Time-resolved immunochromatography test strip and probe for simultaneously detecting rhodamine B and acid orange II

Abstract

The invention discloses a time-resolved immunochromatography test strip, a probe and a detection method for simultaneously detecting rhodamine B and acid orange II, and belongs to the technical field of food safety detection. The time-resolved immunochromatography test strip for simultaneously detecting rhodamine B and acid orange II comprises a test strip base material, and a reaction film of the test strip base material is sequentially provided with a T2 line, a T1 line and a C line from the starting end to the tail end; the T2 line is coated with an acid orange II antigen, the T1 line is coated with a rhodamine B antigen, and the C line is coated with a goat anti-chicken secondary antibody capable of being combined with a chicken IgY antibody. The detection test strip provided by the invention has the advantages of high sensitivity, high specificity, background interference resistance and the like, can realize quantitative detection of rhodamine B and acid orange II in a sample at the same time, and is rapid in detection, low in cost, simple to operate, suitable for professional inspection, customs quarantine, quality detection and the like, and wide in application prospect. Good popularization and application prospects are realized.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a time-resolved immunochromatographic test strip and probe for the simultaneous detection of Rhodamine B and Acid Orange II. Background Technology

[0002] Rhodamine B is a synthetic dye commonly used in the textile, pharmaceutical, and bioanalytical chemistry industries. Studies have shown that Rhodamine B is carcinogenic, has reproductive and developmental toxicity, neurotoxicity, and chronic toxicity in humans and animals. However, due to its bright color, stable coloring, and low price, some unscrupulous vendors still illegally use it in food production, seriously endangering public health. Acid Orange II, also known as Golden Orange II or Orange Yellow II, is a chemical dye mainly used for dyeing leather, wool, silk, and paper. Studies have shown that after being metabolized in the body, it produces aromatic amine compounds that alter DNA, exhibiting potential carcinogenicity and mutagenicity. Therefore, both the EU and China strictly prohibit the use of Acid Orange II as a food additive. However, due to its low cost and good stability, it is still illegally used in the food industry, especially in condiments. Therefore, establishing a rapid detection method that can simultaneously detect Rhodamine B and Acid Orange II is of great significance for controlling their illegal use in food and ensuring food safety.

[0003] Currently, most standard detection methods for Rhodamine B and Acid Orange II in food employ large-scale instrumental methods such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS / MS). While these methods offer high specificity and sensitivity, they require sophisticated equipment, are complex to operate, demand extensive knowledge from personnel, are costly, time-consuming, and labor-intensive, making them unsuitable for widespread application in field settings. Immunochromatography, on the other hand, offers advantages such as simple operation, low cost, high sensitivity, and the elimination of the need for complex instruments. It enables rapid qualitative and quantitative analysis of harmful small molecule compounds in food and is widely recognized as a suitable rapid detection method for on-site testing. This invention proposes a rapid test strip based on the principle of immunological competition, combined with time-resolved fluorescence labeling technology and lateral flow immunochromatography. This strip can rapidly and specifically achieve simultaneous quantitative detection of Rhodamine B and Acid Orange II in food. The test strip is easy to operate, requires no complex instruments, and can be used in laboratories or in real-world settings such as supermarkets and production workshops, making it suitable for large-scale sample screening. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a time-resolved immunochromatographic test strip and probe for the simultaneous detection of Rhodamine B and Acid Orange II. The time-resolved immunochromatographic test strip of this invention can simultaneously achieve quantitative detection of Rhodamine B and Acid Orange II in a sample, and the detection is rapid, low-cost, and does not require complex instruments.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a time-resolved immunochromatographic test strip for the simultaneous detection of Rhodamine B and Acid Orange II, comprising a test strip substrate, wherein the reaction membrane of the test strip substrate has a T2 line, a T1 line, and a C line sequentially from the beginning to the end; the T2 line is coated with Acid Orange II antigen, the T1 line is coated with Rhodamine B antigen, and the C line is coated with goat anti-chicken secondary antibody that can bind to chicken IgY antibody.

[0006] Preferably, the paper strip substrate includes a base plate, a sample pad, a reaction membrane, and an absorbent pad; The sample pad, reaction membrane, and absorbent pad are sequentially attached to the base plate; The end of the sample pad is connected to the beginning of the reaction membrane, and the end of the reaction membrane is connected to the beginning of the absorbent pad.

[0007] Furthermore, the base plate is a polyvinyl chloride backing plate; the sample pad is a glass cellulose membrane; the absorbent pad is ordinary absorbent paper; and the reaction membrane is a nitrocellulose membrane.

[0008] Furthermore, the test strip is 4 mm wide, and the sample pad and reaction membrane have an overlap of 1.5-2.0 mm; the absorbent pad and reaction membrane have an overlap of 1.5-2.0 mm; the beginning of the sample pad is aligned with the beginning of the base plate, and the end of the absorbent pad is aligned with the end of the base plate. The sample pad can be 29 mm long, the reaction membrane can be 25 mm long, and the absorbent paper can be 30 mm long. The vertical distance between the T2 line and the sample pad can be 6 mm, the vertical distance between the T1 line and the T2 line can be 4 mm, and the vertical distance between the C line and the absorbent pad can be 4 mm.

[0009] Preferably, the Acid Orange II antigen coating concentration is 2.0 mg / mL and the coating amount is 0.8 μL / cm; the Rhodamine B antigen coating concentration is 0.5 mg / mL and the coating amount is 0.8 μL / cm. The concentration of the goat anti-chicken secondary antibody coating was 1.0 mg / mL, and the coating amount was 0.8 μL / cm.

[0010] Furthermore, the width of each wrapping can be 1mm.

[0011] The present invention also provides a probe suitable for the time-resolved immunochromatographic test strip of claim 1, which is composed of a rhodamine B time-resolved fluorescent probe, an acid orange II time-resolved fluorescent probe, and a chicken IgY antibody time-resolved fluorescent probe.

[0012] Preferably, the preparation methods for the Rhodamine B time-resolved fluorescent probe, the Acid Orange II time-resolved fluorescent probe, and the chicken IgY antibody time-resolved fluorescent probe are as follows: S1. Place time-resolved fluorescent microspheres in phosphate buffer, add 1-(3-dimethylpropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and react at 37°C for 30 min. S2. Centrifuge the reaction solution at 20000g for 10 min, remove the supernatant, then add the antibody and react at 37℃ for 2 h; then add the blocking solution and block at 37℃ for 1 h, then centrifuge at 20000g for 10 min, remove the supernatant after centrifugation; reconstitute the precipitate with the reconstitution solution to obtain the time-resolved fluorescent probes of each antibody. The antibody is rhodamine B antibody, acid orange II antibody, or chicken IgY antibody.

[0013] Preferably, the mass ratio of time-resolved fluorescent microspheres, 1-(3-dimethylpropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide in S1 is 50:3.5:33.

[0014] Preferably, in S2, the mass ratio of time-resolved fluorescent microspheres to anti-rhodamine B antibody is 1:0.02; the mass ratio of time-resolved fluorescent microspheres to acid orange II antibody is 1:0.08; and the mass ratio of time-resolved fluorescent microspheres to chicken IgY antibody is 1:0.02.

[0015] Preferably, the time-resolved fluorescent microspheres in S2 are carboxyl nanospheres with a diameter between 100-400 nm, an excitation wavelength of 360 nm, and an emission wavelength of 615 nm.

[0016] Preferably, the blocking solution in S2 is a 0.1 M glycine solution, which includes 1% BSA.

[0017] Preferably, the reconstitution solution in S2 is a 0.02 M pH 7.4 phosphate buffer, which further includes 0.5% Tween-20, 1% BSA, 5% trehalose and 0.5% ProClin 300.

[0018] Furthermore, the anti-rhodamine B antibody in S2 is a commercially available specific monoclonal antibody; the chicken IgY antibody is a commercially available antibody.

[0019] The anti-acid orange II antibody is a self-made specific polyclonal antibody, prepared by the following method: 2-Amino-5-sulfobenzoic acid is reacted with sodium nitrite to generate a diazonium salt, which is then reacted with 2-naphthol to generate the hapten of Acid Orange II. The immunogen was obtained by conjugating the hapten of Acid Orange II with bovine serum albumin, and the coating antigen was obtained by conjugating it with ovalbumin. New Zealand white rabbits were immunized with Acid Orange II immunogen, and antibody affinity was determined by indirect competitive enzyme-linked immunosorbent assay (ELISA). Antiserum was collected by collecting blood from the ear vein and heart, and the antibody was purified by affinity chromatography.

[0020] The present invention also provides a detection method for simultaneously determining Rhodamine B and Acid Orange II, wherein the probe described above is mixed with the sample to be tested and incubated, and then the time-resolved immunochromatographic test strip described above is used for rapid quantitative detection of Rhodamine B and Acid Orange II.

[0021] It contains at least the following beneficial technical effects: The test strip provided by this invention is based on the basic principle of immunoassay and uses time-resolved fluorescent microspheres as antibody markers. Compared with colloidal gold and other organic dyes, it has many advantages such as high sensitivity, high specificity, and resistance to background interference. It can simultaneously realize the quantitative detection of Rhodamine B and Acid Orange II in samples. Moreover, the detection is rapid, low-cost, does not require complex instruments, and is simple to operate. It is suitable for professional inspection, customs quarantine, quality inspection, etc., and has a good prospect for widespread application. Attached Figure Description

[0022] Figure 1 A schematic diagram of the structure of a time-resolved fluorescence quantitative PCR strip for the simultaneous detection of Rhodamine B and Acid Orange II.

[0023] Figure 2 This is a competitive inhibition standard curve for time-resolved fluorescence quantitative PCR strips used for the detection of Rhodamine B in samples.

[0024] Figure 3 This is a competitive inhibition standard curve for a time-resolved fluorescent quantitative PCR strip used for the detection of Acid Orange II in samples.

[0025] Figure 4 The reaction kinetics curves of time-resolved fluorescent immunochromatographic test strips for Rhodamine B and Acid Orange II are shown. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0032] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0033] Example 1 Preparation of anti-acid orange II antibody (1) Preparation of Acid Orange II antigen: a. Synthesis of Acid Orange II Hapten: 2-amino-5-sulfobenzoic acid is reacted with sodium nitrite to generate a diazonium salt, which is then reacted with 2-naphthol to generate the Acid Orange II hapten. b. Synthesis of artificial antigen of Acid Orange II: The hapten of Acid Orange II was coupled with bovine serum albumin (BSA) to obtain an immunogen by active ester method, and coupled with ovalbumin (OVA) to obtain a coating antigen. (2) Preparation of anti-acid orange II antibody: The Acid Orange II immunogen prepared above was used to immunize New Zealand white rabbits. Antibody affinity was determined by indirect competitive enzyme-linked immunosorbent assay (ELISA). Antiserum was collected by collecting blood from the ear vein and heart, and antibody was purified by affinity chromatography.

[0034] Example 2 Preparation of time-resolved fluorescence quantitative test strips for simultaneous detection of Rhodamine B and Acid Orange II The preparation method of time-resolved fluorescence quantitative test strips for simultaneous detection of Rhodamine B and Acid Orange II includes the following steps: (1) Preparation of time-resolved fluorescent probes Take 0.05 mL of time-resolved fluorescent microsphere suspension into 1 mL of 0.02 M PBS (pH 7.4), centrifuge at 20000 g for 10 min, discard the supernatant, resuspend the precipitate in 1 mL of PBS, and sonicate to mix well; Add 3.5 μL of activating reagent 1-(3-dimethylpropyl)-3-ethylcarbodiimide hydrochloride (10 mg / mL) and 33 μL of N-hydroxysuccinimide (10 mg / mL) solution, mix quickly, react at 37 °C for 30 min, centrifuge at 20000g to remove supernatant, resuspend the precipitate in 1 mL PBS, and sonicate to mix. Add 10 μg of anti-rhodamine B antibody or 40 μg of anti-acid orange II antibody or 10 μg of chicken IgY antibody, mix well and react at 37℃ for 2 h, then add 0.1 mL of blocking buffer and block at 37℃ for 1 h. Centrifuge at 20000 g for 10 min, discard the supernatant, resuspend the precipitate in 0.5 mL of reconstitution solution, and sonicate to mix. This is the labeled probe.

[0035] (2) Preparation of detection and control zones Rhodamine B and Acid Orange II coating antigens were prepared at concentrations of 0.5 mg / mL and 2.0 mg / mL, respectively, using coating solutions. Goat anti-chicken antibody was prepared at a concentration of 1.0 mg / mL using the same coating solution. The Rhodamine B and Acid Orange II coating antigens were sprayed onto the detection area of ​​the reaction membrane, and the goat anti-chicken secondary antibody was sprayed onto the control area. The membrane was then dried overnight in an oven at 37°C. The coating solution was a 0.02 mol / L phosphate solution (pH 7.4).

[0036] (3) Attach the reaction membrane, sample pad, and absorbent paper to the base plate in sequence, with the detection area on one side of the sample pad and the control area on the other side of the absorbent paper. Cut the test paper into 4mm wide test strips. Seal the test strips in a plastic tube containing desiccant.

[0037] (4) Sample detection: Mix the time-resolved fluorescent probe with the sample, incubate at room temperature for 3 min, and add the mixed solution to the sample pad of the test strip for detection. The content of Rhodamine B and Acid Orange II in the sample can be determined according to the standard curve.

[0038] In step (1), the time-resolved fluorescent microspheres are polystyrene microspheres containing rare earth-based fluorescent dyes, with a diameter of 200 nm, an excitation wavelength of 360 nm, and an emission wavelength of 615 nm.

[0039] The mass ratio of the time-resolved fluorescent microspheres to the coupling agent is m(TRFMs):m(EDC):m(NHS) = 50:3.5:33 The blocking solution is a 0.1M glycine solution containing 1% BSA.

[0040] The reconstitution solution is a 0.02 M phosphate buffer (pH 7.4) containing 0.5% Tween-20, 1% BSA, 5% trehalose and 0.5% ProClin 300.

[0041] The reaction membrane in step (2) is a nitrocellulose membrane.

[0042] The base plate in step (3) is PVC, the sample pad is a glass cellulose membrane, and the absorbent paper is ordinary absorbent paper. The widths of the sample pad, reaction membrane, and absorbent paper are 29, 25, and 30 mm, respectively. They are overlapped and adhered to the base plate, with the sample pad overlapping the reaction membrane by 2 mm and the absorbent paper overlapping the reaction membrane by 2 mm (as shown in the attached figure). Figure 1 (As shown).

[0043] Example 2 Establishment of a standard curve for time-resolved fluorescence immunochromatographic test strips (1) Establishment of the standard curve for detecting Rhodamine B Rhodamine B standard concentrations of 0, 1.25, 2.5, 5, 10, 20, 40, and 80 μg / kg were added to Rhodamine B negative samples (chili powder or chili oil). Each concentration was tested three times using test strips, and the average fluorescence ratio (FI) was recorded. T1 / FI C ), with FI T1 / FI C Plot a standard curve with the vertical axis representing the concentration of the standard and the horizontal axis representing the logarithm of the standard concentration. Calculate the test strip concentration (in terms of IC50). 50 (represented by) and determining the detection linear range of Rhodamine B (in IC50). 20 -IC 80 express).

[0044] (2) Establishment of the standard curve for detecting Acid Orange II Add Acid Orange II standard concentrations of 0, 3.3, 10, 30, 90, 270, 810, and 2430 μg / kg to Acid Orange II negative samples (chili powder or chili oil), respectively. Each concentration is tested three times using test strips, and the average fluorescence ratio (FI) is taken. T2 / FI C ), with FI T2 / FI CPlot a standard curve with the vertical axis representing the concentration of the standard and the horizontal axis representing the logarithm of the standard concentration. Calculate the 50% competitive inhibition rate concentration of the test strip (in IC50). 50 (indicated by) and determining the linear range of detection for Acid Orange II (in IC50). 20 -IC 80 express).

[0045] (3) The results are as follows Figure 2 As shown, the time-resolved fluorescence immunochromatographic assay strip exhibits a linear detection range of 3.2–34.3 μg / kg for rhodamine B in chili powder samples, with an IC50 value of [missing information]. 50 The detection limit is 10.4 μg / kg (using IC50). 10 The linear detection range for Rhodamine B in chili oil samples was 1.6 μg / kg; its IC50 value was 2.3–14.7 μg / kg. 50 The detection limit was 5.8 μg / kg and the limit of detection was 1.3 μg / kg. Figure 3 As shown, the linear detection range of the test strip for Acid Orange II in chili powder is 3.8-766.5 μg / kg, and its IC50 value is [missing information]. 50 The limit of detection (LOD) for Acid Orange II in chili oil was 54.0 μg / kg, and the limit of detection (LOD) was 0.8 μg / kg. The linear detection range for Acid Orange II in chili oil was 2.0–601.1 μg / kg, and its IC50 value was [missing value]. 50 The concentration was 34.6 μg / kg, and the detection limit was 0.38 μg / kg.

[0046] Example 3 Determination of the specificity of time-resolved fluorescence immunochromatographic test strips 1. Using the cross-reactivity (CR) of Rhodamine B and Acid Orange II as 100%, ten structural analogs, including Sunset Yellow, Chromogen FB, Para Red, and Rhodamine 110, were determined. The results are shown in Table 1. When the method uses the cross-reactivity (CR) of Rhodamine B and Acid Orange II as 100%, the cross-reactivity of the other ten analogs is low or non-existent, indicating that the method has good specificity and can simultaneously achieve specific detection of Rhodamine B and Acid Orange II.

[0047] Table 1 Results of cross-reactivity determination of test strips Example 4 Determination of the accuracy and precision of test strips 1. Sample pretreatment Add 5 mL of 40% ethanol-water solution (v / v) to 1 g of chili powder or chili oil sample, vortex vigorously for 3 min, centrifuge at 4000 g for 5 min, take 50 μL of supernatant, dilute 4 times with sample diluent, and use for test strip detection.

[0048] The sample diluent was 0.1M PBS (pH 7.4) containing 1% Tween-20.

[0049] 2. The test results are shown in Table 2. The recovery rate of Rhodamine B on the test strip was 84.7%-111.1%, and the recovery rate of Acid Orange II was 84.5%-116.0%. The relative standard deviations for each sample were all less than 14.5%, indicating that the method has good accuracy and precision.

[0050] Table 2 Results of Sample Addition and Recovery Detection Example 5 Optimization of antibody labeling amount for time-resolved fluorescent probes 1. Referring to Example 2, fluorescent probes were prepared by reacting time-resolved fluorescent microspheres with different gradients of anti-rhodamine B or anti-acid orange II antibody amounts. Test strips were then prepared, and the fluorescence intensity and inhibition rate of the T-line of the test strips were observed (calculated as: FI of positive samples). T / FI C FI values ​​for negative samples T / FI C The optimal reaction ratio is when the fluorescence intensity is good and the inhibition rate is the lowest (ratio of values).

[0051] 2. The results are shown in Table 3. When the mass ratio of anti-rhodamine B antibody to time-resolved fluorescent microspheres is 0.02:1, the test strip exhibits good performance in terms of fluorescence intensity and inhibition rate. Therefore, the optimal mass ratio for the time-resolved fluorescent probe to detect rhodamine B is 0.02:1. When the mass ratio of anti-acid orange II antibody to time-resolved fluorescent microspheres is 0.08:1, the test strip exhibits good performance in terms of fluorescence intensity and inhibition rate. Therefore, the optimal mass ratio for the time-resolved fluorescent probe to detect acid orange II is 0.08:1.

[0052] Table 3 Optimization of the ratio of time-resolved fluorescent microspheres to antibody Example 6 Optimization of antigen coating concentration on test strips 1. The reaction membrane was coated with different gradient concentrations of anti-rhodamine B and acid orange II coating antigens to further prepare test strips. The fluorescence intensity and inhibition rate of the T line of the test strip were observed. The optimal antigen coating concentration was the one with the best fluorescence intensity and the lowest inhibition rate.

[0053] 2. The results are shown in Table 4. When the concentration of Rhodamine B coated antigen was 0.5 mg / mL, the test strip showed good performance in both fluorescence intensity and inhibition rate. Therefore, the optimal antigen coating concentration for detecting Rhodamine B was 0.5 mg / mL. When the concentration of Acid Orange II coated antigen was 2.0 mg / mL, the test strip showed good performance in both fluorescence intensity and inhibition rate. Therefore, the optimal antigen coating concentration for detecting Acid Orange II was 2.0 mg / mL. Table 4 Optimization of antigen coating concentration on test strips Example 7 Optimization of pH value of sample dilution: 1. Select phosphate buffer solutions of different pH values ​​as sample dilution solutions to test the test strips. Observe the fluorescence intensity and inhibition rate of the T line of the test strips. The optimal reaction pH value is when the fluorescence intensity is good and the inhibition rate is the lowest. 2. The results are shown in Table 5. When the pH of the sample dilution solution is 7.4, the test strips show good performance in terms of fluorescence intensity and inhibition rate when detecting Rhodamine B or Acid Orange II. Therefore, phosphate buffer solution with a pH of 7.4 is selected as the optimal sample dilution solution.

[0054] Table 5 Optimization of pH value of sample dilution solution Example 8 Optimization of surfactants in sample dilution solutions: 1. Four different types of surfactants, namely Tween-20, Tween-80, Triton X-100 and Tetronic 1307 (S9), were selected as components of the sample diluent. The fluorescence intensity and inhibition rate of the T line of the test strip were observed. The surfactant with the best fluorescence intensity and the lowest inhibition rate was the best surfactant. 2. The results are shown in Table 6. When the surfactant is 1% Tween-20 (v / v), the test strips have good performance in terms of fluorescence intensity and inhibition rate when detecting Rhodamine B or Acid Orange II. Therefore, 1% Tween-20 is selected as the best surfactant for the sample dilution.

[0055] Table 6 Optimization of Surfactants Example 9 Time-resolved quantitative PCR strip immunochromatographic kinetic analysis 1. The immunochromatographic kinetics curves for detecting Sudan Red and Acid Orange on the test strips were determined separately. The steps are as follows: 50 μL of negative control was added to the sample pad of the test strip. After reacting for 3 min, the fluorescence values ​​FI of the test line and control line of the test strip were read every 1 min using a fluorescence immunoassay analyzer. T1 ,FI T2 and FIC And calculate FI respectively T1 / FI C and FI T2 / FI C And tracked and recorded for 40 minutes.

[0056] 2. Results are as follows Figure 4 As shown, FI T1 FI T2 and FI C The fluorescence ratio FI increased with increasing reaction time. T1 / FI C and FI T2 / FI C The reaction reached a constant value after 30 minutes, indicating that the test strip had reached reaction equilibrium. Therefore, the test strip detection time was set to 30 minutes.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A time-resolved immunochromatographic test strip for simultaneous detection of Rhodamine B and Acid Orange II, characterized in that, The test strip includes a substrate, and the reaction membrane of the substrate has T2 line, T1 line and C line in sequence from the beginning to the end; the T2 line is coated with acid orange II antigen, the T1 line is coated with rhodamine B antigen, and the C line is coated with goat anti-chicken secondary antibody that can bind to chicken IgY antibody.

2. The time-resolved immunochromatographic test strip according to claim 1, characterized in that, The paper strip substrate includes a base plate, a sample pad, a reaction membrane, and an absorbent pad; The sample pad, reaction membrane, and absorbent pad are sequentially attached to the base plate; The end of the sample pad is connected to the beginning of the reaction membrane, and the end of the reaction membrane is connected to the beginning of the absorbent pad.

3. The time-resolved immunochromatographic test strip according to claim 1, characterized in that, The Acid Orange II antigen coating concentration was 2.0 mg / mL, and the coating amount was 0.8 μL / cm; the Rhodamine B antigen coating concentration was 0.5 mg / mL, and the coating amount was 0.8 μL / cm. The concentration of the goat anti-chicken secondary antibody coating was 1.0 mg / mL, and the coating amount was 0.8 μL / cm.

4. A probe suitable for the time-resolved immunochromatographic test strip of claim 1, characterized in that, It consists of a time-resolved fluorescent probe of Rhodamine B, a time-resolved fluorescent probe of Acid Orange II, and a time-resolved fluorescent probe of chicken IgY antibody.

5. The probe according to claim 4, characterized in that, The preparation methods for the Rhodamine B time-resolved fluorescent probe, Acid Orange II time-resolved fluorescent probe, and chicken IgY antibody time-resolved fluorescent probe are as follows: S1. Place time-resolved fluorescent microspheres in phosphate buffer, add 1-(3-dimethylpropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and react at 37°C for 30 min. S2. Centrifuge the reaction solution at 20000g for 10 min, remove the supernatant, then add the antibody and react at 37℃ for 2 h; then add the blocking solution and block at 37℃ for 1 h, then centrifuge at 20000g for 10 min, and remove the supernatant after centrifugation; The time-resolved fluorescent probes of each antibody are obtained by reconstitution with a reconstitution solution to dissolve the precipitate. The antibody is rhodamine B antibody, acid orange II antibody, or chicken IgY antibody.

6. The probe according to claim 4, characterized in that, The mass ratio of time-resolved fluorescent microspheres, 1-(3-dimethylpropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide in S1 is 50:3.5:

33.

7. The probe according to claim 4, characterized in that, In S2, the mass ratio of time-resolved fluorescent microspheres to anti-rhodamine B antibody is 1:0.02; the mass ratio of time-resolved fluorescent microspheres to acid orange II antibody is 1:0.08; and the mass ratio of time-resolved fluorescent microspheres to chicken IgY antibody is 1:0.

02.

8. The probe according to claim 4, characterized in that, The blocking solution in S2 is a 0.1 M glycine solution, which includes 1% BSA.

9. The probe according to claim 4, characterized in that, The reconstitution solution in S2 is a 0.02 M pH 7.4 phosphate buffer, which also includes 0.5% Tween-20, 1% BSA, 5% trehalose and 0.5% ProClin 300.

10. A method for simultaneously determining Rhodamine B and Acid Orange II, characterized in that, After mixing and incubating the probe according to any one of claims 4-9 with the sample to be tested, the rapid quantitative detection of Rhodamine B and Acid Orange II is performed using the time-resolved immunochromatographic test strip according to any one of claims 1-3.

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