paper strip", "test paper sheet", "chromatographic strip" and "test strip" have the same meaning and are used interchangeably.
A preferred test strip housing configuration of the present invention is shown in FIG. 1, which includes:
the plastic ABS shell can be internally provided with the test piece shown in figure 2, and the upper part of the plastic ABS shell is provided with two windows (a sample port and a chromatography window), wherein the sample port is a sample dropping position, and the chromatography window is a sample chromatography and observation detection position.
The test strip structure is shown in FIG. 2, which includes: 1 is a sample pad, 2 is a combination pad, 3 is a chromatography reaction film, 4 is an absorption pad, 5 is a coating antibody, and 6 is a bottom backing, wherein the coating antibody consists of a quality control area a, a quality control area c and a detection area b.
The length of the bottom backing 6 is the same as the test piece.
The sample pad 1 is located at one end of the strip and the absorbent pad 4 is located at the other end of the strip.
The conjugate pad 2 is located between the sample pad 1 and the chromatographic reaction membrane 3, at the proximal end of the sample pad 1 and the distal end of the chromatographic reaction membrane 3, adjacent to the sample pad 1.
The chromatographic reaction membrane 3 is positioned between the conjugate pad 2 and the absorbent pad 4, and the coating antibody 5 is disposed on the chromatographic reaction membrane 3, by which the conjugate pad 2 and the absorbent pad 4 are connected. Preferably, the chromatography reaction membrane 3 is further provided with two quality control regions a and d (also called quality control lines C), the quality control region a is located between the detection regions (also called detection lines T) b and C and the binding pad 2, and the quality control region d is located between the absorption pad 4 and the detection regions b and C.
The test piece is preferably produced by sticking the sample pad 1, the conjugate pad 2, the chromatographic reaction membrane 3 and the absorbent pad 4 to the bottom backing 6 with an adhesive.
In the present invention, each constituent element (or component) of the test strip may be made of a material known in the art.
The bottom backing 6 may be made of any stable, non-porous material that is strong enough to support the material and the constituent elements adhered thereto.
In another preferred embodiment, the base backing 6 is substantially impermeable to water, since many assays use water as a diffusion medium.
In another preferred embodiment, the bottom backing 6 is made of a polymer film, preferably a polyvinyl chloride film (e.g., PVC sheeting).
The sample pad 1 may be made of any absorbent material, preferably cellulose, nitrocellulose, cellulose acetate, glass fiber, nylon, polyelectrolyte ion-exchange membranes, propylene copolymer/nylon, and polyethersulfone, or combinations thereof.
The conjugate pad 2 or the chromatographic reaction membrane 3 may be made of any material as long as the material has sufficient porosity to allow capillary action of fluid to occur at the surface and inside. The conjugate pad 2 or chromatographic reaction membrane 3 should have sufficient porosity to allow the movement of particles coated with the antibody or antigen. The conjugate pad 2 or chromatographic reaction membrane 3 may also be wetted by the liquid used in the sample containing the analyte to be detected (e.g., hydrophilic for aqueous liquids and hydrophobic for organic solvents). The hydrophobicity can be altered to make it hydrophilic for use in aqueous liquids by methods such as those described in U.S. patent No.4,340,482 or No.4,618,533, which describe converting hydrophobic surfaces to hydrophilic surfaces. Materials that can be used to make the conjugate pad 2 or the chromatographic reaction membrane 3 include, but are not limited to: polyester membranes, cellulose, nitrocellulose, cellulose acetate, glass fiber, nylon, polyelectrolyte ion exchange membranes, propylene copolymer/nylon, and polyethersulfone (polyethersulfone).
In another preferred embodiment, the conjugate pad 2 is made of a polyester film, and the chromatographic carrier 3 is made of nitrocellulose.
The absorbent pad 4 may be made of any material capable of absorbing liquid as a sample and a buffer. The absorbent capacity of the absorbent pad 4 should be large enough to absorb the liquid added to the test strip. Examples of suitable materials for the absorbent pad 4 include cellulose and absorbent filter paper.
Detection kit and detection method
The kit of the invention comprises:
(a) a lateral flow test strip according to the first aspect of the present invention; and
(b) optionally a first container, and a first test reagent in the first container for mixing with a sample to be tested.
In another preferred embodiment, the kit comprises instructions for use of the detection method.
Detection device
The detection device of the present invention may include: test piece, detector, light source and computer. An instruction for the method of detection may also be included.
The test strip works according to the principle described above, and any method that can be used for detecting the intensity of light can be used for the detection device of the present invention.
The invention uses a disposable rapid diagnosis test piece, and chromatography test paper is placed in the disposable rapid diagnosis test piece. The gold-labeled pad on the chromatographic strip is coated with n detection binders labeled with a detectable label (preferably colloidal gold, such as a colloidal gold-labeled detection binder).
When a sample of human or animal body fluid is added, the analyte in the sample binds to the detection binding agent (preferably a colloidal gold labelled detection binding agent) and gradually chromatographically diffuses to the detection zone.
The detection area is a chromatography reaction film, a plurality of specific antibodies of analytes and quality control coating antibodies are coated on the chromatography reaction film in advance, and after the coating antibodies are combined with the colloidal gold labeled detection binding agent analyte compound, a plurality of detection zone reaction lines with different colors are formed in the corresponding area.
Particularly, the T detection line position of the chromatographic membrane is cut in an upper arc shape and a lower arc shape to form a saddle shape with a narrower T detection line position and wider two end positions. After the position of the T detection line is narrowed, when the colloidal gold labeled detection binding agent-analyte compound flows through the T detection line, the local concentration of an analysis substance at the position of the T detection line can be obviously improved, so that the binding efficiency of the colloidal gold labeled detection binding agent-analyte compound is improved, the local colloidal gold concentration is improved, the color display of the position of the T detection line is deepened, and the detection sensitivity is finally improved.
A plurality of light sources are arranged above the right angle of the instrument at a certain interval, reflected light irradiated to the test paper by the light sources passes through the lens arranged on the optical axis and then passes through an imaging element such as a CCD (charge coupled device), a detection sample developed by an immunochromatography method is subjected to intensity reading after more than 2 reaction lines related to the test item and quantification appear, and the concentration of the test item is quantitatively calculated by a self-contained CPU (central processing unit).
Cutting mode
The present invention provides a method of preparing a test strip according to the first aspect, the method comprising: cutting the test piece: the test piece was formed into a straight rectangular test piece strip having a width of 28cm and then cut into a test piece strip by a hob cutter (with an arc-shaped blade provided thereon) to obtain a test piece according to the first aspect of the present invention. And after cutting, placing the cut plastic piece into the plastic piece to form a finished test strip.
In the present invention, the test strip can be cut in various ways, including but not limited to: arc-shaped (concentrating analyte), parabolic (optimizing liquid concentration path), spindle-shaped (reducing liquid concentration), multi-curved (improving liquid mixing effect), or combinations thereof.
The main advantages of the invention are:
(1) in the specially cut lateral flow test strip, the special structure of the narrowing region, the narrowing region and the amplifying region can effectively improve the local concentration and the concentration efficiency of the analyte;
(2) the novel cutting method can obviously improve the analysis sensitivity of the test piece;
(3) the invention can flexibly increase the quantity of the substances to be analyzed, can accurately and quantitatively analyze the concentration of the analytes at the same time, and can obtain the concentration data of n analytes in the same time;
(4) the test piece and the method can be widely applied to the fields of tumor diagnosis, drug identification, inflammatory reaction diagnosis and the like.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1: measurement of CRP (C-reactive protein) in blood
And dropwise adding the sample to be detected on the sample pad of the test piece, so that the sample to be detected flows towards the direction of the absorption pad and flows through the combination area, the first quality control area, the detection area and the second quality control area.
The sample flowed from the sample pad to the conjugate zone (containing the colloidal gold substance labeled with a mouse anti-human CRP monoclonal antibody) and dialyzed out the antibody to the chromatographic membrane, where capillary phenomenon occurred at the absorbent pad.
If CRP is present in the sample, it forms a complex with the labeled anti-human CRP colloidal gold substance in the binding zone, and then reacts with the labeled anti-human CRP colloidal gold substance of the detection line T (test zone) to produce a red line.
Meanwhile, the labeled rabbit IgG colloidal gold substance in the colloidal gold pad reacts with the goat anti-rabbit IgG polyclonal antibody regardless of the presence or absence of CRP, and a quality control line C appears at C1 and C2.
In this example, all the lateral flow test strips had a width of 5mm before cutting (i.e., d)0＝d45mm), will be flanked byThe width of the T-test line after the arc cutting of the flow test piece was 2mm, the radius of the cut arc was 57.5mm, and d1 ≈ d3 ≈ 3.5 mm.
As shown in fig. 2, from left to right, the following are sequentially: sample pad 1, binding area 2 (colloidal gold CRP labeled antibody complex, colloidal gold rabbit antibody complex pre-coated in binding area). The coating antibody 5 is arranged on the chromatographic reaction membrane 3, and the T detection line and the C control line are respectively coated with: anti-CRP monoclonal antibody, anti-rabbit polyclonal antibody.
The lateral flow test strip is placed in a housing as shown in FIG. 1 and sample solution may be added through the sample port.
After the sample is added, the analyte antigen in the sample is firstly combined with the colloidal gold labeled detection binding agent-analyte complex, then gradually diffuses to the middle part of the test strip through a chromatography reaction, and the coated antibody in the middle part of the test strip is then diffused to be gradually combined with the colloidal gold labeled detection binding agent-analyte complex. In the process, as the liquid flow path is narrowed, the colloidal gold labeled detection binding agent-analyte compound can be effectively concentrated, and indicator lines with different colors are formed and are respectively named as: test line T, control line C.
After the reaction is finished, the test piece is placed in a reading instrument, and data are directly read. In order to improve the resolution of reading the pixels of the reagent strip, the data of 200-1000 pixels is read and at least 10-50 times of reading is carried out, and after the upper limit value and the lower limit value of the data are removed, the rest of data (the pixels of each point) are averaged and then used as sample integration information. Data formation is shown in table 1 below.
In this example, the test pieces were divided into A, B groups, each group consisting of 6 pieces:
group A: performing arc cutting on A-1 to A-3, and performing no cutting on A-4 to A-6;
group B: the arc-shaped cuts B-1 to B-3 were made, and the cuts B-4 to B-6 were not made (A, B groups cut exactly in the same manner).
Taking the CRP test strip as an example, a diluted plasma CRP standard sample with a final concentration of 20ng/ml was used for the arc cut test strip and the original test strip, respectively, and 3 test strips were measured per group:
group 1: three A groups of arc cutting test pieces, namely A-1 to A-3, are measured;
group 2: three test pieces of the original design of group A, namely A-4 to A-6, were measured;
group 3: three B groups of arc cutting test pieces, namely B-1 to B-3 are measured;
group 4: three test pieces of the original design of group B, namely B-4 to B-6, were tested.
A. The test result data of the test pieces in group B are shown in Table 1.
TABLE 1A, B test piece test result data
Figure BDA0000987471230000111
The resulting average optical density values for the positions of the T-test lines are shown in column 2 of table 1, and the average optical density values for each set of measurements are shown in column 3 of table 1.
As shown in table 1, group a: the average value of the optical density values of the arc-shaped cuts A-1 to A-3 is 0.631, the average value of the optical density values of the original design test pieces A-4 to A-6 of the group A is 0.468, and the signal increment of the cutting group relative to the original design group is 34.89%;
group B: the average values of the optical density values of the arc-shaped cuts B-1 to B-3 were 0.615, the average values of the optical density values of the test pieces B-4 to B-6 of the original design of the group B were 0.508, and the signal increase of the cut group relative to the original design group was 21.26%.
The statistical F test and T test were performed on the test pieces of group A and group B, respectively, and the obtained F value and P value are shown in Table 1, and the two groups of data have significant difference (P < 0.01).
Results and discussion:
as shown in FIG. 3 (left), the conventional test piece has a rectangular shape, the chromatographic carrier is provided in a 3-5mm straight strip, and the concentration of the liquid on the carrier is uniform throughout the chromatographic carrier as the chromatography proceeds.
The test piece is specially cut, the length of the detection line is shortened, the local concentration is effectively improved, and meanwhile, the position of the T detection line is combined with various effects:
(1) local molecular binding efficiency enhancement
The concentration of molecules at the T detection line is increased, the binding efficiency of the molecules and the antibody is obviously increased, and therefore the binding probability of the molecules and the antibody is enhanced. Meanwhile, the probability of collision between the colloidal gold and the antibody is improved due to the higher concentration of the colloidal gold.
(2) Liquid flow rate enhancement
As the flow channel is gradually narrowed, the flow speed of the liquid at the T detection line is gradually increased. In the embodiment, the time for liquid to pass through the whole chromatographic membrane is 20s and the time after arc cutting is 10s on the originally designed test piece, so that the detection time is greatly shortened.
(3) In the embodiment, the antibody is fixed firstly and then cut, so that the detection sensitivity is obviously improved under the condition that the concentration of the detection binding agent is unchanged.
Comparative example 1
The same as example 1, i.e. groups A-4 to A-6 and B-4 to B-6.
As shown in the data in Table 1, the optical density values of the groups A-4 to A-6 and B-4 to B-6 of the original design test pieces without cutting were 0.468 and 0.507, respectively, which are reduced by 34.8% and 21.3% compared to the gray level after cutting, i.e., the sensitivity was reduced by at least 20%.
Comparative example 2
The difference from example 1 was that a rectangular cut was made with a hob cutter to the test piece, and the position of the T-shaped detection line was set to a detection area of 2mm width, as shown in FIG. 4. The assay was carried out as in example 1.
The result shows that in the rectangular cutting mode, the region from the quality control line to the detection line is not buffered, and most of the gold-labeled particles are gathered in the region from the quality control line to the detection line. Meanwhile, the cutting parts on both sides of the narrowing region form a serious edge effect, the sensitivity is greatly reduced (being different from the uncut test piece shown in comparative example 1), and an effective detection effect cannot be achieved.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (8)

1. A lateral-flow test strip, which is an immunoreaction-based test strip and which comprises, in order: a sample pad, a binding region, a first quality control region, a detection region, a second quality control region, and an absorption pad,
wherein the binding zone comprises detection binding agents for detecting n analytes, one for each of the n analytes, which analytes form a "detection binding agent-analyte" complex with the detection binding agents, said detection binding agents being labeled with a detectable label;
the detection area is provided with m detection lines, wherein m is a positive integer and is more than or equal to n, and each detection line is used for capturing a detection binding agent-analyte complex formed by the binding of the analyte and the detection binding agent thereof;
the first quality control area is provided with a first quality control line, and the first quality control line is used for capturing a complex formed by the combination of the first quality control substance and the corresponding detection binding agent;
the second quality control area contains a second quality control line, and the second quality control line is used for capturing a complex formed by the combination of the second quality control substance and the corresponding detection binding agent;
wherein the first quality control area is a narrowing area with a radial width d1And the narrowing region is gradually narrowed;
the detection zone is a narrow zone having a radial width d2；
The second quality control region is an amplification region with a radial width d3；
The radial width of the bonding region is d0；
The radial width of the absorption pad is d4；
And, the radial direction is wideThe degree satisfies:
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>
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>
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and is
Figure DEST_PATH_IMAGE008A
>
Figure DEST_PATH_IMAGE010A
>
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；
D is2In the range of 1-15mm, said d0The range is 3-20 mm;
the narrowing region and the amplifying region are arc-shaped;
the arc line type has the following characteristics:
(a) for the narrowing region, the slope of the tangent to the arc gradually decreases along the direction of sample flow; and
(b) for the amplification zone, the slope of the tangent to the arc increases gradually along the direction of sample flow;
the narrow region
Figure DEST_PATH_IMAGE006AAA
And said amplification region
Figure DEST_PATH_IMAGE004AA
The width ratio of (A) is in the range of 0.1-0.7; and the narrow region
Figure DEST_PATH_IMAGE006AAAA
And said narrowing region
Figure DEST_PATH_IMAGE010AA
The width ratio of (A) is in the range of 0.1-0.7;
the throat region is of an arc type, and the arc comprises: a first constriction region in which the slope of the tangent to the arc gradually decreases and a second constriction region in which the slope of the tangent to the arc gradually increases in the direction along which the sample flows.
2. A lateral flow test strip according to claim 1, wherein said constriction region
Figure DEST_PATH_IMAGE016
And said amplification region
Figure DEST_PATH_IMAGE017
The width ratio of (A) is in the range of 0.2-0.6; and the narrow region
Figure 327969DEST_PATH_IMAGE016
And said narrowing region
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The width ratio of (a) is in the range of 0.2 to 0.6.
3. A lateral flow test strip according to claim 1, wherein said lateral flow test strip
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/
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=1.0 ± 0.2; and said
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/
Figure DEST_PATH_IMAGE026
=1.0±0.2；
And the first quality control line is positioned at the near end, the second quality control line is positioned at the far end, the distance between the first quality control line and the first detection line is 1-10mm, and the distance between the second quality control line and the m detection line is 1-10 mm.
4. A lateral flow test strip according to claim 1, wherein said analyte is selected from the group consisting of procalcitonin, C-reactive protein, troponin, myosin, myoglobin, creatine kinase MB isozyme, B-type natriuretic peptide, D-dimer, N-terminal brain natriuretic peptide precursor, cardiac fatty acid binding protein, microalbumin, neutrophil gelatinase-associated lipocalin, β 2-microglobulin, amyloid, cystatin C, or a combination thereof;
and the detection binding agent is a specific antibody.
5. A method for non-diagnostically detecting n analytes in a sample, n being from 1 to 10, comprising the steps of:
(1) providing a sample to be tested and the test strip of claim 1;
(2) dropwise adding the sample to be detected on the sample pad of the test piece, so that the sample to be detected flows towards the direction of the absorption pad and flows through the combination area, the first quality control area, the detection area and the second quality control area;
(3) respectively reading the optical intensity P of the first quality control line in the first quality control region1And the optical intensity P of the second quality control line in the second quality control region2(ii) a And the optical intensity of the detection line in the detection zone corresponding to the i-th analyte, denoted as PiWherein i is 1 to n; and
(4) the optical intensity Pi and the optical intensity P are measured1And P2And comparing to obtain the qualitative and/or quantitative detection result of the ith analyte respectively.
6. A detection kit, comprising:
(a) a lateral flow test strip according to claim 1; and
(b) optionally a first container, and a first test reagent in the first container for mixing with a sample to be tested.
7. Use of a lateral flow test strip according to claim 1 for the preparation of a kit for detecting the concentration of an analyte in a biological sample.
8. A lateral flow paper, characterized in that the lateral flow paper is provided with a housing, a sample port, and a chromatographic window, the lateral flow test strip of claim 1 is provided inside, and the first quality control region, the detection region, and the second quality control region of the lateral flow test strip are positioned to correspond to the chromatographic window.
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