WO2013082943A1

WO2013082943A1 - Fluorescence assay and device

Info

Publication number: WO2013082943A1
Application number: PCT/CN2012/078715
Authority: WO
Inventors: 李久彤
Original assignee: Li Jiutong
Priority date: 2011-12-06
Filing date: 2012-07-16
Publication date: 2013-06-13

Abstract

A test strip (1) includes: (i) an application zone (3) for receiving a sample; (ii) a binding zone (4) proximal to the application zone (3), which comprises one or more flowable binding reagents, at least one of the binding reagents is labeled with light-absorbing materials, and the binding reagents can bind an analyte or an equivalent thereof to form a complex including the light-absorbing materials; (iii) a detection zone (5) proximal to the binding zone (4) and distal to the application zone (3), in which an immobilized reagent is disposed, and the immobilized reagent is used to capture the complex including the light-absorbing materials, wherein the complex moves from the binding zone (4) to the detection zone (5); and (iv) a sample absorption zone (7) proximal to the detection zone (5) and distal to the binding zone (4). The application zone (3) or the binding zone (4) includes a flowable detection reagent labeled with fluorescent materials. Or, the immobilized reagent disposed in the detection zone (5) is labeled with fluorescent materials when neither the application zone (3) nor the binding zone (4) comprises a flowable detection reagent labeled with fluorescent materials. When the immobilized reagent captures the complex including the light-absorbing materials and the detection reagent, or when the immobilized reagent labeled with fluorescent materials captures the complex including the light-absorbing materials, the light-absorbing materials effect on the fluorescence intensity of fluorescent materials. Provided are a fluorescence measuring device for detecting quantitatively analytes, which comprises a test strip and a detector for measuring the fluorescence intensity, and a fluorescence assay by applying the test strip.

Description

Fluorescence analysis method and device

Technical field

The invention belongs to the field of in vitro detection technology, and in particular relates to an analysis method and device based on measuring fluorescence intensity. Background technique

In the field of immunoassays, it is often necessary to perform qualitative or quantitative detection of various antigens or antibodies. In the prior art, a variety of immune reaction analysis methods are derived based on "competitive inhibition and double-anti-sandwich", such as: radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence assay, time-resolved fluorescence assay, and fluorescence immunoassay. It can be used to identify pathogenic microorganisms, quantitatively detect specific proteins in the human body, and thus assist in the diagnosis or monitoring of diseases. In such an immunological reaction assay method, the capture antibody is usually immobilized on a solid phase carrier, then reacted with an antigen (target protein), washed, reacted with a labeled antibody, washed, and finally detected for radioactivity, solution absorbance or light signal, and thus reported. The concentration of the target protein in the sample is detected. The automation of the above method eliminates the cumbersome manual washing, but because of the automation, the instrument is bulky and expensive, and is generally only used in large laboratories.

In 1990, Beggs et al. colloidal gold immunoassay (GICA) was used to detect HCG in human urine and serum (BEGGS M, NOVOTNY M, SAMPED OS. A selfperforming chromatographic immunoassay for The qualitative determination of human chorionicgonadotrophin (HCG) in urine and serum [J] . Clin Chem, 1990, 36: 1084-1085). For the next 20 years, on the basis of GICA, people have developed pathogens (CN 031 15143.4), hormones (CN 200610014168.3), cardiac markers (CN 20041001 1 165.5), tumor markers (CN 200510104796.6), autoimmune diseases. Detection reagents such as markers (CN 200410027291.X); also applied to food, environmental (CN 031 16692.X) and veterinary (CN 02139704.X) fields. Since this method only needs to be observed by the naked eye, non-professionals can also operate, making it possible to carry out relevant tests in places such as emergency departments, primary hospitals, patient bedssides, and places far away from large laboratories, so the application range is quite extensive.

Although this immunoassay has been developed for more than 20 years, its basic working principle has not changed, which determines that it has only been used for qualitative or semi-quantitative testing (according to the gray scale of the detection line). The sensitivity is not as good as the quantitative immunoassay method described above, and its further application encounters a bottleneck.

Therefore, there is an urgent need in the art to change the existing colloidal gold chromatography method, and to extend its application field while giving it a high sensitivity and a quantitative and accurate new advantage while maintaining its characteristics of being fast, simple, and low in cost. Summary of the invention The present invention provides a test piece for quantitatively detecting a sample to be tested in a sample.

The present invention provides a method for quantitatively detecting a test object, which is highly sensitive and quantitatively accurate.

The invention also provides a detecting device for quantitatively detecting an object to be tested.

A first aspect of the present invention provides a test piece, the test piece comprising:

(i) a sample application area that can be added to the sample;

(ii) a binding zone located at the proximal end of the sample application zone, the binding zone comprising one or more flowable binding agents, at least one of which is labeled by a light absorbing substance, the binding agent being capable of being tested Combining the substance or its equivalent to form a complex containing a light absorbing substance;

(iii) a test zone located at a proximal end of the binding zone and a distal end of the loading zone, the test zone comprising an immobilized reagent for capturing a complex containing a light absorbing substance that moves from the binding zone to the test zone ; with

(iv) a sample absorption zone located at the proximal end of the test zone and distal to the binding zone, wherein the absorption zone has an absorptive capacity such that the sample applied to the sample application zone diffuses from the sample application zone to the terminal sample absorption zone;

Wherein the immobilization reagent is labeled with or not marked by a fluorescent substance; the sample application zone and/or the binding zone comprises a flowable agent that is labeled with a fluorescent substance; or when the addition When both the sample zone and the binding zone do not contain a flowable agent labeled with a fluorescent substance, the immobilization reagent located in the test zone is labeled with a fluorescent substance, and

The light absorbing substance affects the fluorescence intensity of the fluorescent substance when the immobilizing agent captures the complex containing the light absorbing substance and the detecting agent, or when the immobilizing agent labeled with the fluorescent substance captures the complex containing the light absorbing substance.

In another preferred embodiment, the immobilizing agent is not labeled with a fluorescent substance, and the sample loading zone and/or the binding zone comprises a flowable agent that is labeled with a fluorescent substance.

In another preferred embodiment, the immobilizing agent is labeled with a fluorescent substance, and neither the loading zone nor the binding zone contains a flowable agent that is labeled with a fluorescent substance.

In another preferred embodiment, the binding region comprises a first binding region and a second binding region, wherein the first binding region is located at a proximal end of the loading region, and comprises a binding agent labeled by the light absorbing substance, the second The binding zone is located at the distal end of the sample application zone and contains a biotin-labeled analyte equivalent;

Wherein the amount of the binding agent labeled by the light absorbing substance is greater than the amount of the analyte, and the analyte and the equivalent of the biotin-labeled analyte are competitively combined with the binding agent to form a biotin. A labeled complex containing a light absorbing material.

In another preferred embodiment, the analyte comprises: a protein, a nucleic acid or a small molecule compound.

In another preferred embodiment, the fluorescence excitation or emission spectrum of the test zone overlaps all or partially with the absorption spectrum of the light absorbing material.

In another preferred embodiment, the fluorescent substance is selected from the group consisting of fluorescein, carboxyfluorescein, 2-methoxyfluorescein, 4, 5-dimethoxyfluorescein, rhodamine, phycoerythrin, quantum Point, or rare earth ion or Its chelate.

In another preferred embodiment, the light absorbing material is selected from the group consisting of colloidal gold, nanogold rods, nanosilver rods, or combinations thereof.

In another preferred embodiment, at least one control zone is further included between the test zone and the sample absorption zone, the control zone containing an immobilized contrast agent, wherein the control agent is used to specifically bind to the light-absorbing substance. Binding agent.

A second aspect of the present invention provides a fluorescence analysis method for quantitatively detecting a test object, the method comprising the steps of:

(1) adding a sample of the test object to the sample application area of the test piece according to the first aspect of the invention;

(2) The fluorescence intensity of the test area of the test piece is measured and converted into the number of objects to be tested.

In another preferred embodiment, in step (2), the ratio of the fluorescence intensity F2 at the middle of the test zone to the control zone to the fluorescence intensity F1 of the test zone is compared with a standard curve to determine the amount of the analyte; Alternatively, the fluorescence intensity F1 of the test zone is compared with a standard curve or an unimported initial fluorescence intensity F0 to determine the amount of the analyte.

A third aspect of the invention provides a test kit comprising: a test piece according to the first aspect of the invention; and instructions for use.

A fourth aspect of the present invention provides a fluorescence measuring device for quantitatively detecting an object to be tested, the device comprising:

(a) a test piece according to the first aspect of the invention;

(b) a detector for detecting fluorescence intensity; and

(c) A description of the use of the method of the second aspect of the invention.

A fifth aspect of the present invention provides a test piece, the test piece comprising:

(i) a sample application zone that can be added to the sample;

(iiM is located at the proximal end of the loading zone, the binding zone comprising:

One or more flowable binding agents, at least one of which is labeled with a light absorbing substance, the binding agent being capable of combining with the analyte or its equivalent to form a complex comprising a light absorbing substance;

a flowable detecting agent, the detecting agent being labeled with a fluorescent substance;

(iiiM a test zone standing at the proximal end of the binding zone and distal to the loading zone, the test zone comprising an immobilized capture agent for capturing a complex of light absorbing material that moves from the binding zone to the test zone And detection agent; and

(ivM stands in the sample absorption zone at the proximal end of the test zone and at the distal end of the binding zone, wherein the absorption zone has an absorption capacity, so that the sample added to the sample application zone is diffused from the sample application zone to the end sample absorption zone;

Wherein, when the capturing agent captures the complex containing the light absorbing substance and the detecting agent, the light absorbing substance affects the fluorescence intensity of the test area.

In another preferred embodiment, the fluorescence of the test zone is the fluorescence of the detector. In another preferred embodiment, the light absorbing material affects the fluorescence intensity of the test zone resulting in a decrease in the fluorescence intensity of the test zone.

In another preferred embodiment, the analyte is an antigen or an antibody.

In another preferred embodiment, the binding agent specifically binds to the analyte or an equivalent thereof; preferably, the binding agent is an antigen, an antibody or an oligonucleotide.

In another preferred embodiment, the binding region may comprise two binding agents, wherein one binding agent labeled by the light absorbing substance and the other binding agent labeled with biotin, the two binding agents simultaneously The analytes are combined to form a biotin-labeled complex containing light absorbing material.

In another preferred embodiment, the light-absorbing substance-containing composite may contain the analyte or an equivalent thereof.

In another preferred embodiment, the flowable detector is a fluorescently labeled biotin or capture antibody. In another preferred embodiment, the capture agent specifically binds to a complex containing a light absorbing substance and a detection agent. In another preferred embodiment, the capture agent is an antibody to streptavidin or a test substance.

In another preferred embodiment, the amount of the binding agent is greater than the amount of the analyte; the amount of the detecting agent is greater than the amount of the complex containing the light absorbing material; the amount of the capturing agent is greater than the composite containing the light absorbing material The total number of substances and detectors.

In another preferred embodiment, the binding region comprises a first binding region and a second binding region, wherein the first binding region is located at a proximal end of the loading region, and comprises a binding agent labeled with a light absorbing substance and is labeled with a fluorescent substance. The second binding zone is located at the distal end of the loading zone, and comprises a biotin-labeled analyte equivalent; wherein the amount of the binding agent labeled by the light absorbing substance is greater than the number of the analytes, The analyte and the biotin-labeled analyte equivalent compete with the binding agent to form a biotin-labeled complex containing the light absorbing material.

In another preferred embodiment, after the binding agent is combined with the analyte, the remaining binding agent moves to the second binding region, and combines with the biotin-labeled analyte equivalent to form a biotin-labeled inclusion. A composite of light absorbing materials.

In another preferred embodiment, the detection agent is a fluorescently labeled biotin or a capture agent antibody.

In another preferred embodiment, the analyte is a liquid phase (solution), a suspension or a solid phase.

In another preferred embodiment, the analyte comprises a specific protein such as a tumor marker or a myocardial marker.

In another preferred embodiment, the fluorescent substance is selected from the group consisting of fluorescein, carboxyfluorescein, 2-methoxyfluorescein, ₄ , 5-dimethoxyfluorescein, rhodamine, phycoerythrin, quantum a point, or a rare earth element ion or a chelate thereof. In another preferred embodiment, the light absorbing material is selected from the group consisting of colloidal gold, nanogold rods, nanosilver rods, or combinations thereof.

In another preferred embodiment, the colloidal gold is colloidal gold particles having an average particle diameter of 10 to 70 nm.

In another preferred embodiment, the control zone is used to reveal the validity of the test result of the test object.

A sixth aspect of the present invention provides a fluorescence analysis method for quantitatively detecting a test object, comprising the steps of: (1) adding a sample of the test object to a sample application area of the test piece according to the fifth aspect of the present invention;

In another preferred embodiment, when the analyte is a solid phase, the step (1) further comprises adding a solvent such as water or a buffer.

In another preferred embodiment, in step (2), the ratio of the fluorescence intensity F2 at the middle of the test zone to the control zone to the fluorescence intensity F1 of the test zone is compared with a standard curve to determine the amount of the analyte; Alternatively, the fluorescence intensity F 1 of the test zone is compared with a standard curve to determine the amount of the analyte. In another preferred embodiment, the method further comprises the step of making a standard curve by measuring with a known concentration of the test object standard.

A seventh aspect of the invention provides a test kit comprising: a test piece according to the fifth aspect of the invention; and an instruction manual.

According to an eighth aspect of the invention, there is provided a fluorescence measuring device for quantitatively detecting an object to be tested, the device comprising:

(a) a test piece according to the fifth aspect of the invention;

(b) a detector for detecting fluorescence intensity; and

(c) Instructions for using the method of use.

In another preferred embodiment, the apparatus further includes a light source and a computer.

The light source is irradiated to the detection line through the optical fiber, and the excited fluorescence enters the detector through the optical fiber, and is processed and analyzed by a computer.

A ninth aspect of the present invention provides a test piece for quantitatively detecting a sample to be tested in a sample, the test piece comprising:

a sample loading zone that can be added to the sample;

a binding zone located at a proximal end of the sample application zone, the binding zone comprising one or more flowable binding agents capable of binding to the analyte, at least one of the binding agents being labeled by a light absorbing substance, the binding The agent can be combined with the analyte or its equivalent to form a complex containing the light absorbing material;

a test zone located at a proximal end of the binding zone and a distal end of the sample application zone, the test zone comprising an immobilized detection agent, the detection agent being labeled by a fluorescent substance, the detection agent being used to capture movement from the binding zone to the test zone Containing suction a complex of light substances; and

a sample absorption zone located at a proximal end of the test zone and at a distal end of the binding zone, wherein the absorption zone has an absorption capacity, thereby allowing the sample added to the sample application zone to diffuse from the sample application zone to the terminal sample absorption zone;

Wherein, when the detecting agent captures a complex containing a light absorbing substance, the light absorbing substance affects the fluorescence intensity of the fluorescent substance.

In another preferred embodiment, the analyte is an antigen or an antibody.

In another preferred embodiment, the binding agent specifically binds to the test substance or its equivalent; preferably, the binding agent is an antigen, an antibody or an oligonucleotide.

In another preferred embodiment, the composite containing the light absorbing material may contain the analyte or the equivalent thereof.

In another preferred embodiment, the detecting agent specifically binds to the complex containing the light absorbing substance; preferably, the detecting agent is a fluorescently labeled antigen or antibody.

In another preferred embodiment, the binding region may comprise two binding agents, wherein one binding agent labeled by the light absorbing substance and the other binding agent labeled with biotin, the two binding agents simultaneously The analytes are combined to form a biotin-labeled complex containing a light absorbing material.

In another preferred embodiment, the detection agent of the test zone is a fluorescently labeled streptavidin.

In another preferred embodiment, the amount of the binding agent is greater than the amount of the analyte; the amount of the detecting agent is greater than the amount of the complex containing the light absorbing material.

In another preferred embodiment, the binding region comprises a first binding region and a second binding region, wherein the first binding region is located at a proximal end of the loading region, and comprises a binding agent labeled by a light absorbing substance; The binding zone is located at the distal end of the sample application zone and contains a biotin-labeled analyte equivalent;

Wherein the amount of the binding agent labeled by the light absorbing substance is greater than the amount of the analyte, and after the binding agent is combined with the analyte, the remaining binding agent moves to the second binding region, and the biotin-labeled The analyte equivalent combines to form a biotin-labeled complex containing a light absorbing material.

In another preferred embodiment, the amount of the biotin-labeled analyte equivalent is greater than the amount of the remaining binder.

In another preferred embodiment, when the analyte is a solid phase, the step (2) further comprises adding a solvent such as water or a buffer. In another preferred embodiment, the analyte comprises a specific protein such as a tumor marker, a myocardial marker, or the like. In another preferred embodiment, the binding of the binding agent to the analyte or its equivalent, the detecting agent and the complex is a specific combination.

In another preferred embodiment, the analyte or the equivalent thereof is an antigen, and the binding agent and the detection agent are antibodies that bind to the antigen simultaneously; or The test substance or its equivalent is an antibody, and the binding agent and the detection agent are antibodies (anti-antibodies) which can bind to the antigen of the antibody or the antibody at the same time.

In another preferred embodiment, the binding agent is a biotin-labeled binding agent, and the detection agent is a fluorescently-labeled streptavidin.

In another preferred embodiment, the analyte equivalent is labeled with biotin, and the detection reagent is a fluorescently labeled streptavidin.

In another preferred embodiment, the excitation or emission spectrum of the fluorescent substance overlaps all or partially with the absorption spectrum of the light absorbing substance.

In another preferred embodiment, the fluorescent substance is selected from the group consisting of fluorescein, carboxyfluorescein, 2-methoxyfluorescein, 4, 5-dimethoxyfluorescein, rhodamine, phycoerythrin, quantum a point, or a rare earth element ion or a chelate thereof.

In another preferred embodiment, the light absorbing material is a nano gold rod.

In another preferred embodiment, the nano-gold rod has an aspect ratio of from 1.5 to 10, preferably from 1.5 to 5.

In another preferred embodiment, the nanogold rod has a length of 10 to 200 nm, preferably 20 to 100 nm. In another preferred embodiment, the colloidal gold is colloidal gold particles having an average particle diameter of 10 to 70 nm.

In another preferred embodiment, the control zone is used to control the validity of the test result of the test object.

A tenth aspect of the present invention provides a fluorescence analysis method for quantitatively detecting a test object, comprising the steps of:

(1) providing a test piece according to the ninth aspect of the invention;

(2) adding a sample of the sample to the sample application area;

(3) the analyte in the sample moves to the binding zone and combines with the binding agent to form a complex containing the light absorbing substance;

(4) Step (3) The obtained composite containing the light absorbing substance is moved to the test area, and combined with the detecting agent therein to form a composite containing the light absorbing material and the fluorescent substance;

(5) Measurement step (4) The fluorescence intensity F of the composite containing the light absorbing material and the fluorescent material is converted into the number of analytes.

In another preferred embodiment, the binding zone in the step (3) comprises a first binding zone and a second binding zone, wherein the first binding zone is located at a proximal end of the loading zone and comprises a binding agent labeled by the light absorbing substance. The second binding zone is located at a distal end of the sample application zone and comprises a biotin-labeled analyte equivalent;

Wherein the amount of the binding agent labeled by the light absorbing substance is greater than the amount of the analyte, and after the binding agent is combined with the analyte, the remaining binding agent moves to the second binding region, and the biotin-labeled Equivalent object Combines to form a biotin-labeled complex containing a light absorbing material.

In another preferred embodiment, the step (5) comprises: fluorescence intensity of the complex containing the light absorbing material and the fluorescent material

The F and the standard curve are compared with the unsampled initial fluorescence intensity F0 to determine the amount of the analyte.

In another preferred embodiment, the method further comprises the step of making a standard curve by measuring with a known concentration of the test object standard.

According to an eleventh aspect of the present invention, there is provided a fluorescence measuring device for quantitatively detecting an object to be tested, the device comprising:

(a) a test piece according to the ninth aspect of the invention;

(b) a detector for detecting fluorescence intensity; and

(c) A description of the use of the method of the tenth aspect of the invention.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here. DRAWINGS

Figure 1 is a schematic diagram of a test piece.

Figure 2 is a schematic view of the detecting device.

Fig. 3 is a graph showing the standard concentration (C) of the AFP standard series and the corresponding fluorescence intensity ratio (F2/F1) of Example 1.

Fig. 4 is a graph showing the logarithm of the logarithmic value (lgC) of the CRP standard series concentration of Example 2 and the logarithmic value of the corresponding fluorescence intensity value lg (F2/Fl).

Fig. 5 is a graph showing the AFP standard series concentration (C) and the corresponding fluorescence intensity (F) standard chart of Example 3.

Figure 6 is a graph showing the AFP standard series concentration (C) and the corresponding fluorescence intensity (F) standard curve of Example 4.

Figure 7 is a graph showing the CRP standard series concentration (C) and the corresponding fluorescence intensity (F) standard curve of Example 5. detailed description

Through long-term and in-depth research, the present inventors have found a detection method based on the principle of fluorescence quenching, which can not only determine whether the object to be tested is contained by visually measuring the color of the test area, but also measure the fluorescence intensity of the test area. The method for quantitatively detecting the analyte, the flowable fluorescent substance is added to the above method, and then the amount of the analyte is determined by quenching the influence of the substance on the fluorescence intensity of the fluorescent substance. The method not only has the advantages of high sensitivity and accurate quantification, but also is simple, fast, and low in cost. The method is based on a test piece provided by the present invention, through a device containing a light source and a detector The quantitative detection of the object to be tested is realized.

The inventors have also discovered a detection method based on the principle of fluorescence quenching, which can not only determine whether the object to be tested is contained by visually measuring the color of the test area, but more preferably by measuring the fluorescence intensity of the test area. Quantitative detection of the analyte is not only fast, simple, and low-cost, but also has the advantages of high sensitivity and accurate quantitation. The method is based on a test piece provided by the present invention, and the quantitative detection of the object to be tested is realized by a device containing a light source and a detector. On this basis, the inventors have completed the present invention. Test piece

The methods and materials used to make the test pieces of the present invention are now described in more detail. It should be noted that the specific configuration of the test piece may vary depending on the specific test in which the test piece is intended to be performed. It is also within the scope of the invention to make variations of test pieces outside of the examples.

As shown in Fig. 1, the test piece 1 may include a backing sheet 2 having the same length as the test piece.

The sample application zone 3 is located at one end of the test piece, and the sample pad 31 is located in the sample application area and can be attached to the sample application area by an adhesive.

The absorption zone 7 is located at the other end of the test piece, and at the distal end of the application zone, the water absorbing pad 71 is located in the absorption zone. The bonding zone 4 is located between the application zone and the absorption zone. At the proximal end of the sample application zone and the distal end of the absorption zone, the bonding pad 41 is located in the bonding zone.

A test zone 5 (also referred to as a test line) is located between the bond zone and the absorber zone. Typically, the test zone is disposed on the diaphragm 56 through which the bond pad and the absorbent pad are attached. Preferably, the membrane is further provided with a control zone 6 (also called a quality control line), and the control zone is located between the test zone and the absorption zone, or between the test zone and the bonding zone, and between the test zone and the test zone. Have the right space.

The test piece manufacturing method, preferably, as shown in FIG. 1, the sample pad, the bonding pad, the film, and the water absorbing pad are respectively adhered to the backing plate by an adhesive, that is, the test piece is obtained.

The backing sheet can be made of any stable, non-porous material that is strong enough to support the material and the test piece adhered thereto. Because many assay waters are used as diffusion media, the backing sheet is preferably substantially water impermeable. In a preferred embodiment, the backing sheet is made of a polymer film, more preferably a polyvinyl chloride film (e.g., a PVC board).

The sample gasket can be made of any absorbent material. Examples of materials that can be used include: cellulose, nitrocellulose, cellulose acetate, glass fiber, nylon, polyelectrolyte ion exchange membrane, propylene copolymer/nylon, and polyethersulfone.

The bonding pad or diaphragm may be made of any material as long as the material has sufficient porosity to allow capillary action of the fluid on the surface and inside. The bonding pad or membrane should have sufficient porosity to allow the particles coated with antibodies or antigens to move. The bonding pad or membrane may also be wetted by the liquid used in the sample containing the analyte to be detected (e.g., hydrophilic for aqueous liquids, hydrophobic for organic solvents). The method described in, for example, U.S. Patent No. 4,340,482 or No. 4,618,533 By converting a hydrophobic surface to a hydrophilic surface, its hydrophobicity can be altered to render it hydrophilic for use in aqueous liquids. Examples of materials that can be used to make bonded gaskets or membranes include: polyester film, cellulose, nitrocellulose, cellulose acetate, glass fiber, nylon, polyelectrolyte ion exchange membrane, propylene copolymer/nylon, and polyethersulfone. (polyether _S ulfon _e ). In a preferred embodiment, the bonding pad is made of a polyester film and the diaphragm is made of nitrocellulose.

The absorbent pad can be made of any material that absorbs the liquid as a sample and buffer. The absorption capacity of the absorbent pad should be large enough to absorb the liquid added to the test piece. Examples of materials suitable for the absorbent pad include cellulose and glass fibers.

The control area is used to reveal the validity of the test result of the test object. The control zone contains an immobilized contrast agent, wherein the control agent is used to specifically bind to a binding agent labeled with a light absorbing substance, free or bound to a test substance. Detection method

The test strips of the present invention can be used in a number of different lateral flow assays, and these assays or assays preferably include the steps of:

(1) adding a sample of the test object to the sample application area of the test piece of the present invention;

Preferably, the analysis or detection method comprises the following method:

Test Method A: One or more flowable binders and a flowable detector are used, as well as an immobilized capture agent.

After the sample is applied, the analyte in the sample is moved to a binding zone, the binding zone containing a binding agent and a detecting agent, and the analyte is combined with the binding agent therein to form a complex containing the light absorbing substance; The detection agent in the binding zone and the resulting complex containing the light absorbing material are moved to the test zone and combined with the capture agent therein to form a test zone enriched with the light absorbing material and the fluorescent material.

Test Method B: One or more flowable binders and an immobilized detector are used.

Wherein at least one of the binding agents is labeled with a light absorbing substance, and the binding agent can be combined with the analyte or an equivalent thereof to form a complex containing a light absorbing substance; the detecting agent is labeled with a fluorescent substance. The detecting agent is for capturing a complex containing a light absorbing substance that moves from the binding zone to the test zone, thereby forming a complex containing the light absorbing material and the fluorescent substance.

Specifically, when the light absorbing substance affects (partially quenches or completely quenches) the fluorescence intensity of the test zone, the detection method of the present invention can determine the amount of the analyte by detecting the fluorescence intensity of the test zone.

A preferred detection method comprises: measuring the fluorescence intensity F2 at the middle of the test zone and the control zone and the fluorescence intensity F1 of the test zone, and determining the number of analytes based on the ratio of the fluorescence intensities F2 and F1. It should be understood that the ratio of the fluorescence intensity F2 to F 1 includes (but is not limited to): F 1 /F2, F2/F 1 , F 1 /(F 1 +F2), F2/(F 1 +F2), (F1 +F2)/ Fl, (Fl+F2)/F2, F1/(|F1-F2|), F2/(|F1-F2|), (|F1-F2|)/ Fl, (|F1-F2| )/F2 And similar ratios. Test object or its equivalent

As used herein, the terms "subject", "its equivalent" or "analyte" refer to any component of a sample to be tested with a test piece and optionally quantified, the analyte or equivalent thereof. Examples of substances or analytes include: proteins, such as hormones or other secreted proteins, enzymes and cell surface proteins; glycoproteins; peptides; small molecules; polysaccharides; antibodies (including monoclonal or polyclonal antibodies and fragments thereof); Drugs (including cardiotonic drugs such as digoxin); toxins; drugs; viral or viral particles; cell wall components; or other compounds with epitopes.

Preferably, the analyte comprises a specific protein such as a tumor marker, a myocardial marker, and the tumor marker is selected from the group consisting of: alpha fetoprotein (AFP), C-reactive protein (CRP), carcinoembry Antigen (CEA), cancer antigen 125 (CA125), carbohydrate antigen 19-9 (CA19-9), total prostate specific antigen (PSA), free prostate specific antigen (f-PSA), neuron-specific enolase (NSE), sugar chain antigen (CA242), cancer antigen (CA15-3), or human chorionic gonadotropin (β-HCG). Binding agent

The binding agent used in the present invention may be any substance capable of binding to the analyte or its equivalent. Specifically, it is a substance that specifically binds the analyte or its equivalent. At least one of the binding agents is labeled with a light absorbing substance, and the binding agent can be combined with the analyte or an equivalent thereof to form a complex containing a light absorbing substance;

There are a variety of different types of molecules that can be used as analyte binding agents, including, for example, oligonucleotides, antibodies, engineered proteins, peptides, haptens, or antigens that have an analyte binding site. Lysate of the source mixture. P. Holliger et al, Trends in Biotechnology 13: 7-9 (1995); S. M. Chamow et al, Trends in Biotechnology 14: 52-60 (1996). If the analyte to be detected is a ligand, a receptor that binds to the ligand can be used, and vice versa.

Preferably, the test substance or its equivalent is an antigen, and the binding agent is an antibody that binds to the antigen; or the test substance or its equivalent is an antibody, and the A binding agent is an antigen (anti-antibody) that binds to an antigen of the antibody or the antibody. Detecting agent

The detecting agent of the present invention may preferably be the following detecting agent:

Detection agent A according to the invention

The detection agent A of the present invention can be applied to the detection method A, and the detection agent A is fluid, and can be any substance capable of binding the test zone capture agent, including, for example, an antibody, an engineered protein, a peptide, a hapten. Or a lysate of a heterologous mixture of antigens (the antigen has an analyte binding site).

Preferably, the binding of the antigen and the antibody (or the antibody to the antibody and the antibody) or the oligonucleotide may be The detection agent is bound to the capture agent by means of a specific binding of the nucleic acid single strand or by a combination of biotin and streptavidin (SA).

The detection agent is labeled with a fluorescent substance and does not bind directly or indirectly to the binding agent; the "intermediate bonding" means that the detecting agent and the binding agent are attached to the same substance. Detection agent B according to the invention

The detecting agent B of the present invention is fixed and can be applied to the detecting method B, and the detecting agent B can be any substance capable of combining the complex containing the light absorbing substance from the binding zone to the test zone, including, for example: An antibody, engineered protein, peptide, hapten, or lysate containing a heterologous mixture of antigens (the antigen has an analyte binding site).

Preferably, the fluorescently labeled substance is bound to the substance containing the label of the light absorbing substance by binding of the antigen and the antibody or the specific binding of the oligonucleotide to the complementary nucleic acid single strand; and biotin and chain can also be used. The combination of Streptavidin (SA) combines a fluorescently labeled substance with a substance containing a label for the light absorbing substance. Capture agent

The capture agent used in the present invention can be applied to the detection method A, and can be any substance capable of binding to a complex and/or a detection agent containing a light-absorbing substance, including, for example, an antibody, an engineered protein, a peptide, a hapten, or an antigen ( The antigen has a lysate of a heterologous mixture of analyte binding sites.

The capture agent is used to capture a light-absorbing substance-containing complex and a detector that moves from the binding zone to the test zone. Thereby, the complex containing the light absorbing substance and the detecting agent are concentrated in the test area.

Preferably, the capture agent can be combined with the binding of the antigen and the antibody or the specific binding of the oligonucleotide to the complementary nucleic acid single strand or by the combination of biotin and Streptavidin (SA). The complex containing the light absorbing material and/or the detector is combined to enrich the complex and/or the detection agent containing the light absorbing material in the test zone.

The binding of the capture agent to the complex or detector comprising the light absorbing material is specific binding. Preferably, the capture agent is an antibody, and the detection agent is an antibody (anti-antibody) that can bind to the antibody; or the capture agent is streptavidin, and the detection agent is Fluorescently labeled biotin. Marking substance

A detectable label attached to a binding agent or detection agent includes a large number of different substances as long as the label can be detected. Examples of detectable labels include, but are not limited to: particles, luminescent labels, colorimetric labels, fluorescent labels, chemical labels, enzymes, radioactive labels, or radio frequency labels, metal colloids, and chemiluminescence Mark. Examples of commonly used detection methods include (but are not limited to): optical methods such as measuring light scattering, single reflection, photometers or photomultiplier tubes; radioactivity (measured with Geiger counters, etc.); conductivity Or dielectric (capacitance); electrochemically detecting the released electroactive species, such as indium, bismuth, gallium, strontium ions [as described in Hayes et al. (Analytical Chem. 66: 1860-1865 (1994)], Or ferrocyanide [as in the method proposed by Roberts and Durst ( Analytical Chem. 67: 482-491 (1995). The ferrocyanide encapsulated in the liposome is added by adding detergent to the test area. It is released, and then the released ferrocyanide is detected electrochemically. Other conventional methods can be used as appropriate.

In a preferred embodiment, the detectable label is a particle. Examples of particles that can be used include, but are not limited to, colloidal gold particles, colloidal sulfur particles, colloidal selenium particles, colloidal barium sulfate particles, colloidal iron sulfate particles, metal iodate particles, chemical silver particles, silica particles. , colloidal (hydrated) metal oxide particles, colloidal metal sulfide particles, colloidal selenium selenide particles, colloidal cadmium selenide particles, colloidal metal phosphate particles, colloidal metal ferrite particles, coated with an organic or inorganic layer A colloidal particle, a protein or peptide molecule, a liposome, or an organic polymer latex particle, such as a polystyrene latex bead.

As the label for the detecting agent, a fluorescent label (abbreviated as a fluorescent substance) is preferred in the present invention; a label for a binding agent, and a light-absorbing substance label (abbreviated as a light-absorbing substance) is preferred in the present invention. When the fluorescent substance and the light absorbing substance are combined in a specific manner, if the excitation or emission spectrum of the fluorescent substance partially or completely overlaps with the absorption spectrum of the light absorbing substance, the light absorbing substance may affect (partially quenching or completely quenching) the fluorescence of the fluorescent substance. Thus, the amount of the analyte is determined by detecting the fluorescence intensity of the fluorescent substance. Selection of fluorescent substances and light absorbing substances

The fluorescent substance and the light absorbing substance used for the labeling of the present invention should be used in combination, and the basic principle is that the excitation or emission spectrum of the fluorescent substance partially or completely overlaps with the absorption spectrum of the light absorbing substance.

The best is the complete overlap, which has a higher detection sensitivity.

Representative fluorescent substances include, but are not limited to, fluorescein, carboxyfluorescein, 2-methoxyfluorescein, 4, 5-dimethoxyfluorescein, rhodamine, phycoerythrin (PE) A combination of a quantum dot, a rare earth element ion (such as Eu ³⁺ ), and a chelate thereof, as long as the excitation or emission spectrum of the selected fluorescent substance overlaps with the absorption spectrum of the selected light absorbing material.

Representative light absorbing materials include, but are not limited to, a combination of colloidal gold, nanogold rods, nanosilver rods, and the like, as long as the absorption spectrum of the selected light absorbing material overlaps with the excitation or emission spectrum of the selected fluorescent material.

The colloidal gold particles can be produced by any conventional method, for example, as summarized in G. Frens, 1973 Nature Physical Science, 241: 20 (1973). Other methods are described in U.S. Patent Nos. 5,578,577, 5,141,850, 4,775,636, 4,853,335, 4,859,612, 5,079, 172, 5,202,267, 5,514,602, 5,616,467, 5,681,775 c

As used herein, the term "nano-gold rod" refers to gold particles having a certain aspect ratio and having a horizontal axis and a longitudinal axis in the range of 5-200 nm.

A particularly preferred light absorbing material is colloidal gold, especially colloidal gold having a particle size of 20-40 nm.

Preferably, those skilled in the art commonly use fluorescent labeling materials including PE, and the absorption spectrum of PE is Between 450 and 600 nm, the maximum emission wavelength is 575 nm, and the absorption spectrum of 30 nm colloidal gold is 300 to 800 nm. The broad absorption peak is 525 to 530 nm, which overlaps with the absorption and emission spectra of PE. Therefore, 30 nm is selected. Colloidal gold acts as a corresponding light absorbing material. Principle of fluorescence quenching

The fluorescence quenching principle of the present invention may include the following principles:

Fluorescence quenching principle A: It can be applied to detection method A. The substance containing the light-absorbing substance labeling, after being captured by the capture reagent in the test area, the substance containing the light-absorbing substance label will be enriched in the test area and quench the fluorescence intensity of the test area. .

When the inventor adds a substance containing a fluorescent label and a substance containing a light-absorbing substance (or a binding agent) in the binding region, as with the substance containing the light-absorbing substance, the substance containing the fluorescent label is also captured by the capturing agent in the test area. Enriched in the test zone, but the substance containing the light-absorbing substance label and the substance containing the fluorescent label are in a mixed state without forming a complex. When the excitation or emission spectrum of the fluorescent substance overlaps with the absorption spectrum of the light-absorbing substance as the fluorescence quencher in whole or in part, the light-absorbing substance quenches the fluorescence of the fluorescent substance due to the resonance energy transfer.

Fluorescence quenching principle B: It can be applied to the detection method B. When the fluorescent label-containing substance is combined with the substance containing the light-absorbing substance label, the excitation spectrum of the fluorescent substance and the absorption spectrum of the light-absorbing substance as the fluorescence quencher are all or When partially overlapping, the light absorbing material quenches the fluorescence of the fluorescent material due to resonance energy transfer.

In the present invention, the cause of fluorescence quenching includes two aspects: one is the quenching of the fluorescent substance by the internal light absorbing substance of the composite; the other is the mutual quenching between the stacked composites, such as: The light absorbing material quenches the fluorescence of the complex B, the light absorbing material of the complex B quenches the fluorescence of the complex C, and the light absorbing material of the complex C quenches the fluorescence of the complex A, and so on. working principle

The working principle of the detection method (including detection method A and detection method B) of the present invention will now be described with reference to FIG. 1 and specific embodiments:

Working principle A: It can be applied to the detection method A. The detection line is fixed at the same time as the binding agent and the detection agent (ie, the capturing agent), and the detecting agent and the bonding agent are both located in the bonding pad and can flow. After the binding agent and the detecting agent are dissolved by the sample, the binding agent combines with the analyte or the equivalent thereof in the sample to form a complex containing the light absorbing substance during the chromatography, and the complex flows through the test area. (or the detection line) is captured, the more the analytes are, the more the complex is captured; at the same time, the detection agent is also captured when passing through the detection line during the chromatography (the detection line is a mixture of the complex and a detection agent).

Specifically, when the complex affects (partially quenches or completely quenches) the fluorescence intensity of the detecting agent, the amount of the analyte can be determined by detecting the fluorescence intensity thereof. The present invention herein provides the following preferred methods based on the above principles:

Method 1: Fix a capture agent (such as streptavidin, SP Streptavidin or SA) at the test line.

(1.1) The sample is applied to the glass fiber sample gasket, and the analyte (such as antigen) in the sample moves toward the water absorbing pad under capillary action, and carries a binding agent (such as gold that can bind to different epitopes of the antigen). After the polyester film of the labeled antibody and the biotin-labeled antibody is combined with the spacer, the binding agent is completely reconstituted, and the antigen and the binding agent form a "gold standard antibody-antigen-biotin antibody" ternary complex during the chromatography. , captured by Streptavidin at the detected line;

At the same time, the sample will be completely reconstituted in combination with the detector (such as biotin-PE) on the pad. During the chromatography, the detector is also captured by Streptavidin at the detection line;

The fluorescence of PE in the gold quenching detector in the ternary complex at the detection line.

(1.2) The excess free gold-labeled antibody continues to move forward, and the control agent (such as the antibody against the gold-labeled antibody) immobilized at the quality control line is captured in red, indicating that the test is effective.

(1.3) Determine the fluorescence intensity F1 at the detection line of the film strip and the fluorescence intensity F2 at the middle of the detection line and the quality control line, and calculate the ratio of F2 and F1, preferably the value of F2/F1. The larger the value, the object to be tested The higher the concentration, the lower the opposite. Method 2: A capture agent (such as a capture antibody against an antigen in a test object) is immobilized at a detection line; and the antibody-labeled antibody of the capture antibody is used as a detection agent.

(1.1) The sample is applied to the glass fiber sample pad, and the test object (such as antigen) in the sample moves toward the water absorbing pad under capillary action, and passes through the bonding pad carrying the binding agent (such as the gold standard antibody of the antigen). After the tablet, the binding agent is completely reconstituted, and the antigen and the binding agent form a "gold standard antibody-antigen" 2-ary complex during the chromatography, and is captured by the capture antibody at the detection line;

At the same time, the sample will also be completely reconstituted in combination with the detection agent on the shims. During the chromatography, the detection agent is also captured by the capture antibody at the detection line;

The fluorescence of PE in the gold quenching detector in the 2-member complex at the detection line.

(1.3) Determine the fluorescence intensity F1 at the detection line of the film strip and the fluorescence intensity F2 at the middle of the detection line and the quality control line, and calculate the ratio of F2 and F1, preferably the value of F2/F1. The larger the value, the object to be tested The higher the concentration, the lower the opposite. The invention is also applicable to the competition inhibition method:

(1.1) A mixture of a binding agent (such as a gold standard antibody), a detecting agent (such as biotin-PE) and a test substance equivalent (such as a biotin labeled antigen) are separately added to the bonding pad of Fig. 1, the binding agent. The detector mixture is close to the sample The product mat, the equivalent of the object to be tested is close to the detection line, and the two are separated by a suitable space.

(1.2) The sample is added to the glass fiber sample gasket, wherein the object to be tested (such as the antigen) moves toward the water absorbing pad, and passes through the bonding pad carrying the bonding agent and the detecting agent to completely reconstitute the two. , the analyte in the sample and the equivalent of the analyte on the binding spacer competitively form a "1nd binary complex" with the gold-labeled antibody-antigen and a gold-labeled antibody-antigen-biotin" 2 binary complex".

(1.3) The above two complexes continue to the detection line of the membrane 56, and the "second binary complex" is captured by a trapping agent (such as Streptavidin) immobilized thereon, and the higher the concentration of the analyte is formed, The fewer "second binary complexes", the fewer "second binary complexes" are captured;

At the same time, the detector is also captured by the capture agent (such as Streptavidin) at the detection line of the membrane 56 during the chromatography;

Excess free gold-labeled antibody or substance containing gold-labeled antibody (such as gold-labeled antibody-antigen) continues to move forward, and is captured by a control agent (such as an antibody against gold-labeled antibody) immobilized at the quality control line, showing a red color. The test is valid.

(1.4) Determine the fluorescence intensity F1 at the detection line of the film strip and the fluorescence intensity F2 at the middle of the detection line and the quality control line, and calculate the ratio of F2 and F1, preferably the value of F2/F1. The larger the value, the object to be tested The smaller the concentration, the greater the opposite. Working principle B: It can be applied to the detection method B. The working principle of the detection method of the invention will be described with reference to Fig. 1 and specific embodiments:

method one,

(1.1) The sample is applied to the glass fiber sample pad, and the sample (such as antigen) in the sample moves toward the water absorbing pad under capillary action, and passes through a binding agent (such as an antigen colloidal gold-labeled antibody, referred to as After the polyester membrane of the antigen is combined with the gasket, the gold-labeled antibody is completely reconstituted, and the analyte and the binding agent (such as the antigen and its gold-labeled antibody) in the sample form a binary element such as a gold-labeled antibody-antigen. Complex.

(1.2) The above 2-ary complex continues to the detection line of the nitrocellulose membrane, and the fluorescent substance-labeled antigen-detecting agent (such as the fluorescently labeled second antibody) immobilized thereon captures the 2-ary complex. A ternary complex is formed, a "sandwich complex" in which the gold quenches the fluorescence of the fluorescent material at the detection line.

(1.3) The excess free gold-labeled antibody continues to move forward, and the control agent (such as the antibody against the gold-labeled antibody) immobilized at the quality control line is captured and appears red, indicating that the test is effective.

(1.4) Determine the fluorescence intensity of the fluorescent substance at the detection line. The stronger the fluorescence, the lower the target antigen concentration, and vice versa. If there is no antigen in the sample, the "sandwich complex" cannot be formed, then the fluorescence intensity at the detection line is It is the original fluorescence intensity. Method 2: Other fluorescent labeling substances capable of capturing the "sandwich complex" can also be fixed at the detection line.

(2.1) The sample is applied to the glass fiber sample gasket, and the sample (such as antigen) in the sample is directed to the water absorption gasket. Directional movement, after the polyester membrane with the two binding agents (such as gold-labeled antibody and biotin-labeled antibody) is combined with the gasket, after the gold-labeled antibody and the biotin-labeled antibody are completely reconstituted, the sample is tested. And the two binding agents (such as an antigen and a gold-labeled antibody, a biotin-labeled antibody) form a ternary complex such as a gold-labeled antibody-antigen-antibody-biotin.

(2.2) The above complex continues to the detection line of the nitrocellulose membrane strip, and the fluorescently labeled detection agent (such as fluorescently labeled SA) immobilized on the fluorescent marker is captured to capture the ternary complex to form 4 yuan. The complex, wherein the gold quenches the fluorescence of the fluorescent material at the detection line.

(2.3) The excess free gold-labeled antibody continues to move forward, and the control agent (such as the antibody against the gold-labeled antibody) immobilized at the quality control line is captured and appears red, indicating that the test is effective.

(2.4) Determine the fluorescence intensity of the fluorescent substance at the detection line. The stronger the fluorescence, the lower the target antigen concentration, and vice versa. If there is no antigen in the sample, the "sandwich complex" cannot be formed, then the fluorescence intensity at the detection line is It is the original fluorescence intensity. Method 3, the present invention is also applicable to the competition inhibition method:

(3.1) Add the binding agent (such as gold standard antibody) and the equivalent of the analyte (such as biotin labeled antigen) to the polyester film of Figure 1, respectively, and the binding agent is close to the sample pad, and the equivalent of the analyte is close to the detection. Line, 2 separate the appropriate space distance.

(3.2) The sample is applied to the glass fiber sample gasket, and the analyte (such as antigen) moves toward the water absorbing pad and passes through the polyester film carrying the binding agent (such as the gold standard antibody of the antigen). The agent is completely reconstituted, and the analyte and the binding agent in the sample form a "first binary complex" such as a gold-labeled antibody-antigen, and the remaining binding agent forms a gold equivalent with the analyte on the polyester film. The "second binary complex" of the antibody-antigen-biotin.

(3.3) The above two compounds continue to the detection line of the nitrocellulose membrane, and the detection agent (such as fluorescently labeled SA) immobilized here only captures the "second binary complex", in which the gold is quenched. The fluorescence of the fluorescent substance at the detection line is extinguished.

(3.4) Determine the fluorescence intensity of the fluorescent substance at the detection line. The more target antigen in the sample, the resulting "number

The less the 2 binary complex, the weaker the fluorescence quenching at the detection line, and accordingly, the stronger the fluorescent signal, conversely, the less the target antigen in the sample, the weaker the fluorescent signal at the detection line.

The detection device of the present invention will now be described with reference to FIG. 2:

As shown in FIG. 2, the apparatus may include: a test piece, a detector, a light source, an optical fiber, and a computer. A description of the use of the test method can also be included. The working principle of the test piece is as described above, and the method of detecting the fluorescence intensity can be as follows (but not limited to), and any method which can be used for detecting the fluorescence intensity can be used for the detecting device of the present invention. Fluorescence intensity detection

The light source is irradiated to the detection line through the optical fiber, and the excited fluorescence enters the detector through the optical fiber, and the obtained data is processed and analyzed by a computer.

The optical fibers may be Y-shaped and connected to a light source, a detection line, and a detector, respectively.

In the present invention, the light source is used to provide light of a certain emission wavelength, thereby exciting the fluorescent substance to emit fluorescence. Any source that provides the appropriate wavelength can be used, including (but not limited to, LED, xenon, tungsten halogen, laser, etc.).

A preferred source of light is a laser source that can be produced by methods and equipment conventional in the art, such as a laser. Representative lasers include, but are not limited to, semiconductor lasers, helium neon lasers, argon ion lasers, wavelength selective lasers, multi-wavelength lasers, and dual wavelength lasers.

The laser wavelength generated by the laser is related to the laser medium. The common laser wavelengths are shown in the following table 1:

Table 1

The detector in the present invention may be, but not limited to, a photomultiplier tube, a CCD or a photovoltaic cell or the like. standard curve line

In the present invention, the amount of the analyte can be determined directly by measuring the fluorescence intensity at the detection line; or by measuring the fluorescence intensity F1 at the detection line and the fluorescence intensity F2 at the middle of the detection line and the quality control line. Calculate the ratio of F2 and F1 to determine the number of objects to be tested.

In a preferred embodiment, quantitative results can be obtained by comparison with a standard curve.

The standard curve can be obtained in the following ways:

After passing the known samples of different concentrations (C) of the sample to be tested, the fluorescence intensity (F) at the detection line is measured, and each concentration (C) or its logarithm (log C or lg C) is measured. ) and the corresponding fluorescence intensity (F) or its logarithmic value (log F or lg F) to obtain a standard curve; or

After the known samples of different concentrations (C) of the sample are passed through the above detection method, they are respectively measured and detected. Fluorescence intensity at the line (Fl), fluorescence intensity F2 at the middle of the test line and the control line, calculate the ratio of F2 and F1, and each concentration (C) or its logarithm (log C or lg C) and corresponding fluorescence The intensity ratio (eg F2/F1) or its logarithmic value (eg log F2/F 1 or lg F2/F 1 ) is plotted to obtain a standard curve. The main advantages of the invention are:

(1) The present invention provides a test piece which can be based on the working principle A or the working principle B.

(2) The present invention provides a method for detecting the above test piece, which is based on the principle of fluorescence quenching, and measures the fluorescence intensity of the fluorescent substance to determine the amount of the object to be tested, which is quick, simple, and low in cost. Moreover, the sensitivity is high and the quantitative is accurate.

(3) The present invention also provides a detection kit and a detection device which are widely used in the field of quantitative detection based on the above detection method. The invention will be further elucidated below in conjunction with specific implementations. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually produced according to the conditions described in the conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Reagents and equipment:

1 pair of monoclonal antibodies against AFP (Abl and Ab2), commercially available; monoclonal antibodies against C-reactive protein (CRP), commercially available;

CRP antigen, commercially available; AFP antigen was purchased from Biodesign; 30 nm colloidal gold, commercially available;

PE was purchased from Invitrogen; Streptavidin (SA), commercially available; SA-PE was purchased from Invitrogen; Dig, commercially available; BSA, commercially available; Biotin, City Sales; Detector: USB4000-FL (US Ocean Optics); Light source: 532nm laser source. Example 1: Detection of alpha-fetoprotein (AFP) in serum (Detector in reaction system contains fluorescent substance)

1. Preparation of gold standard antibody

1.1 Colloidal gold-antibody preservation solution

After adding water, adjust the pH to 7.4 with 6N HC1, hydrate to 250ml, filter with 0.45 μm filter, 4~

Store at 8 ° C.

1.2 working fluid

After adding water, the pH was adjusted to 7.0 to 7.5 with 6N HCL to 1000 ml, filtered through a 0.45 μm filter, and stored at 4 to 8 °C.

1.3 Preparation of gold standard antibody (Gold-Ab l)

1.3.1 Take 20ml of 20~30nm granular colloidal gold solution, slowly add 1.0ml (0.6mg/ml) of purified Abl antibody under magnetic stirring, and stir at room temperature for 30min;

1.3.2 Add 10% BSA 0.8ml (final concentration 0.4%), stir at room temperature for 5min;

1.3.3 Add 10% PEG 0.4ml (final concentration 0.2%), stir at room temperature for 5min;

1.3.4 Centrifuge at 12000~1500r/min for 60~40min, carefully centrifuge the supernatant, and dissolve the precipitate in 0.5ml preservation solution. The optical density (OD) of the gold standard antibody is about 100 OD, and the concentration of Ab l antibody is 1mg. /ml, set to 4 ° C to save spare;

2, biotin standard i has Ab2 antibody (biotin-Ab2)

2.1 Ab2 pretreatment

2.2 Take the above pretreated Ab2 10 μL, add 25 μL of lmg/ml NHSS-Biotin DMSO solution, mix well, avoid the light reaction in the refrigerator at 4 °C for 2 hours, and dialyze overnight for use.

3. biotin label PE (biotin-PE)

The biotin-PE was prepared in the same manner as in step 2.2, except that the pretreated A2 was replaced with purified PE, and the final concentration of PE was 0.65 mg/ml.

4, SA film and upload Gold-Abl, biotin- Ab2 Take SA lmg/ml phosphate buffer (ρΗ7.2) 0.5 μ 1 point in the detection line position of Figure 1, and dry at room temperature in the dark;

0.5 μl of each of Gold-Abl, biotin-Ab2, and biotin-PE was added to the bonding pad of Fig. 1.

5, standard curve preparation

5.1 Prepare AFP series standard solution with working fluid (see Table 2 for concentration);

5.2 Take 6 test pieces and place them horizontally. Add 6 kinds of 50 μl standard solution to each sample pad. After 5.3 lOmin, measure the fluorescence intensity F1 of 575 nm and the detection line and quality control line on the test line of each test piece. The fluorescence intensity F2 at 575 nm in the middle is calculated as the value of F2/F1. The data is shown in Table 2. The standard curve (with the concentration C as the abscissa and F2/F1 as the ordinate) is shown in Fig. 3.

AFP standard series concentration and corresponding fluorescence intensity

6, sample testing

Replace the standard series of solutions with serum samples, repeat steps 5.2 and 5.3, and substitute F2/F1 into the standard curve. The AFP concentrations of the six samples are shown in Table 3.

Table 3 Test results of the method of the present invention and Roche electrochemiluminescence detection results

The detection result of the method of the invention has a good correlation with the detection result of Roche electrochemiluminescence, and R ² = 0.9856. Example 2: Detection of C-reactive protein (CRP) based on competitive inhibition assay

1, CRP monoclonal antibody gold (Gold-CRP)

The procedure was the same as in Example 1, except that the purified CRP monoclonal antibody was used in place of the Abl antibody, and the labeled colloidal gold had an OD value of 75 and a CRP monoclonal antibody concentration of 1 mg/ml. 2. CRP antigen label biotin (biotin-CRP)

The procedure was the same as in step 2.2 of Example 1, except that the pretreated A2 was replaced with purified CRP, and the labeled CRP antigen concentration was 0.4 mg/ml.

3, biotin standard i self PE

The biotin-PE of Example 1 was used.

4, SA film and upload Gold-CRP, biotin-CRP and biotin-PE

Take SA lmg/ml phosphate buffer (ρΗ7.2) 0.5 μ 1 at the test line position in Figure 1, and dry at room temperature in the dark;

0.5 μL of Gold-CRP and biotin-PE were added to the position of the polyester film-bonding pad of Figure 1 near the sample pad; take biotin-CRP0.5 w bribes to the position of the polyester film near the detection line in Figure 1. , dry at room temperature in the dark. 5, standard curve preparation

5.1 Prepare CRP series standard solution of 0, 1, 10, 50, 200 μg/ml with working fluid (see Table 4 for concentration); 5.2 Take 5 test pieces, place horizontally, add 5 kinds of concentration on each sample pad 50 μ 1 standard solution; After 5.3 lOmin, measure the fluorescence intensity F1 at 575 nm on the test line and the fluorescence intensity F2 at 575 nm between the detection line and the control line. Calculate the value of F2/F1 as shown in Table 4. Standard curve (With lg(C) as the abscissa and lg(F2/Fl) as the ordinate), see Figure 4.

Table 4 Standard series concentration and corresponding fluorescence intensity

6, sample testing

Replace the standard series of solutions with serum samples, repeat steps 5.2 and 5.3, and substitute F2/F1 into the standard curve. The CRP values of the six samples are shown in Table 5.

Table 5 Test results of the method of the present invention and enzyme-linked immunosorbent assay results

3 32.36 46.40

4 74.23 55.80

5 108.64 133.34

6 178.6 188.70

The correlation between the detection results of the method of the present invention and the results of the enzyme-linked immunosorbent assay is good, R ² = 0.9615. Conclusions of Examples 1 to 2:

The method of the present invention adds a flowable fluorescent substance-labeled detecting agent, and measures the amount of the test object by measuring the influence of the quenching substance (ie, the light-absorbing substance or the substance labeled by the light-absorbing substance) on the fluorescence intensity of the fluorescent substance; The quenching substance and the fluorescent substance do not need to form a complex, but only the two substances are enriched and mixed in the test area, and fluorescence quenching can occur, thereby eliminating the formation of the complex between the quenching substance and the fluorescent substance. The necessary processing steps are very easy to operate.

It can be seen that the method of the invention not only has the advantages of high sensitivity and accurate quantification, but also is quick, easy to operate and low in cost. Example 3: Detection of alpha-fetoprotein (AFP) in serum (PE-labeled antibody is spotted at the detection line)

1. Preparation of gold standard antibody

1.1 Colloidal gold-antibody preservation solution

After adding water, adjust the pH to 7.4 with 6N HC1, hydrate to 250ml, filter with 0.45μιη filter, 4

Store at 8 ° C.

1.2 working fluid

1.3 Preparation of gold standard antibody (Gold-Abl)

1.3.1 Take 20ml of 20~30nm granular colloidal gold solution, slowly add 1.0ml (0.6mg/ml) of purified Abl antibody under magnetic stirring, and stir at room temperature for 30min; 1.3.2 Add 10% BSA 0.8ml (final concentration 0.4%), stir at room temperature for 5min;

1.3.4 Centrifuge at 12000~1500r/min for 60~40min, carefully centrifuge the supernatant, and dissolve the precipitate in 0.5ml preservation solution. The optical density (OD) of the gold standard antibody is about 60 OD, and the concentration of Abl antibody is lmg/ Ml, set at 4 ° C for storage;

2. Preparation of PE label Ab2 antibody (Ab2-PE)

2.1 Ab2 pretreatment

2.2 biotin marker Ab2

Take the pretreated Ab2 ΙΟμΙ^, add 25 L of lmg/ml NHSS-Biotin DMSO solution, mix well, avoid the light reaction in the refrigerator at 4 °C for 2 hours, and dialyze overnight for use.

2.3 biotin- Ab2 label PE

Label biotin-Ab2, add pH7.4 phosphate buffer, the final concentration of Ab2 is 5 g/ml, add SA-PE, the final concentration of PE is 60 g/ml, Ab2-PE (ie Ab2-biotin- SA-PE) has a total volume of 5 ml and is stored at 4 ° C in the dark.

3, Ab2-PE film and upload gold antibody

Take Ab2-PE Ιμΐ at the test line position in Figure 1. Add Gold-Abl 0.5μ1 drop to the polyester film-bonding pad of Figure 1 and dry at room temperature.

4, standard curve preparation

4.1 Prepare AFP series standard solution with working fluid (see Table 6 for concentration);

4.2 Take 5 test pieces and place them horizontally. Add 5 kinds of 50μ1 standard solution to each sample pad. 4.3 After 10 minutes, measure the fluorescence intensity at the test line on each test piece. The data is shown in Table 2. The standard curve is shown in Figure 5.

Table 6: AFP Standard Series Concentrations and Corresponding Fluorescence Intensities

Standard series concentration ng/ml) Fluorescence intensity (F) 10.58 22500

32.45 18600

95.32 15400

301.56 13600

5, sample testing

Replace the standard series of solutions with serum samples, repeat steps 4.2 and 4.3, and substitute the fluorescence intensity values into the standard curve. The AFP values of the six samples are: 6.2, 8.9, 12.6, 40.8, 112.3, 235.6 ng/ml, and the enzyme. The correlation between the corresponding test results of the combined immunoassay was good, R ² = 0.9128.

Example 4: Detection of alpha-fetoprotein (AFP) in serum (SA-PE spot at the detection line)

1. Preparation of Gold-Abl

The Gold-Abl of Example 3 was used. 2. biotin standard i self Ab2

The biotin-Ab2 of Example 3 was used.

3. Mix of Gold- Abl and biotin-Ab2

Mix 5μ1 Gold-Abl and biotin-Ab2, respectively.

4. SA-PE film and upload mixed antibody

Take SA-PE (100 g/ml) Ι μΐ at the detection line position in Figure 1. Take 0.5μ1 of the mixed antibody and add it to the polyester membrane bonding pad of Figure 1, and dry at room temperature. 5. Standard curve preparation

5.1 Prepare AFP series standard solution with working fluid (see Table 7);

5.2 Take 5 test pieces and place them horizontally. Add 5 kinds of 50μ1 standard solution to each sample pad. 5.3 After 10min, measure the fluorescence intensity at the test line on each test piece. The data is shown in Table 7. The standard curve is shown in Figure 6.

Table 7: AFP Standard Series Concentrations and Corresponding Fluorescence Intensities

Standard Series Concentration (C ng/ml) Fluorescence Intensity (F)

0 18500

10.58 16500

32.45 12300

95.32 8500

301.56 4350 6. Sample testing

Replace the standard series with serum samples, repeat steps 5.2 and 5.3, and substitute the fluorescence intensity values into the standard curve. The AFP values of the six samples are: 7.2, 13.2, 15.6, 44.8, 102.3, 255.6 ng/ml, and the enzyme. The correlation between the corresponding test results of the combined immunoassay was good, R ² =0.8985. Example 5: Competitive inhibition assay for detection of C-reactive protein (CRP)

1. CRP monoclonal antibody gold (Gold-CRP)

The procedure was the same as in Example 3 except that the purified CRP monoclonal antibody was used in place of the Abl antibody, and the labeled colloidal gold had an O.D value of 75 and a CRP monoclonal antibody concentration of 1 mg/ml.

2. CRP antigen label biotin (biotin-C P )

The procedure was the same as in step 2.2 of Example 3, except that the pretreated A2 was replaced with purified CRP, and the labeled CRP antigen concentration was 0.4 mg/ml. 3, SA-PE film and upload Gold-CRP and biotin-CRP

Take SA-PE (100 g/ml) Ι μΐ at the test line position in Figure 1. Take Gold-CRP 0.5μ1 drop to the position of the polyester film-bonding pad of Figure 1 near the sample pad, and take biotin-CRP 0.5μ1. Dropped on the position of the polyester film in Figure 1 near the detection line, and dried at room temperature in the dark. 4, standard curve preparation

4.1 Prepare CRP series standard solution of 0, 1, 5, l O g/ml with working fluid;

4.2 Take 4 test pieces, place them horizontally, and add 4 kinds of 50μ1 standard solution to each sample pad;

4.3 After 10 min, the fluorescence intensity at the test line on each test piece was measured. The data is shown in Table 8. The standard curve is shown in Figure 7.

Table 8: Standard series concentration and corresponding fluorescence intensity

5, sample testing

Replace the standard series of solutions with serum samples, repeat steps 4.2 and 4.3, and substitute the fluorescence intensity values into the standard curve to determine the CRP values of the six samples: 0.53, 1.2, 1.88, 5.8, 4.3, 9.6 g/ml, linked to the enzyme. The correlation of the corresponding test results of the immunization method was good, R ² = 0.9336. Example 6: Determination of digoxin concentration in serum by competitive inhibition method

1, BSA-Dig

Digo is attached to the BSA using methods well known to those skilled in the art.

2, biotin-BSA-Dig

Biotin-BSA-Dig was prepared as described in Example 3, except that the purified BSA-Dig was used in place of the pretreated Ab2.

3. Gold-Dig- Ab

Gold-Dig-Ab was prepared as described in Example 3, except that the purified Dig-Ab was used in place of the Abl antibody.

4. SA-PE film and upload biotin-BSA-Dig and Gold-Dig-Ab

Take SA-PE (100 g/ml) Ι μΐ at the test line position in Figure 1. Take Gold-Dig-Ab 0.5μ1 and add it to the position of the polyester film-bonding pad of Figure 1 near the sample pad. Take biotin-BSA. -Dig 0.5 μl was added to the position of the polyester film of Fig. 1 near the detection line, and dried at room temperature in the dark.

5, the standard curve

Reference Example 5 was made to prepare a standard curve of the digoxin standard series concentration and its corresponding fluorescence intensity.

6, sample testing

Referring to Example 5, 8 actual samples were tested, and the concentrations of digoxin in 8 samples were determined to be: 1.32, 0.85, 2.44, 5.35, 3.36, 1.44, 4.18, 4.76 ng/ml, corresponding to the microparticle enzyme exemption method. The correlation of values is good, ² = 0.9012 o Discussions in Examples 3 to 6:

The test method of the invention measures the fluorescence intensity of the fluorescent substance to determine the quantity of the object to be tested, and the method only needs to add the sample to be tested, no need to wash, the operation is quick, simple, low cost, high sensitivity, accurate quantitative . All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

A test piece, characterized in that the test piece comprises:

(i) a sample application area that can be added to the sample;

2. The test piece according to claim 1, wherein the immobilization reagent is not labeled with a fluorescent substance, and the application zone and/or the binding zone comprises a flowable substance and is labeled with a fluorescent substance. Detector.

3. The test piece according to claim 1, wherein the immobilization reagent is labeled with a fluorescent substance, and the application zone and the binding zone do not contain flowable and are marked by a fluorescent substance. Detection agent.

The test piece according to claim 1, wherein the bonding zone comprises a first bonding zone and a second bonding zone, wherein the first bonding zone is located at a proximal end of the loading zone, and comprises a light-absorbing substance a labeled binding agent, the second binding region is located at a distal end of the sample loading region, and comprises a biotin-labeled analyte equivalent; wherein the amount of the binding agent labeled by the light absorbing material is greater than the number of the analyte And the analyte and the biotin-labeled analyte equivalent are competitively combined with the binding agent to form a biotin-labeled complex containing the light absorbing substance.

The test piece according to claim 1, wherein the analyte comprises: a protein, a nucleic acid or a small molecule compound.

The test piece according to claim 1, wherein the fluorescence excitation or emission spectrum of the test zone overlaps with the absorption spectrum of the light absorbing material in whole or in part.

7. The test piece according to claim 1, wherein the fluorescent substance is selected from the group consisting of: fluorescence , carboxyfluorescein, 2-methoxyfluorescein, 4, 5-dimethoxyfluorescein, rhodamine, phycoerythrin, quantum dots, or rare earth ions or their chelate complexes.

The test piece according to claim 1, wherein the light absorbing material is selected from the group consisting of colloidal gold, nano gold bars, nano silver bars, or a combination thereof.

The test piece according to claim 1, further comprising at least one control zone between the test zone and the sample absorption zone, wherein the control zone contains an immobilized contrast agent, wherein the control agent is used A binding agent that specifically binds to the light-absorbing substance.

10. A method for quantitatively detecting a fluorescence analysis of a test object, comprising the steps of:

(1) adding a sample of the test object to the sample application area of the test piece according to any one of claims 1-9;

1 1. A fluorescence analysis method for quantitatively detecting a test object, comprising the steps of:

(2) measuring the fluorescence intensity of the test zone of the test piece, thereby being converted into the number of analytes, wherein in step (2), the fluorescence intensity F2 between the test zone and the control zone and the fluorescence intensity of the test zone The ratio of F1 is compared with a standard curve to determine the number of analytes; or the fluorescence intensity F1 of the test zone is compared with a standard curve or an unapplied initial fluorescence intensity F0 to determine the number of analytes

A test kit, comprising: the test piece according to any one of claims 1-9; and instructions for use.

13. A fluorescence measuring device for quantitatively detecting a test object, wherein the device comprises:

(a) a test piece according to any one of claims 1-9;

(b) a detector for detecting fluorescence intensity; and

(c) A description of the use of the method of claim 10.

A test piece, characterized in that the test piece comprises:

(i) a sample application area that can be added to the sample;

(ii) a binding zone located at the proximal end of the loading zone, the binding zone comprising:

(iii) a test zone located at the proximal end of the binding zone and distal to the loading zone, the test zone comprising an immobilized capture agent for capturing a complex of light absorbing material that moves from the binding zone to the test zone And detection agent;

(iv) a sample absorption zone located at the proximal end of the test zone and at the distal end of the binding zone, wherein the absorption zone has an absorptive capacity, Thereby diffusing the sample added to the sample application zone from the sample application zone to the end sample absorption zone;

A test piece for quantitatively detecting a sample to be tested in a sample, the test piece comprising: a sample application zone to which a sample can be added;

a test zone located at a proximal end of the binding zone and a distal end of the sample application zone, the test zone comprising an immobilized detection agent, the detection agent being labeled by a fluorescent substance, the detection agent being used to capture movement from the binding zone to the test zone a composite containing a light absorbing material; and