Spectrum detection method based on immunochromatography

Abstract

The invention provides a detection system, in particular, a background fluorescence enhanced quenching immunochromatography method is adopted, laser with smaller light spots is used as an excitation source, and compared with a common background fluorescence quenching immunochromatography method for quantitative detection of the content of a certain antigen, better analysis performance is achieved, and sensitivity is remarkably improved. When the quenching agent colloidal gold is replaced by the nano microspheres with the Raman spectrum signals, the positive and negative properties can be quickly judged by observing the color intensity of the T line with naked eyes, accurate quantification can be realized by measuring the fluorescence signals on the T line, and the detection technology of three signal output modes of measuring the Raman spectrum signals on the T line by using a Raman spectrometer can be used; the detection sensitivity is further improved. In addition, by arranging two detection lines, the effect of joint detection is achieved while high sensitivity is achieved. Therefore, the test piece and the corresponding detection system have very excellent analysis performance.

Description

Technical Field

[0001] The invention relates to the technical field of immunochromatography, and in particular to an immunochromatography rapid detection method with three signal readout modes. Background Art

[0002] Patent 201210051830.8 and the paper by Xiangjun Chen et al. (Analytica Chimica Acta 841 (2014) 44–50, Antigen detection based on background fluorescence quenching immunochromatographic assay) disclose a “background fluorescence quenching immunochromatographic assay (bFQICA) technique”, which is an analytical technique established on the basis of colloidal gold immunochromatography (GICA). This method can not only quickly judge the positive or negative result by observing the color intensity of the T line with the naked eye, but also perform quantitative detection by detecting the background fluorescence of the membrane strip and the relative fluorescence intensity of the detection line (T line).

[0003] Since the detection is relative fluorescence intensity, the interference of factors such as protein binders and drug metabolites in the blood on the fluorescence intensity is significantly suppressed, and quantitative detection is more accurate than GICA. However, due to the low intensity of background fluorescence in the public literature, the fluorescence quenching space is limited. In addition, the light source spot size and quenching substances in the measuring instrument are not optimized, which is not conducive to the detection of target analytes with high detection sensitivity requirements and large concentration dynamic range. Therefore, it is necessary to improve the existing bFQICA to further improve the detection sensitivity and measurement range to improve the analytical performance. Summary of the invention

[0004] The purpose of the present invention is to provide a test piece and a corresponding test system, which can detect HPV E7 protein or Helicobacter pylori antigen with high efficiency and high sensitivity.

[0005] In a first aspect of the present invention, a test system is provided, the system comprising a test piece and a detection device,

[0006] The test sheet comprises a substrate, and a sample adding area, a binding area, a test area and a sample absorption area are sequentially arranged on the substrate from one end to the other end of the substrate.

[0007] Wherein, the binding zone contains one or more flowable tracers, the tracers are labeled with light-absorbing substances, and the tracers can bind to the analyte to form a binary complex of analyte-tracer; wherein the light-absorbing substances are colloidal gold particles or Raman microspheres;

[0008] The test area comprises a background enhanced fluorescence area, the background enhanced fluorescence area contains one or more detection lines located at the proximal end of the binding area and the distal end of the sample absorption area, and a quality control line located at the proximal end of the sample absorption area and the distal end of the binding area, each of the detection lines independently contains different immobilized capture antibodies, the quality control lines contain immobilized quality control antibodies, the different capture antibodies are independently used to capture different binary complexes of analytes-tracers that move from the binding area to the test area, and the quality control antibodies are used to bind to tracers labeled with light absorbing substances that are not bound to the analytes;

[0009] The background enhanced fluorescence region has enhanced fluorescence; and F1>30000, wherein F1 refers to the fluorescence intensity at the middle of the detection line and the quality control line in the background enhanced fluorescence region;

[0010] The sample absorption zone has absorption capacity, so that the sample added to the sample adding zone diffuses from the sample adding zone to the terminal sample absorption zone;

[0011] Wherein, when the capture antibody captures the binary complex of the analyte-tracer, the fluorescence intensity of the test area decreases;

[0012] When the light-absorbing substance is colloidal gold particles, the detection device is a laser excitation source; when the light-absorbing substance is Raman microspheres, the detection device is a laser excitation source or a Raman detector.

[0013] In the laser excitation source, the spot size of the laser is less than 1.0 mm or smaller than the T-line width.

[0014] In a preferred embodiment, the test sheet comprises a sample pad, a conjugation pad, an NC membrane and an absorption pad arranged in sequence, and a rubber sheet backing for fixing the sample pad, the conjugation pad, the fluorescent NC membrane and the absorption pad;

[0015] The conjugate pad contains a tracer labeled with a light-absorbing substance, which can form a binary complex of the analyte-tracer with the analyte; the light-absorbing substance is colloidal gold or Raman microspheres;

[0016] The fluorescent NC film comprises a background enhanced fluorescent area, wherein one or more detection lines and quality control lines are arranged in the background enhanced fluorescent area, wherein the one or more detection lines are located at the proximal end of the conjugation pad and the distal end of the absorption pad, and the quality control line is located at the distal end of the conjugation pad and the proximal end of the absorption pad.

[0017] Each of the detection lines is independently coated with a different capture antibody, and the capture antibodies are independently used to capture different binary complexes of analytes-tracers that move from the conjugate pad to the detection line, thereby forming a ternary complex of capture antibody-analyte-tracer;

[0018] The quality control line is coated with a quality control antibody, which is used to bind to a tracer labeled with a light-absorbing substance that is not bound to the capture antibody and the analyte; and

[0019] The background enhanced fluorescence region has enhanced fluorescence, and F1>30000, wherein F1 refers to the fluorescence intensity at the middle of the detection line and the quality control line in the background enhanced fluorescence region.

[0020] In a preferred embodiment, the test piece is a test strip.

[0021] In a preferred embodiment, the sample application area is a sample pad.

[0022] In a preferred embodiment, the binding area is a binding pad.

[0023] In a preferred embodiment, the analyte is selected from the following group: one or more of HPV16 E7 protein, Helicobacter pylori, brucellosis IgG, and brucellosis IgM.

[0024] In a preferred embodiment, when the analyte is HPV16 E7 protein, the tracer is an HPV16 E7 protein-specific detection antibody, such as HPV16 E7 antibody 2.

[0025] In a preferred embodiment, when the analyte is HPV16 E7 protein, the light absorbing substance is colloidal gold particles.

[0026] In a preferred embodiment, the colloidal gold is colloidal gold particles with an average particle size of 10-70 nm (eg, 40 nm).

[0027] In a preferred embodiment, when the analyte is Helicobacter pylori, the tracer is a Helicobacter pylori-specific detection antibody.

[0028] In a preferred embodiment, when the analyte is brucellosis IgG, the tracer is brucellosis antigen.

[0029] In a preferred embodiment, when the analyte is brucellosis IgM, the tracer is brucellosis antigen.

[0030] In a preferred embodiment, when the analyte is Helicobacter pylori, brucellosis IgG and / or brucellosis IgM, the light absorbing substance is Raman microspheres.

[0031] In a preferred embodiment, the Raman microspheres are nanometer-sized Raman microspheres.

[0032] In a preferred embodiment, the test area is a NC membrane, ie, a nitrocellulose membrane.

[0033] In a preferred embodiment, the background enhanced fluorescence area occupies a part or the whole of the test area.

[0034] In a preferred embodiment, the background enhanced fluorescent area is a fluorescent nitrocellulose membrane.

[0035] In a preferred embodiment, the fluorescence enhancement refers to increasing the concentration of fluorescent substances on the surface of the background fluorescence enhancement region, thereby increasing the fluorescence intensity of the background fluorescence enhancement region.

[0036] In a preferred embodiment, the fluorescence enhancement refers to increasing the fluorescence intensity of the background fluorescence enhancement area by adding a high concentration of fluorescent substance.

[0037] In a preferred embodiment, the background fluorescence enhanced region is a background fluorescence enhanced region with a fluorescence intensity exceeding 30,000, preferably exceeding 40,000, 50,000, or 60,000.

[0038] In a preferred embodiment, the fluorescent NC film is obtained by mixing nitrocellulose casting solution with a fluorescent substance solution and then casting the mixture during the preparation of the fluorescent NC film.

[0039] In a preferred embodiment, the fluorescent NC membrane is obtained by adding fluorescent substances to a finished nitrocellulose membrane.

[0040] In a preferred embodiment, the fluorescent NC film is obtained by covering the NC film with a fluorescent pad containing a fluorescent substance.

[0041] In a preferred embodiment, the fluorescent substance is fluorescent microspheres or fluorescent dyes.

[0042] In a preferred embodiment, the fluorescent substance is uniformly dispersed in the fluorescence enhancement area.

[0043] In a preferred embodiment, F1>40000, such as>45000,>50000,>55000.

[0044] In a preferred embodiment, when the analyte is HPV16 E7 protein, the capture antibody is an HPV16 E7 protein-specific detection antibody, such as HPV16 E7 antibody 1.

[0045] In a preferred embodiment, when the analyte is Helicobacter pylori, the capture antibody is a Helicobacter pylori-specific detection antibody.

[0046] In a preferred embodiment, when the analyte is brucellosis IgG, the capture antibody is a brucellosis IgG-specific detection antibody.

[0047] In a preferred embodiment, when the analyte is brucellosis IgM, the capture antibody is a brucellosis IgM-specific detection antibody.

[0048] In a preferred embodiment, when the test area contains multiple test lines, the multiple test lines are separated, and F1 refers to the fluorescence intensity of the test line closest to the quality control line in the background enhanced fluorescence area and the middle of the quality control line. In a preferred embodiment, when the analyte is brucellosis IgG and brucellosis IgM, the test area contains two test lines, including a first test line located at the proximal end of the binding area and the distal end of the quality control line, and a second test line located at the proximal end of the quality control line and the distal end of the binding area.

[0049] In a preferred embodiment, the first detection line contains immobilized brucellosis IgG-specific detection antibodies for capturing the binary complex of brucellosis IgG-tracer moving from the binding zone to the test zone; the second detection line contains immobilized brucellosis IgM-specific detection antibodies for capturing the binary complex of brucellosis IgM-tracer moving from the binding zone to the test zone.

[0050] In a preferred embodiment, the first detection line contains immobilized brucellosis IgM-specific detection antibodies for capturing the binary complex of brucellosis IgM-tracer moving from the binding zone to the test zone; the second detection line contains immobilized brucellosis IgG-specific detection antibodies for capturing the binary complex of brucellosis IgG-tracer moving from the binding zone to the test zone.

[0051] In a preferred embodiment, the quality control antibody is an IgG or IgM antibody, preferably a goat anti-chicken antibody.

[0052] In a preferred embodiment, the amount of the tracer antibody labeled with the light absorbing substance is greater than the amount of the analyte.

[0053] In a preferred embodiment, the amount of the capture antibody is greater than the sum of the binary complex of the analyte-tracing antibody and the tracing antibody labeled with the light absorbing substance that moves from the binding zone to the test zone.

[0054] In a preferred embodiment, the excitation or emission spectrum of the enhanced fluorescence of the test piece completely or partially overlaps with the absorption spectrum of the light-absorbing substance.

[0055] In a preferred embodiment, the sample absorption area is an absorption pad.

[0056] In a preferred embodiment, the spot size is <0.8 mm, preferably <0.5 mm.

[0057] In a preferred embodiment, the system further comprises instructions for use describing the method of use.

[0058] In a preferred embodiment, the system further comprises a computer.

[0059] In a preferred embodiment, in the system, the laser light source irradiates the detection area through the optical fiber, and the excited fluorescence enters the detector through the optical fiber, and the computer performs data processing and analysis.

[0060] In a preferred embodiment, when the test system uses HPV E7 protein as the analyte, at a concentration of 4000-5000 ng / mL, F1 / F2 reaches 15 or more, more preferably 18 or more, more preferably 20 or more, wherein F1 refers to the fluorescence intensity in the middle of the detection line and the quality control line in the background enhanced fluorescence area, and F2 refers to the fluorescence intensity of the test area.

[0061] In a preferred embodiment, when Helicobacter pylori is used as the test object and the detection equipment is used as the laser excitation source, at a concentration of 3 ng / mL, F1 / F2 reaches above 2, more preferably above 2.5, more preferably above 3, wherein F1 refers to the fluorescence intensity in the middle of the detection line and the quality control line in the background enhanced fluorescence area, and F2 refers to the fluorescence intensity of the test area.

[0062] In a preferred embodiment, when Helicobacter pylori is used as the test object in the test system and the detection device is a Raman detector, at a concentration of 3 ng / mL, the Raman signal value is above 20,000, preferably above 22,000.

[0063] In a preferred embodiment, when the test system uses HPV E7 protein as the test object, the system includes a test piece and a detection device, wherein the test piece includes a substrate, and a sample addition area, a binding area, a test area and a sample absorption area are sequentially provided on the substrate from one end to the other end of the substrate.

[0064] Wherein, the binding area contains a flowable tracer, the tracer is labeled with colloidal gold particles, and the tracer can bind to the HPV E7 protein to form a binary complex of HPV E7 protein-tracer; wherein, the tracer is a first specific detection antibody for the HPV16E7 protein;

[0065] The test area includes a background enhanced fluorescence area, wherein the background enhanced fluorescence area includes a detection line located at the proximal end of the binding area and the distal end of the sample absorption area, and a quality control line located at the proximal end of the sample absorption area and the distal end of the binding area.

[0066] The detection line contains an immobilized capture antibody, which is used to capture the binary complex of HPV E7 protein-tracer that moves from the binding area to the test area, and the capture antibody is a second specific detection antibody for HPV16 E7 protein;

[0067] The quality control line contains an immobilized quality control antibody, and the quality control antibody is used to bind to the tracer labeled with colloidal gold particles that is not bound to the HPV E7 protein;

[0068] The background enhanced fluorescence region has enhanced fluorescence; and F1>40000, wherein F1 refers to the fluorescence intensity at the middle of the detection line and the quality control line in the background enhanced fluorescence region;

[0069] The sample absorption zone has absorption capacity, so that the sample added to the sample adding zone diffuses from the sample adding zone to the terminal sample absorption zone;

[0070] Wherein, when the capture antibody captures the binary complex of HPV E7 protein-tracer, it causes the fluorescence intensity of the test area to decrease;

[0071] The detection device is a laser excitation source, wherein the spot size of the laser is less than 1.0 mm or smaller than the T-line width.

[0072] In a preferred embodiment, when Helicobacter pylori is used as the test object, the test system comprises a test piece and a detection device, wherein the test piece comprises a substrate, and a sample addition area, a binding area, a test area and a sample absorption area are sequentially arranged on the substrate from one end to the other end of the substrate.

[0073] Wherein, the binding area contains a flowable tracer, the tracer is labeled with Raman microspheres, and the tracer can bind to Helicobacter pylori to form a binary complex of Helicobacter pylori-tracer; wherein, the tracer is a first specific detection antibody for Helicobacter pylori;

[0074] The test area includes a background enhanced fluorescence area, wherein the background enhanced fluorescence area includes a detection line located at the proximal end of the binding area and the distal end of the sample absorption area, and a quality control line located at the proximal end of the sample absorption area and the distal end of the binding area.

[0075] The detection line comprises an immobilized capture antibody, the capture antibody is used to capture the binary complex of Helicobacter pylori-tracer that moves from the binding zone to the test zone, and the capture antibody is a second specific detection antibody for Helicobacter pylori;

[0076] The quality control line contains an immobilized quality control antibody, and the quality control antibody is used to bind to the tracer labeled with Raman microspheres that is not bound to Helicobacter pylori;

[0077] The background enhanced fluorescence region has enhanced fluorescence; and F1>40000, wherein F1 refers to the fluorescence intensity at the middle of the detection line and the quality control line in the background enhanced fluorescence region;

[0078] The sample absorption zone has absorption capacity, so that the sample added to the sample adding zone diffuses from the sample adding zone to the terminal sample absorption zone;

[0079] wherein, when the capture antibody captures the binary complex of Helicobacter pylori-tracer, the fluorescence intensity of the test area decreases;

[0080] The detection device is a laser excitation source or a Raman detector, wherein the spot size of the laser is less than 1.0 mm or smaller than the T-line width.

[0081] In a preferred embodiment, when brucellosis IgG and brucellosis IgM are used as test objects, the test system comprises a test piece and a detection device, wherein the test piece comprises a substrate, and a sample addition area, a binding area, a test area and a sample absorption area are sequentially provided on the substrate from one end to the other end of the substrate.

[0082] Wherein, the binding area contains a flowable tracer, the tracer is labeled with Raman microspheres, and the tracer can bind to brucellosis IgG and brucellosis IgM to form a binary complex of Helicobacter pylori-tracer; wherein, the tracer is a brucellosis antigen;

[0083] The test area includes a background enhanced fluorescence area, wherein the background enhanced fluorescence area includes a first detection line and a second detection line located at the proximal end of the binding area and the distal end of the sample absorption area, and a quality control line located at the proximal end of the sample absorption area and the distal end of the binding area.

[0084] The first detection line comprises an immobilized first capture antibody, the first capture antibody is used to capture the binary complex of brucellosis IgG-tracer moving from the binding area to the test area, and the first capture antibody is a brucellosis IgG-specific detection antibody;

[0085] The second detection line comprises an immobilized second capture antibody, the second capture antibody is used to capture the binary complex of brucellosis IgM-tracer that moves from the binding area to the test area, and the second capture antibody is a brucellosis IgM-specific detection antibody;

[0086] The quality control line comprises an immobilized quality control antibody, and the quality control antibody is used to bind to a tracer labeled with a Raman microsphere that is not bound to brucellosis IgG or brucellosis IgM;

[0087] The background enhanced fluorescence region has enhanced fluorescence; and F1>40000, wherein F1 refers to the fluorescence intensity at the middle of the second detection line and the quality control line in the background enhanced fluorescence region;

[0088] The sample absorption zone has absorption capacity, so that the sample added to the sample adding zone diffuses from the sample adding zone to the terminal sample absorption zone;

[0089] Wherein, when the first capture antibody captures the binary complex of brucellosis IgG-tracer, the fluorescence intensity of the test area decreases, and when the second capture antibody captures the binary complex of brucellosis IgM-tracer, the fluorescence intensity of the test area decreases;

[0090] The detection device is a laser excitation source or a Raman detector, wherein the spot size of the laser is less than 1.0 mm or smaller than the T-line width.

[0091] In a second aspect of the present invention, a test sheet is provided, the test sheet comprising a substrate, wherein from one end to the other end of the substrate, a sample addition area, a binding area, a test area and a sample absorption area are sequentially provided on the substrate,

[0092] Wherein, the binding zone contains one or more flowable tracers, the tracers are labeled with light-absorbing substances, and the tracers can bind to the analyte to form a binary complex of analyte-tracer; wherein the light-absorbing substance is colloidal gold or Raman microspheres;

[0093] The test area comprises a background enhanced fluorescence area, the background enhanced fluorescence area contains one or more detection lines located at the proximal end of the binding area and the distal end of the sample absorption area, and a quality control line located at the proximal end of the sample absorption area and the distal end of the binding area, each of the detection lines independently contains different immobilized capture antibodies, the quality control lines contain immobilized quality control antibodies, the different capture antibodies are independently used to capture different binary complexes of analytes-tracers that move from the binding area to the test area, and the quality control antibodies are used to bind to tracers labeled with light absorbing substances that are not bound to the analytes;

[0094] The background enhanced fluorescence region has enhanced fluorescence; and F1>30000, wherein F1 refers to the fluorescence intensity at the middle of the detection line and the quality control line in the background enhanced fluorescence region;

[0095] The sample absorption zone has absorption capacity, so that the sample added to the sample adding zone diffuses from the sample adding zone to the terminal sample absorption zone;

[0096] When the capture antibody captures the binary complex of the analyte-tracer, the fluorescence intensity of the test area decreases.

[0097] In a preferred embodiment, the sample addition area, test area, binding area, background enhanced fluorescence area, detection line, quality control line, sample absorption area, and test piece are as described in the first aspect of the present invention.

[0098] In a third aspect of the present invention, a fluorescence analysis method for quantitatively detecting an analyte is provided, comprising the steps of:

[0099] (1) adding a sample to be tested to the sample adding area of ​​the test piece according to the second aspect of the present invention;

[0100] (2) using a detection device to measure the fluorescence intensity or Raman signal value of the test area of ​​the test piece, thereby converting it into the amount of the object to be tested;

[0101] Among them, when the light-absorbing substance is colloidal gold particles, the detection device is a laser excitation source, and when the light-absorbing substance is Raman microspheres, the detection device is a laser excitation source or a Raman detector. In the laser excitation source, the spot size of the laser is <1.0 mm or smaller than the T-line width.

[0102] In a preferred embodiment, the analyte is selected from the following group: one or more of HPV16 E7 protein, Helicobacter pylori, brucellosis IgG, and brucellosis IgM.

[0103] In a preferred embodiment, when the analyte is HPV16 E7 protein, the light absorbing substance is colloidal gold particles.

[0104] In a preferred embodiment, when the analyte is Helicobacter pylori, brucellosis IgG and / or brucellosis IgM, the light absorbing substance is Raman microspheres.

[0105] In a preferred embodiment, when the analyte is HPV16 E7 protein, the detection device is a laser excitation source; when the analyte is one or more of Helicobacter pylori, brucellosis IgG, and brucellosis IgM, the detection device is a laser excitation source or a Raman detector.

[0106] In a preferred embodiment, the method is an in vitro method for non-diagnostic and non-therapeutic purposes.

[0107] In a preferred embodiment, the spot size is <0.8 mm, preferably <0.5 mm.

[0108] In a preferred embodiment, the test piece, the sample addition area, the test area, the object to be tested, and the detection device are each independently as described in the first aspect of the present invention.

[0109] In a preferred embodiment, in step (2), the ratio F1 / F2 of the fluorescence intensity F1 at the middle of the detection line and the quality control line in the test area to the fluorescence intensity F2 of the detection line in the test area is compared with the standard curve to determine the amount of the analyte.

[0110] In a preferred embodiment, in step (2), when there are multiple test lines, F1 refers to the fluorescence intensity between the test line closest to the quality control line in the test area and the middle of the quality control line.

[0111] In a preferred embodiment, in step (2), the Raman signal value on the detection line is compared with the standard curve to determine the amount of the analyte.

[0112] In a fourth aspect of the present invention, a fluorescence analysis method for quantitatively detecting an analyte is provided, comprising the steps of:

[0113] (a) providing a test piece according to the second aspect of the present invention;

[0114] (b) adding a sample to be tested to the sample adding area of ​​the test piece;

[0115] (c) The analyte in the sample moves to the binding zone and binds to the tracer labeled with the light-absorbing substance therein, thereby forming a mobile analyte-tracer binary complex;

[0116] (d) the tracer antibody labeled with the light-absorbing substance remaining in step (c) or the flowable binary complex of the analyte-tracer obtained in step (c) moves to the background enhanced fluorescence area in the test area and combines with the capture antibody of the detection line therein, thereby forming a ternary complex of the capture antibody-analyte-tracer fixed to the detection line;

[0117] (e) using a detection device to measure the fluorescence intensity or Raman signal value of the test area of ​​the test piece, thereby converting it into the amount of the object to be tested;

[0118] Among them, when the light-absorbing substance is colloidal gold particles, the detection device is a laser excitation source, and when the light-absorbing substance is Raman microspheres, the detection device is a laser excitation source or a Raman detector. In the laser excitation source, the spot size of the laser is <1.0 mm or smaller than the T-line width.

[0119] In a preferred embodiment, the analyte is selected from the following group: one or more of HPV16 E7 protein, Helicobacter pylori, brucellosis IgG, and brucellosis IgM.

[0120] In a preferred embodiment, when the analyte is HPV16 E7 protein, the light absorbing substance is colloidal gold particles.

[0121] In a preferred embodiment, when the analyte is Helicobacter pylori, brucellosis IgG and / or brucellosis IgM, the light absorbing substance is Raman microspheres.

[0122] In a preferred embodiment, when the analyte is HPV16 E7 protein, the detection device is a laser excitation source; when the analyte is one or more of Helicobacter pylori, brucellosis IgG, and brucellosis IgM, the detection device is a laser excitation source or a Raman detector.

[0123] In a preferred embodiment, the method is an in vitro method for non-diagnostic and non-therapeutic purposes.

[0124] In a preferred embodiment, the spot size is <0.8 mm, preferably <0.5 mm.

[0125] In a preferred embodiment, the method further comprises the step of measuring with a standard substance of known concentration to prepare a standard curve.

[0126] In a preferred embodiment, the fluorescence in the background enhanced fluorescence area is the fluorescence generated by the self-fluorescence of the test piece plus the fluorescence generated by the fluorescent substance.

[0127] In a preferred embodiment, in step (e), the ratio F1 / F2 of the fluorescence intensity F1 between the detection line and the quality control line in the test area and the fluorescence intensity F2 of the detection line in the test area is compared with the standard curve to determine the amount of the analyte.

[0128] In a preferred embodiment, in step (e), the Raman signal value on the detection line is compared with the standard curve to determine the amount of the analyte.

[0129] The fifth aspect of the present invention provides a detection kit, which comprises: a test piece as described in the second aspect of the present invention; and instructions for use.

[0130] The sixth aspect of the present invention provides a use of the test system as described in the first aspect of the present invention or the test piece as described in the second aspect of the present invention, for preparing a detection kit, wherein the kit is used to detect an object to be detected.

[0131] In a preferred embodiment, the analyte is selected from the following group: one or more of HPV16 E7 protein, Helicobacter pylori, brucellosis IgG, and brucellosis IgM.

[0132] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1 The principle of background fluorescence enhanced quenching immunochromatography is shown.

[0134] Figure 2 The Raman microsphere immunochromatography principle is shown.

[0135] Figure 3 The standard curve of the common background fluorescence quenching immunochromatography method in Example 2 is shown.

[0136] Figure 4 The background fluorescence enhanced quenching immunochromatography standard curve in Example 2 is shown.

[0137] Figure 5 The standard curve of the large spot, background fluorescence enhanced quenching immunochromatography method in Example 3 is shown.

[0138] Figure 6 The standard curve of the small spot, background fluorescence enhanced quenching immunochromatography method in Example 3 is shown.

[0139] Figure 7 A schematic diagram of an immunoassay test strip using colloidal gold as a quencher in Example 4 is shown.

[0140] Figure 8 The standard curve of the immunoassay using colloidal gold as a quencher in Example 4 is shown.

[0141] Fig. 9 A schematic diagram of an immunoassay test strip using Raman microspheres as a quencher in Example 4 is shown.

[0142] Fig.10 The F1 / F2 value comparison concentration immunoassay standard curve of Raman microspheres as quenchers in Example 4 is shown.

[0143] Fig.11 The Raman immunoassay standard curve of Raman signal value versus concentration in Example 4 is shown. DETAILED DESCRIPTION

[0144] After extensive and in-depth research, the inventors have discovered for the first time a method for detecting a test object by a background fluorescence enhanced quenching immunochromatography method. When detecting high-risk HPV E7 protein in cervical exfoliated cells, the color intensity of the T line can be observed with the naked eye to quickly determine the positive and negative, and the fluorescence signal on the T line can be accurately quantified. When the quencher colloidal gold used in the test strip is replaced with nano-microspheres with Raman spectral signals, when detecting Helicobacter pylori antigens, the color intensity of the T line can be observed with the naked eye to quickly determine the positive and negative, and the fluorescence signal on the T line can be accurately quantified. The Raman spectral signal on the T line can also be measured by a Raman spectrometer to further improve the detection sensitivity. When the test object is IgG and IgM related to brucellosis, two detection lines can be set in the same test strip to detect IgG and IgM at the same time, and the color intensity of the T line can be observed with the naked eye to quickly determine the positive and negative, and the fluorescence signal on the T line can be accurately quantified. The Raman spectral signal on the T line can also be measured by a Raman spectrometer to achieve the effect of high sensitivity and combined detection. The present invention was completed on this basis.

[0145] the term

[0146] 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.

[0147] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0148] As used herein, when used in reference to a specific recited numerical value, the term "about" 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 all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0149] How it works

[0150] The test strip of the present invention uses background fluorescence enhanced quenching immunochromatography to detect high-risk HPV E7 protein in cervical exfoliated cells. The color intensity of the T line can be observed by naked eyes to quickly judge the positive or negative, and the fluorescence signal on the T line can be measured to accurately quantify.

[0151] The quencher colloidal gold used in the test strip is replaced with nano-microspheres with Raman spectral signals to detect Helicobacter pylori antigen. The color intensity of the T line can be observed with the naked eye to quickly determine the positive or negative, the fluorescence signal on the T line can be measured, and the Raman spectral signal on the T line can be measured with a Raman spectrometer. There are three signal output methods, which can not only achieve accurate quantification but also further improve the detection sensitivity.

[0152] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0153] Option 1:

[0154] The E7 protein-specific capture antibody (test line T) and quality control antibody (quality control line C) are coated on the nitrocellulose membrane that significantly enhances the background fluorescence, and another E7 protein-specific detection antibody (tracing antibody) labeled with colloidal gold (quencher) is coated on the conjugate pad. After adding the sample, the E7 protein in the sample and the E7 protein-specific detection antibody form an immune complex, which can be observed by the naked eye. A purple-red band appears on the test line T, and the test result is positive; otherwise, the test result is negative. Regardless of whether the sample contains E7 protein, the quality control line C shows a purple-red band, indicating that the test is effective.

[0155] Another example Figure 1 It can be seen that the high-risk E7 protein in the cervical exfoliated cells specifically binds to the anti-E7 monoclonal tracer antibody labeled with a quencher to form a "binary complex", which flows along the fluorescently labeled NC membrane to the monoclonal capture antibody fixed with anti-E7 through capillary action and is specifically captured to form a "ternary complex", thereby quenching the background fluorescence on the NC membrane at that location. The degree of quenching is related to the amount of E7. The more E7 there is, the more quenchers are gathered at the T line, and the greater the decrease in fluorescence F2 at the T line relative to the background fluorescence F1 on the membrane surface, the larger the F1 / F2 ratio is, and vice versa, until F1=F2. Figure 1 The lower middle picture shows a scan from line C to line T. The trough F3 formed at line C is only used to locate the trough F2 of line T. The concentration of HPV16 E7 protein in the sample can be detected by the adapter instrument (Model TIC-Q100N) of Shanghai Xinpu Biotechnology Co., Ltd. The instrument detection results are in ng / ml.

[0156] Option 2:

[0157] The quencher colloidal gold used in the test strip in Scheme 1 was replaced with nano-microspheres with Raman spectral signals, namely Raman microspheres. The Helicobacter pylori-specific capture antibody (test line T) and quality control antibody (quality control line C) were coated on the nitrocellulose membrane that significantly enhanced the background fluorescence, and another Helicobacter pylori-specific detection antibody (tracing antibody) labeled with Raman microspheres was coated on the conjugate pad. After adding the sample, the Helicobacter pylori in the sample and the Helicobacter pylori-specific detection antibody formed an immune complex, which can be seen by naked eye observation, showing a black strip on the test line T, and the test result is positive; otherwise, the test result is negative. Regardless of whether the sample contains Helicobacter pylori, the quality control line C shows a black strip, indicating that the test is effective.

[0158] Another example Figure 2 It can be seen that Helicobacter pylori specifically binds to the Helicobacter pylori monoclonal tracer antibody labeled with Raman microspheres to form a "binary complex". Through capillary action, it flows forward along the NC membrane to the fixed Helicobacter pylori monoclonal capture antibody and is specifically captured to form a "ternary complex". The detection line T presents a black strip, so the color of the T line becomes darker as the concentration of the Helicobacter pylori sample increases, so the Raman signal will increase accordingly. Regardless of whether the sample contains Helicobacter pylori, the quality control line C presents a black strip, indicating that the test is effective. The concentration of Helicobacter pylori in the sample can be detected by the adapter instrument (portable Raman spectrometer) of Shanghai Xinpu Biotechnology Co., Ltd. The instrument detection results are in ng / ml.

[0159] The main advantages of the present invention include:

[0160] The present invention firstly uses colloidal gold as a quencher, adopts background fluorescence enhanced quenching immunochromatography, and uses a laser with a smaller spot as an excitation source. Compared with the common background fluorescence quenching immunochromatography, it is used for quantitative detection of a certain antigen content and has better analytical performance.

[0161] At the same time, the quencher colloidal gold is replaced with nano-microspheres with Raman spectral signals, so that the color intensity of the T line can be observed by the naked eye to quickly judge the positivity, the fluorescence signal on the T line can be accurately quantified, and the Raman spectral signal on the T line can be measured by a Raman spectrometer. The three signal output detection technologies further improve the detection sensitivity.

[0162] Afterwards, when the quencher colloidal gold was replaced with nano-microspheres with Raman spectral signals, two detection lines were set up in the detection area, which can simultaneously detect IgG and IgM of the same antigen, achieving three signal output modes and the effect of combined detection.

[0163] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0164] Example

[0165] 1. Instruments and reagents

[0166] 1.1 Instruments

[0167] FIC-Q100N dry immunofluorescence analyzer (Shanghai Xinpu Biotechnology Co., Ltd.);

[0168] CT14RD desktop high-speed refrigerated centrifuge (Shanghai Tianmei Biochemical Instrument Equipment Engineering Co., Ltd.);

[0169] Portable Raman detector (Shanghai Liqiong Optoelectronics Technology Co., Ltd.);

[0170] FA1004 electronic balance (Shanghai Sunyu Hengping Scientific Instrument Co., Ltd.).

[0171] 1.2 Drug testing

[0172]

[0173]

[0174]

[0175] Example 1 Qualitative detection of high-risk HPV E7 protein

[0176] 1 Preparation of 40nm colloidal gold

[0177] 1) Before preparing the colloidal gold solution, make sure the container and rotor are clean. After cleaning, rinse them three times with ultrapure water before use.

[0178] 2) Use a measuring cylinder to measure 980 ml of ultrapure water and add it to a 2L conical flask with a magnetic stirrer. Use clean plastic wrap or tin foil to seal the bottle mouth to avoid contamination.

[0179] 3) Place the conical flask on a constant temperature magnetic stirrer, adjust the temperature to 110°C, and heat until the water in the conical flask boils.

[0180] 4) Accurately measure 10 ml of 1% chloroauric acid solution.

[0181] 5) Accurately measure 10 ml of 1% trisodium citrate solution.

[0182] 6) Quickly pour the measured chloroauric acid solution and trisodium citrate solution into the conical flask.

[0183] 7) Observe the changes in the solution. When the solution turns reddish brown, continue to boil for 10 minutes.

[0184] 8) Remove the conical flask from the constant temperature magnetic stirrer, place it on another magnetic stirrer, stir at low speed, and cool to room temperature.

[0185] 9) Add ultrapure water to make the solution dilute to 1L.

[0186] 10) Complete the spectrum scan of 40nm gold and record the wavelength of the maximum absorption peak and the absorption value of the maximum absorption peak.

[0187] 11) Label the prepared gold and record the preparation date, name, preparer and expiration date.

[0188] 2 Preparation of high-risk HPV E7 antibody 2 gold labeling

[0189] 1) Dilute E7 antibody 2 with pure water to 1 mg / ml and precool the low-temperature high-speed centrifuge to 4°C.

[0190] 2) Select a beaker that corresponds to the volume of the solution to be labeled, add a rotor, and use a measuring cylinder to measure 1000 ml of 40 nm gold and pour it into the beaker.

[0191] 3) Add 7 ml of 0.1 M potassium carbonate solution to the colloidal gold solution and stir evenly on a magnetic stirrer for 5 min.

[0192] 4) Add 5 ml of 1 mg / ml E7 antibody 2 solution into the beaker and stir evenly on a magnetic stirrer for 15 min.

[0193] 5) Add 20 ml of 10% BSA solution into the beaker and stir evenly on a magnetic stirrer for 15 minutes.

[0194] 6) Centrifuge the solution in step 5) in a refrigerated centrifuge at 4°C, 6000rpm for 10 minutes to collect the precipitate; continue to centrifuge the supernatant at 4°C, 9000rpm for 15 minutes to collect the precipitate (if the supernatant is dark in color, continue to centrifuge until it is almost colorless). The precipitate collected twice is the labeled colloidal gold, and the volume is measured.

[0195] 7) Complete the absorbance measurement of the labeled gold solution and record the OD value.

[0196] 3 High-risk HPV E7 antibody 2 Gold label conjugate pad coated

[0197] 1) Dilute the labeled E7 antibody 2 gold solution with Base1 and Base2 to OD 4, where the amount of Base1 accounts for 30% of the total.

[0198] 2) Spread the glass fiber KB50 (200mm×300mm) flat on a glass plate, pour in 19ml of E7 antibody 2 gold solution diluted to OD4, and roll repeatedly with a roller until the gold solution is completely and evenly absorbed, and transfer the gold-covered glass fiber to a stainless steel grid.

[0199] 3) Place the coated gold pad in a drying room to dry and set aside.

[0200] 4. Preparation of coating solution

[0201] 4.1 Preparation of coating solution for quality control line (C line): Dilute GXM to 1.0 mg / ml with coating buffer, mix well and set aside.

[0202] 4.2 Preparation of the coating solution for the detection line (T line): Dilute the E7 antibody 1 to 1.5 mg / ml with the coating buffer, mix well, and set aside.

[0203] 5. Preparation of Chromatographic Test Strips

[0204] Take a PVC base plate, such as Figure 2 As shown, the fluorescent NC membrane is attached to position g, and a 1.0 mg / ml goat anti-chicken antibody (quality control line (C line) coating solution) is drawn to position d and 1.5 mg / ml HPV16 E7 antibody (detection line (T line) coating solution) is drawn to position c using a film drawer. After drying for 2 hours, the absorbent pad is attached to position e, the E7 antibody 2 gold-labeled binding pad prepared in step 3 is attached to position b, and the prepared sample pad is attached to position a to obtain a high-risk HPV E7 protein detection chromatography test strip; the prepared chromatography strip is placed in an appropriate amount of desiccant and heat-sealed in an aluminum foil bag, a label is affixed, the preparation date, name and expiration date are recorded, and it is stored at room temperature with a validity period of 12 months.

[0205] 6High-risk HPV E7 protein antigen qualitative detection steps and result interpretation

[0206] The test reagents, sample diluents, samples to be tested and other test materials need to be equilibrated to room temperature. Use a pipette to draw 60μl of sample from the test tube where the sample has been processed, and drop it into the sample well of the test strip; after adding the sample, as the test proceeds, you will see the purple-red color moving in the test area of ​​the test strip, wait for 15 minutes, and then read the results with the naked eye.

[0207] Explanation of test results:

[0208] (1) Positive result: Color bands appear on both the test line (T) and the control line (C), indicating that the test result of this sample is positive;

[0209] (2) Negative result: The color band appears only on the quality control line (C), which indicates that the test result of this sample is negative;

[0210] (3) Invalid result: When no red line appears on the control line (C), the result is considered invalid regardless of whether the test line (T) is colored. This indicates that the test instructions may not have been followed correctly. The test needs to be repeated.

[0211] Example 2 Quantitative detection of high-risk HPV E7 protein

[0212] The high-risk HPV E7 protein test strips in Example 1 were used to compare the differences in the measurement range and high-value samples between the common background fluorescence quenching immunochromatography and the background fluorescence enhanced quenching immunochromatography. Quantitative detection was performed using the matching detection instrument designed by the present invention, and a standard curve test was performed as follows.

[0213] 1. Preparation of HPV E7 protein series standard solutions

[0214] Use 50mM Tris-HCL solution with pH 9.0 as sample diluent and dilute its working concentration to 5ng / ml, 10ng / ml, 50ng / ml, 100ng / ml, 200ng / ml, 500ng / ml, 1000ng / ml, 2000ng / ml, 4000ng / ml, 5000ng / ml and 10000ng / ml for use.

[0215] 2. Standard curve establishment of common background fluorescence quenching immunochromatography (membrane surface F1 value is about 25000)

[0216] Use a nitrocellulose membrane with a membrane surface fluorescence intensity F1 value of about 25000 to obtain a test strip according to the method of Example 1, take out the test strip (note: do not touch the surface of the middle membrane of the test strip), and lay it flat on the table; use a pipette to draw 60μl of a series of standard solutions from the test tube that has processed the sample, and drip it into the sample loading hole of the test strip; after adding the sample, wait for 15 minutes, and use the matching detection instrument for detection (the laser spot diameter is 0.5mm or less than the T line width). After 30 minutes, the test result is invalid. The test data of the three parallel experimental results of the F1 / F2 value on the T line of the obtained chromatography strip as the concentration C changes are shown in Table 1. The standard curve is plotted with the concentration (ng / ml) of the high-risk HPV E7 protein series standard solution as the horizontal axis and the F1 / F2 value on the T line of the chromatography strip as the vertical axis. Figure 3 .

[0217] Table 1: Common background fluorescence quenching immunochromatography test data

[0218]

[0219] 3. Establishment of standard curve for enhanced background fluorescence quenching immunochromatography (F1 value on membrane surface is about 60000)

[0220] Use a nitrocellulose membrane with a membrane surface fluorescence intensity F1 value of about 60,000 to obtain a test strip according to the method of Part 6 of Example 1, take out the test strip (note: do not touch the surface of the middle membrane of the test strip), and place it flat on the table; use a pipette to draw 60 μl of a series of standard solutions from the test tube that has processed the sample, and add it dropwise to the sample loading hole of the test strip; after adding the sample, wait for 15 minutes, and use the matching detection instrument for detection (the laser spot diameter is 0.5 mm or less than the T line width). After 30 minutes, the test result is invalid. The test data of the three parallel experimental results of the F1 / F2 value on the T line of the obtained chromatography strip as the concentration C changes are shown in Table 2. The standard curve is plotted with the concentration (ng / ml) of the high-risk HPV E7 protein series standard solution as the horizontal axis and the F1 / F2 value on the T line of the chromatography strip as the vertical axis. Figure 4 .

[0221] Table 2: Background fluorescence enhanced quenching immunochromatography detection data

[0222]

[0223]

[0224] According to the comparison results of Example 2, when the test strip using background fluorescence enhanced quenching (membrane surface F1 value is about 60000), when the concentration range of the high-risk HPV E7 protein series standard solution is 10ng / ml-10000ng / ml, there is a good correlation between the high-risk HPV E7 protein concentration and the T-line F1 / F2 value on the chromatography strip, and the F1 / F2 value is higher, and there is no decrease in the high-value concentration signal. Therefore, the background fluorescence enhanced quenching immunochromatography method can achieve quantitative detection of higher concentrations of high-risk HPV E7 proteins.

[0225] Example 3 Quantitative detection of high-risk HPV E7 protein

[0226] The same test strip after the chromatographic reaction of the background fluorescence enhanced quenching immunochromatography (membrane surface F1 value of about 60000) in Example 2 was used for standard curve test to compare the significant effect of laser spot size on analytical performance. Quantitative detection was performed using a matching detection instrument, and the standard curve test was performed as follows.

[0227] 1. Establishment of standard curve for large spot (diameter greater than 1.0 mm or greater than T line width) detection

[0228] Take out the test strip (note: do not touch the surface of the middle film of the test strip), and lay it flat on the table; use a pipette to draw 60μl of the series of standard solutions from the test tube after the sample is processed, and add it dropwise to the sample well of the test strip; after adding the sample, wait for 15 minutes, and use the matching detection instrument for detection. After 30 minutes, the test result is invalid. The test data of the three parallel experimental results of the F1 / F2 value on the T line of the obtained chromatographic strip as the concentration C changes are shown in Table 3. The standard curve is plotted with the concentration (ng / ml) of the high-risk HPV E7 protein series standard solution as the horizontal axis and the F1 / F2 value on the T line of the chromatographic strip as the vertical axis. Figure 5 .

[0229] Table 3: Large spot, background fluorescence enhanced quenching immunochromatography detection data

[0230]

[0231] 2. Establishment of standard curve for small spot (diameter less than 0.5mm or less than T line width) detection

[0232] Take out the test strip (note: do not touch the surface of the middle film of the test strip), and lay it flat on the table; use a pipette to draw 60μl of the series of standard solutions from the test tube that has processed the sample, and add it dropwise to the sample well of the test strip; after adding the sample, wait for 15 minutes, and use the matching detection instrument for detection. After 30 minutes, the test result is invalid. The test data of the three parallel experimental results of the F1 / F2 value on the T line of the obtained chromatographic strip as the concentration C changes are shown in Table 4. The standard curve is plotted with the concentration (ng / ml) of the high-risk HPV E7 protein series standard solution as the horizontal axis and the F1 / F2 value on the T line of the chromatographic strip as the vertical axis. See the attached figure. Figure 6 .

[0233] Table 4: Small spot, background fluorescence enhanced quenching immunochromatography detection data

[0234]

[0235] According to the laser spot comparison results of Example 3, when using a test strip with background fluorescence enhancement quenching (membrane surface F1 value of about 60,000) and a small spot detection (diameter less than 0.5 mm or less than the T-line width), when the concentration range of the high-risk HPV E7 protein series standard solution is 10 ng / ml-10000 ng / ml, there is a good correlation between the high-risk HPV E7 protein concentration and the F1 / F2 value of the T line on the chromatography strip, the F1 / F2 variation range is large, and there is basically no background signal in the negative state, the sensitivity is higher, and there will be no decrease in high-value concentration signals. Therefore, the laser spot should be controlled to have a diameter less than 0.5 mm or less than the T-line width to achieve quantitative detection of high-risk HPV E7 protein.

[0236] Example 4 Quantitative Detection of Helicobacter pylori

[0237] Helicobacter pylori antibodies were labeled with quencher colloidal gold and nano-microspheres with Raman spectral signals, namely Raman microspheres, to prepare Helicobacter pylori detection reagent strips, and the background fluorescence enhanced quenching immunochromatography method in Example 2 (the membrane surface F1 value was about 60,000) was used to perform chromatography reaction test standard curves under bovine serum matrix, and the significant effects of the two nano-materials on analytical performance were compared. Quantitative detection was performed using the matching detection instrument designed by the present invention, and the standard curve test was performed as follows.

[0238] 1. Preparation of Helicobacter pylori series standard solutions

[0239] Use 50 mM Tris-HCL solution at pH 9.0 as sample diluent and dilute its working concentration to 0.03 ng / ml, 0.1 ng / ml, 0.5 ng / ml, 1 ng / ml, and 5 ng / ml respectively for use.

[0240] 2 Establishment of standard curve for Helicobacter pylori antigen detection using colloidal gold as quencher

[0241] Take out the test strip (note: do not touch the surface of the middle film of the test strip), and lay it flat on the table; use a pipette to draw 60μl of the series of standard solutions from the test tube after the sample is processed, and add it dropwise to the sample addition hole of the test strip; after adding the sample, wait for 15 minutes, and use the matching detection instrument to test. After 30 minutes, the test result is invalid. The test data of the three parallel experiments on the F1 / F2 value on the T line of the obtained chromatographic strip with the change of concentration C are shown in Table 5 and the test strip is shown in Figure 7 The standard curve is plotted with the concentration of Helicobacter pylori series standard solution (ng / ml) as the horizontal axis and the F1 / F2 value on the chromatographic strip T line as the vertical axis. Figure 8 .

[0242] Table 5: Immunoassay data using colloidal gold as a quencher

[0243]

[0244] 3 Establishment of standard curve for Helicobacter pylori antigen detection using Raman microspheres as quenchers

[0245] Take out the test strip (note: do not touch the surface of the middle film of the test strip), and lay it flat on the table; use a pipette to draw 60μl of the series of standard solutions from the test tube that has processed the sample, and add it dropwise to the sample well of the test strip; after adding the sample, wait for 15 minutes, and use the matching detection instrument for detection. After 30 minutes, the test result is invalid. The test data of the three parallel experimental results of the F1 / F2 value on the T line of the obtained chromatographic strip as the concentration C changes are shown in Table 6, and the test data of the three parallel experimental results of the Raman signal value on the T line of the obtained chromatographic strip as the concentration C changes are shown in Table 7. The photo of the test strip is shown in Fig. 9 The standard curve is plotted with the concentration of Helicobacter pylori series standard solution (ng / ml) as the horizontal axis and the F1 / F2 value and Raman signal value on the chromatographic strip T line as the vertical axis. Fig.10 , 11 .

[0246] Table 6: Immunoassay data using Raman microspheres as quenchers

[0247]

[0248] Table 7: Raman immunoassay data

[0249]

[0250]

[0251] According to the comparison results of the two reagents in Example 4, when the Raman microsphere reagent is used for immunoassay, when the concentration range of the Helicobacter pylori series standard solution is 0.03ng / ml-3ng / ml, there is a good correlation between the Helicobacter pylori antigen concentration and the T line F1 / F2 value on the chromatographic strip, the F1 / F2 variation range is large, and there is basically no background signal in the negative state, the sensitivity is higher, and there is no phenomenon of signal decrease at high concentrations. Therefore, Raman microspheres can be used as labeling reagents to achieve quantitative detection of Helicobacter pylori, which not only can have three signal output modes, but also can further improve the detection sensitivity.

[0252] As can be seen from the above embodiments, the present invention first uses colloidal gold as a quencher, adopts background fluorescence enhanced quenching immunochromatography, and uses a laser with a smaller spot as an excitation source, which is used for quantitative detection of a certain antigen content compared to the ordinary background fluorescence quenching immunochromatography, and has better analytical performance. At the same time, the quencher colloidal gold is replaced with nano-microspheres with Raman spectral signals, so that the color intensity of the T line can be observed by the naked eye to quickly judge the positive and negative, and the fluorescence signal on the T line can be accurately quantified, and the Raman spectral signal on the T line can be measured by a Raman spectrometer. The three signal output detection technologies further improve the detection sensitivity.

[0253] Example 5 Combined detection of brucellosis IgG / IgM using the color-fluorescence-Raman triple signal mode

[0254] 1 Brucella antigen labeled Raman microspheres

[0255] 1) Dilute brucellosis antigen to 1 mg / ml with pure water.

[0256] 2) In 1mL of Raman microspheres 013 Add 7.5 μg of brucellosis antigen, mix well and let stand for 15 minutes.

[0257] 3) Add 20 ml of 10% BSA solution and allow to react for 15 minutes.

[0258] 4) Centrifuge at 8000 rpm for 10 minutes at 4°C, discard the supernatant, and reconstitute with 100 μL of water.

[0259] 5) Complete the absorbance measurement of the labeled Raman microsphere solution and record the OD value.

[0260] 2 Brucella antigen labeled Raman microsphere binding pad coating

[0261] 1) Dilute the labeled brucellosis Raman microsphere solution with Base1 and Base2 to OD20, where the amount of Base1 accounts for 30% of the total amount.

[0262] 2) Spread glass fiber KB50 (2.50 cm × 5.0 cm) flat on a glass plate, pour in 400 μL of Raman microsphere solution labeled with brucellosis antigen diluted to OD20, and roll repeatedly with a roller until the Raman microsphere solution is completely and evenly absorbed, and transfer the glass fiber with the Raman microsphere solution to a stainless steel grid.

[0263] 3) Dry the coated binding pad in a drying room and set aside.

[0264] 3. Preparation of coating solution

[0265] 3.1 Preparation of coating solution for quality control line (C line): Dilute goat anti-chicken polyclonal antibody to 0.8 mg / ml with coating buffer, mix well and set aside.

[0266] 3.2 Preparation of coating solution for detection line (T1 line): Dilute mouse anti-human IgG to 1 mg / ml with coating buffer, mix well and set aside.

[0267] 3.3. Preparation of coating solution for detection line (T2 line): Dilute mouse anti-human IgM to 1 mg / ml with coating buffer, mix well and set aside.

[0268] 4. Preparation of Chromatographic Test Strips

[0269] Take a PVC base plate, stick a fluorescent NC film on it, use a film marker to draw 0.8 mg / ml goat anti-chicken antibody to the quality control line (C line), and draw 1 mg / ml mouse anti-human IgG and IgM to the T1 line and T2 detection line respectively. After drying for 2 hours, stick an absorbent pad on the upper edge, and stick the Raman microsphere binding pad labeled with brucellosis antigen prepared in the above step 2 on the lower edge. Stick the prepared sample pad on the top of the binding point to obtain the brucellosis IgG / IgM joint detection chromatography test strip; put the prepared chromatography strip into an appropriate amount of desiccant and heat-seal it in an aluminum foil bag, stick a label, record the preparation date, name and expiration date, and store it at room temperature. The validity period is 12 months.

[0270] 5. Test steps and result interpretation methods

[0271] The test reagents, sample diluents, samples to be tested and other test materials need to be equilibrated to room temperature. Use a pipette to draw 60μl of sample from the test tube where the sample has been processed, and drop it into the sample well of the test strip; after adding the sample, react for 15 minutes as the test proceeds, and then read the result with the naked eye.

[0272] Interpretation method:

[0273] (4) Positive result: Color bands appear on both the test line (T1) and the quality control line (C), indicating that the test result of this sample is positive for brucellosis IgG; color bands appear on both the test line (T2) and the quality control line (C), indicating that the test result of this sample is positive for brucellosis IgM; color bands appear on both T1, T2 and the quality control line (C), indicating that the test result of this sample is double positive for brucellosis IgG and IgM.

[0274] (5) Negative result: The color band appears only on the quality control line (C), which indicates that the test result of this sample is negative;

[0275] (6) Invalid result: When no red line appears on the control line (C), the result is considered invalid regardless of whether the test line (T) is colored. This indicates that the test instructions may not have been followed correctly. The test needs to be repeated.

[0276] 6. Combined detection and analysis of brucellosis IgG / IgM by fluorescence and Raman methods

[0277] Using the fluorescent immunochromatographic test strip prepared in step 4 (the F1 value on the membrane surface is about 60000), the positive serum samples with different dilution multiples were subjected to chromatography reaction test, and the Raman signal and fluorescence signal were measured by a portable Raman analyzer and a fluorescent immunoassay analyzer after 15 minutes of reaction. The test data of the three parallel experimental results of the F1 / F2 value on the T line of the obtained chromatographic strip as the dilution multiple changes are shown in Tables 8 and 9, and the test data of the three parallel experimental results of the Raman signal value on the T line of the obtained chromatographic strip as the dilution multiple changes are shown in Tables 10 and 11.

[0278] Table 8: Fluorescence detection data on T1 line (IgG)

[0279]

[0280]

[0281] Table 9: Fluorescence detection data on T2 line (IgM)

[0282]

[0283] Table 10: Raman detection data at T1 (IgG)

[0284]

[0285] Table 11: Raman detection data at T2 (IgM)

[0286]

[0287] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A test system, comprising a test piece and a detection device, in, The test sheet comprises a substrate, and a sample adding area, a binding area, a test area and a sample absorption area are sequentially arranged on the substrate from one end to the other end of the substrate. Wherein, the binding zone contains one or more flowable tracers, the tracers are labeled with light-absorbing substances, and the tracers can bind to the analyte to form a binary complex of analyte-tracer; wherein the light-absorbing substances are colloidal gold particles or Raman microspheres; The test area comprises a background enhanced fluorescence area, the background enhanced fluorescence area contains one or more detection lines located at the proximal end of the binding area and the distal end of the sample absorption area, and a quality control line located at the proximal end of the sample absorption area and the distal end of the binding area, each of the detection lines independently contains different immobilized capture antibodies, the quality control lines contain immobilized quality control antibodies, the different capture antibodies are independently used to capture different binary complexes of analytes-tracers that move from the binding area to the test area, and the quality control antibodies are used to bind to tracers labeled with light absorbing substances that are not bound to the analytes; The background enhanced fluorescence region has enhanced fluorescence; and F1>30000, wherein F1 refers to the fluorescence intensity at the middle of the detection line and the quality control line in the background enhanced fluorescence region; The sample absorption zone has absorption capacity, so that the sample added to the sample adding zone diffuses from the sample adding zone to the terminal sample absorption zone; Wherein, when the capture antibody captures the binary complex of the analyte-tracer, the fluorescence intensity of the test area decreases; When the light-absorbing substance is colloidal gold particles, the detection device is a laser excitation source; when the light-absorbing substance is Raman microspheres, the detection device is a laser excitation source or a Raman detector. In the laser excitation source, the spot size of the laser is less than 1.0 mm or smaller than the T-line width.

2. The test system according to claim 1, characterized in that: The test sheet comprises a sample pad, a conjugation pad, an NC membrane and an absorption pad arranged in sequence, and a rubber sheet backing for fixing the sample pad, the conjugation pad, the fluorescent NC membrane and the absorption pad; The conjugate pad contains a tracer labeled with a light-absorbing substance, which can form a binary complex of the analyte-tracer with the analyte; the light-absorbing substance is colloidal gold or Raman microspheres; The fluorescent NC film comprises a background enhanced fluorescent area, wherein one or more detection lines and quality control lines are arranged in the background enhanced fluorescent area, wherein the one or more detection lines are located at the proximal end of the conjugation pad and the distal end of the absorption pad, and the quality control line is located at the distal end of the conjugation pad and the proximal end of the absorption pad. Each of the detection lines is independently coated with a different capture antibody, and the capture antibodies are independently used to capture different binary complexes of analytes-tracers that move from the conjugate pad to the detection line, thereby forming a ternary complex of capture antibody-analyte-tracer; The quality control line is coated with a quality control antibody, which is used to bind to a tracer labeled with a light-absorbing substance that is not bound to the capture antibody and the analyte; and The background enhanced fluorescence region has enhanced fluorescence, and F1>30000, wherein F1 refers to the fluorescence intensity at the middle of the detection line and the quality control line in the background enhanced fluorescence region.

3. The test system according to claim 1, characterized in that: The test object is selected from the following group: one or more of HPV16 E7 protein, Helicobacter pylori, brucellosis IgG, and brucellosis IgM.

4. The test system according to claim 1, characterized in that: When the analyte is HPV16 E7 protein, the light-absorbing substance is colloidal gold particles; when the analyte is Helicobacter pylori, brucellosis IgG and / or brucellosis IgM, the light-absorbing substance is Raman microspheres.

5. The test system according to claim 1, characterized in that: When the test system uses HPV E7 protein as the analyte, at a concentration of 4000-5000 ng / mL, F1 / F2 reaches 15 or more, more preferably 18 or more, and more preferably 20 or more, wherein F1 refers to the fluorescence intensity at the middle of the detection line and the quality control line in the background enhanced fluorescence area, and F2 refers to the fluorescence intensity of the test area; When the test system uses Helicobacter pylori as the test object and the detection device as the laser excitation source, at a concentration of 3 ng / mL, F1 / F2 reaches 2 or more, more preferably 2.5 or more, more preferably 3 or more, wherein F1 refers to the fluorescence intensity between the detection line and the quality control line in the background enhanced fluorescence area, and F2 refers to the fluorescence intensity of the test area; When the test system uses Helicobacter pylori as the test object and the detection device is a Raman detector, at a concentration of 3 ng / mL, the Raman signal value is above 20,000, preferably above 22,000.

6. A test piece, comprising a substrate, wherein from one end to the other end of the substrate, a sample addition area, a binding area, a test area and a sample absorption area are sequentially arranged on the substrate, in, The binding zone contains one or more flowable tracers, the tracers are labeled with light-absorbing substances, and the tracers can bind to the analyte to form a binary complex of analyte-tracer; wherein the light-absorbing substance is colloidal gold or Raman microspheres; The test area comprises a background enhanced fluorescence area, the background enhanced fluorescence area contains one or more detection lines located at the proximal end of the binding area and the distal end of the sample absorption area, and a quality control line located at the proximal end of the sample absorption area and the distal end of the binding area, each of the detection lines independently contains different immobilized capture antibodies, the quality control lines contain immobilized quality control antibodies, the different capture antibodies are independently used to capture different binary complexes of analytes-tracers that move from the binding area to the test area, and the quality control antibodies are used to bind to tracers labeled with light absorbing substances that are not bound to the analytes; The background enhanced fluorescence region has enhanced fluorescence; and F1>30000, wherein F1 refers to the fluorescence intensity at the middle of the detection line and the quality control line in the background enhanced fluorescence region; The sample absorption zone has absorption capacity, so that the sample added to the sample adding zone diffuses from the sample adding zone to the terminal sample absorption zone; When the capture antibody captures the binary complex of the analyte-tracer, the fluorescence intensity of the test area decreases.

7. A fluorescence analysis method for quantitatively detecting an analyte, comprising the steps of: (1) Adding a sample to be tested to the sample application area of ​​the test piece according to claim 6; (2) using a detection device to measure the fluorescence intensity or Raman signal value of the test area of ​​the test piece, thereby converting it into the amount of the object to be tested; in, When the light-absorbing substance is colloidal gold particles, the detection device is a laser excitation source; when the light-absorbing substance is Raman microspheres, the detection device is a laser excitation source or a Raman detector. In the laser excitation source, the spot size of the laser is <1.0 mm or smaller than the T-line width.

8. A fluorescence analysis method for quantitatively detecting an analyte, comprising the steps of: (a) providing a test piece according to claim 6; (b) adding a sample to be tested to the sample adding area of ​​the test piece; (c) The analyte in the sample moves to the binding zone and binds to the tracer labeled with the light-absorbing substance therein, thereby forming a mobile analyte-tracer binary complex; (d) the tracer antibody labeled with the light-absorbing substance remaining in step (c) or the flowable binary complex of the analyte-tracer obtained in step (c) moves to the background enhanced fluorescence area in the test area and combines with the capture antibody of the detection line therein, thereby forming a ternary complex of the capture antibody-analyte-tracer fixed to the detection line; (e) using a detection device to measure the fluorescence intensity or Raman signal value of the test area of ​​the test piece, thereby converting it into the amount of the object to be tested; in, When the light-absorbing substance is colloidal gold particles, the detection device is a laser excitation source; when the light-absorbing substance is Raman microspheres, the detection device is a laser excitation source or a Raman detector. In the laser excitation source, the spot size of the laser is <1.0 mm or smaller than the T-line width.

9. A detection kit, characterized in that: The kit comprises: a test piece as claimed in claim 6; and instructions for use.

10. Use of the test system according to any one of claims 1 to 5 or the test piece according to claim 6 for preparing a detection kit for detecting an analyte.

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