Ratio-dependent immunochromatographic test strip based on fluorescence technology and detection method of ratio-dependent immunochromatographic test strip

Abstract

The invention discloses a ratio-dependent immunochromatographic test strip based on a fluorescence technology and a detection method thereof, relates to the technical field of immunodetection analysis, and provides a use method of the test strip in detection work by using the test strip as a detection platform, the test strip is simple in reaction condition and high in repeatability. The invention provides a fluorescent material which can present red, green and intermediate colors during color development, and solves the problem of unnatural color transition in the prior art, and in the preparation of the test strip, a simulation peptide and an anti-IC peptide of chlorothalonil are utilized, and an immunochromatography test strip with wide identifiability and applicability is used as an analysis platform, so that the immunochromatography test strip can be used for detecting the content of chlorothalonil. The quantum dots with green and red emission are determined as ratio fluorescence tracers, and the multicolor fluorescent test strip has excellent quantitative and qualitative detection capability on food and environmental samples under the requirement of on-site rapid detection and has a good development prospect.

Description

Technical Field

[0001] The invention relates to the field of environmental water body detection, in particular to a ratiometric immunochromatographic test strip based on fluorescence technology and a detection method thereof. Background Art

[0002] Rapid immunoassay detection is characterized by being fast, simple, low-cost and capable of on-site detection. Among immunoassay methods, immunochromatographic analysis (test strips) can complete the screening of targets in a short time and is a practical rapid immunoassay method. At present, test strips have been widely used in food safety and environmental safety monitoring, identification and quarantine of animal and plant pathogens, and medical diagnosis. In immunoassay, ratiometric signals are widely used to improve detection accuracy, sensitivity and anti-interference ability due to their self-calibration capabilities. Traditional ratiometric signal generation methods are usually based on fluorescence / bioluminescence resonance energy transfer, but these methods have some limitations, such as strict requirements for spectral overlap between donors and acceptors, and sensitivity to the distance between the two. Therefore, it is of great significance to explore new ratiometric signal generation methods. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a ratiometric immunochromatographic test strip based on fluorescence technology and a detection method thereof.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solution: a ratio-type immunochromatographic test strip based on fluorescence technology and a detection method thereof, comprising:

[0005] The object to be detected includes a competitive immunoassay reagent and a non-competitive immunoassay reagent;

[0006] A conjugate pad, wherein a sample pad and a chromatographic membrane are respectively arranged on both sides of the conjugate pad, a detection line and a control line are sequentially arranged on the surface of the chromatographic membrane, an absorbent paper is arranged at one end of the chromatographic membrane, and a labeled tracer is coated on the conjugate pad, wherein the tracer includes a first tracer and a second tracer, and the first tracer and the second tracer are respectively labeled with a competitive immunoassay reagent and a non-competitive immunoassay reagent of the object to be detected;

[0007] A detection line package, wherein the detection line package is used to capture antibodies of the object to be detected;

[0008] A control wire package is provided for capturing the labeled tracer.

[0009] Preferably, the competitive immunoassay reagent is a mimicking epitope polypeptide or a competitive antigen of the analyte, and the non-competitive reagent is an anti-immune complex polypeptide or antibody of the analyte.

[0010] Preferably, the tracer is labeled on the peptide-SA-QD conjugate, and then the competitive immunoassay reagent and non-competitive immunoassay reagent of the complex are labeled by the tracer through antigen-antibody binding. The tracer is directly labeled on the competitive immunoassay reagent or the non-competitive immunoassay reagent using a covalent bond or a weak force.

[0011] Preferably, the first tracer and the second tracer are luminescent materials having different quantum dots, and the luminescent quantum dots are red quantum dots and green quantum dots, which can form an intermediate color yellow after compounding.

[0012] Preferably, the sample pad is a glass fiber pad, and the chromatography membrane is a cellulose acetate membrane.

[0013] Preferably, 100 μL of thiophanate-methyl standard or sample solution containing 10% methanol, 5% BSA and 2 μL of peptide mimetic-SA-QD, 2 μL of anti-IC peptide-SA-QD, the mixture was dropped onto the sample pad and allowed to flow through the NC membrane by capillary action for 10 min, and the images were recorded using a smartphone camera and analyzed using ImageJ software on a computer.

[0014] The present invention provides a ratiometric immunochromatographic test strip detection method based on fluorescence technology, comprising the following steps:

[0015] (1) labeling the first tracer and the second tracer with a competitive immunoassay reagent and a non-competitive immunoassay reagent for an analyte, respectively;

[0016] (2) Chromatographic membrane pretreatment: Place the NC membrane, disperse the anti-mouse IgG antibody and rabbit anti-SApAb on the NC membrane as the test line and control line, respectively, and dry the chromatographic membrane at 37°C for 1-2 hours;

[0017] (3) Treatment of the conjugate pad: The NC membrane was assembled on a semi-rigid polyvinyl chloride sheet with a sample pad and an absorbent pad;

[0018] (4) The assembled test strips were cut into 4 mm width and stored in a zipper bag with desiccant.

[0019] Beneficial effects: The present invention integrates negative readout competitive and positive readout non-competitive immunoassays into a single assay, and creates a new unconstrained ratio signal method by adopting different color tracers, labeled peptide mimetics and anti-immune complex peptides. It utilizes RGB fluorescence, which is red and green when developing, and yellow in the intermediate state. The color is positively correlated with the drug concentration, and the results can be read directly by the naked eye and a mobile phone. The test line of the test strip will show different colors as the control line combines with different amounts of markers, which can be used to correct the test results. In quantitative analysis, multi-color signals can serve as internal references to each other, thereby improving the accuracy of quantitative detection of multi-color test strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0021] In the attached picture:

[0022] Figure 1 It is a schematic diagram of the test paper for semi-quantitative and quantitative detection of thiophanate-methyl of the present invention;

[0023] Figure 2 Schematic diagram of competitive and non-competitive signal standard curves extracted by ImageJ software in the present invention;

[0024] Figure 3 It is the image and ratio signal of RFICS and RCICS for detecting thiophanate-methyl in the buffer of the present invention;

[0025] Figure 4 It is a schematic diagram of the fluorescence emission colors of RFICS and RCICS of thiophanate-methyl of the present invention;

[0026] Figure 5 These are the RFICS and RCICS images of the spiked sample analysis of the present invention. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following text is only used to describe an implementation method of a ratio-type immunochromatographic test strip based on fluorescence technology and a detection method thereof of the present invention, and does not strictly limit the scope of protection specifically requested by the present invention.

[0028] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of such features.

[0029] In Example 1: Figure 1-Figure 5 As shown, Figure 2 (D) Standard curve of RFICS extracted using ImageJ software; Figure 5 (A) RFICS and RCICS images corresponding to the analysis of the spiked sample. The measurement number is marked in the frame.

[0030] A ratiometric immunochromatographic test strip based on fluorescence technology and a detection method thereof, comprising:

[0031] The object to be detected includes a competitive immunoassay reagent and a non-competitive immunoassay reagent;

[0032] A conjugate pad, wherein a sample pad and a chromatographic membrane are respectively arranged on both sides of the conjugate pad, a detection line and a control line are sequentially arranged on the surface of the chromatographic membrane, an absorbent paper is arranged at one end of the chromatographic membrane, and a labeled tracer is coated on the conjugate pad, wherein the tracer includes a first tracer and a second tracer, and the first tracer and the second tracer are respectively labeled with a competitive immunoassay reagent and a non-competitive immunoassay reagent of the object to be detected;

[0033] A detection line package, wherein the detection line package is used to capture antibodies of the object to be detected;

[0034] A control wire package is provided for capturing the labeled tracer.

[0035] In Example 1, the competitive immunoassay reagent is a mimicking epitope polypeptide or a competitive antigen of the analyte, and the non-competitive reagent is an anti-immune complex polypeptide or an antibody of the analyte.

[0036] In Example 1, the tracer is labeled on the peptide-SA-QD conjugate, and then the competitive immunoassay reagent and non-competitive immunoassay reagent of the complex are labeled by the tracer through antigen-antibody binding. The tracer is directly labeled on the competitive immunoassay reagent or the non-competitive immunoassay reagent using a covalent bond or a weak force.

[0037] In Example 1, the first tracer and the second tracer are luminescent materials having different quantum dots, and the luminescent quantum dots are red quantum dots and green quantum dots, which can form an intermediate color yellow after compounding.

[0038] In Example 1, the sample pad is a glass fiber pad, and the chromatography membrane is a cellulose acetate membrane.

[0039] The present invention provides a ratiometric immunochromatographic test strip detection method based on fluorescence technology, comprising the following steps:

[0040] (1) labeling the first tracer and the second tracer with a competitive immunoassay reagent and a non-competitive immunoassay reagent for an analyte, respectively;

[0041] (2) Chromatographic membrane pretreatment: Place the NC membrane, disperse the anti-mouse IgG antibody and rabbit anti-SApAb on the NC membrane as the test line and control line, respectively, and dry the chromatographic membrane at 37°C for 1-2 hours;

[0042] (3) Treatment of the conjugate pad: The NC membrane was assembled on a semi-rigid polyvinyl chloride sheet with a sample pad and an absorbent pad.

[0043] (4) The assembled test strips were cut into 4 mm width and stored in a zipper bag with desiccant.

[0044] In Example 2: Biotinylated peptide mimetics and anti-IC peptides were conjugated to QD525 and QD623, respectively. First, QDs were coated with SA using the following procedure: QDs, 25 μL EDC, and 25 μL sulfo-NHS were added to 2 mL MES buffer and stirred in the dark for 15 min; 52.8 μL SA was then added and stirred for 1 h. The remaining active sites on QDs were blocked with 10 μL 10% BSA and 50 μL glycine for 1 h, and biotinylated peptide mimetics and anti-IC peptides were added to the above SA-coated QD solution, respectively. The mixture was stirred for another 1 h, and unreacted reagents were removed using an ultrafiltration tube at 3000 g for 5 min. Finally, the peptide-SA-QD conjugate was suspended in 200 μL 10 mM phosphate-buffered saline and stored at 4 °C.

[0045] In Example 2: goat anti-mouse IgG antibody and rabbit anti-SApAb were dispersed on the NC membrane as the test line and the control line, respectively, then the NC membrane was dried at 37°C for 1 hour and assembled on a semi-rigid polyvinyl chloride sheet with a sample pad and an absorption pad, finally, the assembled test strips were cut into 4 mm width and stored in a zipper bag with a desiccant.

[0046] In Example 2: A ratiometric immunochromatographic test strip based on fluorescence technology is used to detect added samples.

[0047] (1) Preparation and treatment of spiked samples

[0048] Blank samples of soil, rice, cucumber, and wheat were characterized by HPLC with UV detection. Samples were collected from local farms and markets in Nanjing, China. The samples were homogenized and spiked with chlorothalonil standard solution to final concentrations of 10, 50, 200, 500, and 1000 ng mL -1 ,The sample was mixed with 5 mL of deionized water, and then 20 mL of methanol and 5 g of anhydrous sodium sulfate were added to the sample. The mixture was shaken thoroughly for 5 min, ultrasonicated for 15 min, and centrifuged at 1660 g for 5 min. The separated supernatant was filtered and diluted for ICS analysis. To evaluate the accuracy of ion chromatography, the content of thiophanate-methyl in blind soil samples was determined simultaneously by HPLC and ion chromatography.

[0049] (2) Ratio immunochromatographic test strips based on fluorescence technology

[0050] 100 μL of thiophanate-methyl standard or sample solution was mixed with 2 μL of peptide mimetic-SA-QD and 2 μL of anti-IC peptide-SA-QD, and the mixture was dropped onto the sample pad and allowed to flow through the NC membrane by capillary action for 10 min.

[0051] (3) Analysis and determination of test results

[0052] Method 1: Semi-quantitative analysis is performed by observing the color of the T line with the naked eye. When there is no thiocarbamide in the sample or its concentration is low, a bright green fluorescence or red colorimetric T line is produced. Under medium concentrations of thiocarbamide, yellow fluorescence or purple colorimetric T line is produced. Under high concentrations of thiocarbamide, bright red fluorescence or blue colorimetric T line is produced. A C line with mixed colors is produced, which indicates that the test is valid. Colorless and single-color C lines represent invalid tests. The effect diagram can be seen. Figure 4 .

[0053] Method 2: Use a smartphone camera to record ICS images, and use ImageJ software on a computer to extract competitive and non-competitive signals of RFICS. Import the images to establish analysis relationship curves. The results are shown in Figure 2 , Figure 3 and Figure 5 .

[0054] Finally, it should be noted that the above is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A ratiometric immunochromatographic test strip based on fluorescence technology, characterized in that: include: The object to be detected includes a competitive immunoassay reagent and a non-competitive immunoassay reagent; A conjugate pad, wherein a sample pad and a chromatographic membrane are respectively arranged on both sides of the conjugate pad, a detection line and a control line are sequentially arranged on the surface of the chromatographic membrane, an absorbent paper is arranged at one end of the chromatographic membrane, and a labeled tracer is coated on the conjugate pad, wherein the tracer includes a first tracer and a second tracer, and the first tracer and the second tracer are respectively labeled with a competitive immunoassay reagent and a non-competitive immunoassay reagent of the object to be detected; A detection line package, wherein the detection line package is used to capture antibodies of the object to be detected; A control wire package is provided for capturing the labeled tracer.

2. The ratiometric immunochromatographic test strip based on fluorescence technology according to claim 1, characterized in that: The competitive immunoassay reagent is a mimicking epitope polypeptide or a competitive antigen of the analyte, and the non-competitive reagent is an anti-immune complex polypeptide or an antibody of the analyte.

3. The ratiometric immunochromatographic test strip based on fluorescence technology according to claim 1, characterized in that: The tracer is labeled on the peptide-SA-QD conjugate, and then the competitive immunoassay reagent and non-competitive immunoassay reagent of the complex are labeled by the tracer through antigen-antibody binding. The tracer is directly labeled on the competitive immunoassay reagent or the non-competitive immunoassay reagent using a covalent bond or a weak force.

4. The ratiometric immunochromatographic test strip based on fluorescence technology according to claim 1, characterized in that: The first tracer and the second tracer are luminescent materials with different quantum dots, and the luminescent quantum dots are red quantum dots and green quantum dots, which can form an intermediate color yellow after compounding.

5. The ratiometric immunochromatographic test strip based on fluorescence technology according to claim 1, characterized in that: The sample pad is a glass fiber pad, and the chromatography membrane is a cellulose acetate membrane.

6. A detection method for a ratiometric immunochromatographic test strip based on fluorescence technology according to any one of claims 1 to 5, characterized in that: The steps include: (1) labeling the first tracer and the second tracer with a competitive immunoassay reagent and a non-competitive immunoassay reagent for an analyte, respectively; (2) Chromatographic membrane pretreatment: Place the NC membrane, disperse the anti-mouse IgG antibody and rabbit anti-SApAb on the NC membrane as the test line and control line, respectively, and dry the chromatographic membrane at 37°C for 1 to 2 hours; (3) Treatment of the conjugate pad: The NC membrane was assembled on a semi-rigid polyvinyl chloride sheet with a sample pad and an absorbent pad; (4) The assembled test strips were cut into 4 mm width and stored in a zipper bag with desiccant.

7. The detection method of a ratiometric immunochromatographic test strip based on fluorescence technology according to claim 6, characterized in that: 100 μL of thiophanate-methyl standard or sample solution, which contained 10% methanol, 5% BSA and 2 μL of peptide mimetic-SA-QD, 2 μL of anti-IC peptide-SA-QD, was dropped onto the sample pad and allowed to flow through the NC membrane by capillary action for 10 min. Images were recorded using a smartphone camera and analyzed using ImageJ software on a computer.

Images