Double-line ratio fluorescence competitive immunochromatographic detection device, preparation method and application thereof

Abstract

The application discloses a double-line ratio fluorescent competitive immunochromatographic detection device and a preparation method and application thereof, and relates to the technical field of immunochromatographic detection.The detection device comprises a double-specificity recognition unit mixed-labeled quenched fluorescent probe, a background fluorescent material and a solid phase substrate, the solid phase substrate is provided with a T line on which a target object binding ligand is immobilized and a C line on which a quality control ligand is immobilized; and the application also provides application of the detection device in detection of exogenous harmful substances.The application solves the technical problem of low detection sensitivity of the existing method, and adjusts the binding amount of the probe on the T line and the C line through an immune competition reaction, adjusts the quenching degree of the background fluorescence based on an internal filter effect, takes (T line quenched fluorescent signal / background fluorescent signal) / (C line quenched fluorescent signal / background fluorescent signal) as a quantitative signal, and takes the C line to participate in ratio calculation to improve the detection sensitivity, so that the application is suitable for high-sensitivity qualitative and quantitative detection of small molecule compounds and the detection sensitivity is improved.

Description

Technical Field

[0001] This invention relates to the field of immunochromatographic detection technology, specifically to a dual-line ratio fluorescence competitive immunochromatographic detection device, its preparation method, and its application. Background Technology

[0002] Competitive fluorescence immunochromatography is a simple, rapid, and non-invasive technique for on-site screening of trace small molecules. It is widely used in food safety, environmental monitoring, and other fields, enabling rapid detection of pesticides, veterinary drugs, and fungal toxins. Detection sensitivity is the core key to adapting this technology to the needs of trace small molecule detection and has been a long-standing technical bottleneck in the industry.

[0003] Traditional competitive fluorescence immunochromatography (CIM) detection methods have significant limitations in signal output, mostly relying solely on the fluorescence intensity of the T-line or the T / C ratio for quantification. This single-dimensional signal output approach cannot amplify effective signal differences, resulting in low detection sensitivity and failing to meet the detection requirements of ultra-trace small molecule targets. To address this issue, ratiometric fluorescence detection strategies have been introduced, using the intensity ratio of two fluorescence signals as the output signal. This optimizes signal stability to some extent and achieves a preliminary improvement in detection sensitivity.

[0004] However, existing ratiometric fluorescence competitive immunochromatography technology has not fundamentally overcome the sensitivity bottleneck. Its core deficiency lies in the fact that existing ratiometric fluorescence schemes still use the fluorescence ratio of a single T-line as the core quantitative signal, while the C-line only serves as a quality control function to verify the effectiveness of the detection system and does not participate in the core quantitative calculation. It is impossible to further amplify the effective signal and reduce background noise through the synergistic effect of the two-line signals. At the same time, the signal probes of existing technologies mostly use single recognition element labels, which cannot form a competitive capture relationship between the T-line and C-line for the probe. It is difficult to construct a ratiometric fluorescence output mechanism with a synergistic response of the two lines, so the potential for improving the sensitivity of ratiometric fluorescence has not been fully explored, and it still cannot meet the needs of accurate detection of ultra-trace small molecules.

[0005] To address the shortcomings of existing technologies, there is an urgent need to develop a novel competitive immunochromatographic detection method with a new signal output mode. By innovating probe labeling methods and using a dual-line ratio fluorescence calculation mechanism, this method can further overcome sensitivity bottlenecks, achieve highly sensitive and accurate detection of trace small molecule targets, and fill the gap in existing technologies. Summary of the Invention

[0006] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a dual-line ratio fluorescence competitive immunochromatographic detection device, its preparation method and application, which is particularly suitable for the high-sensitivity detection of small molecule compounds and can be widely used in the rapid detection of trace small molecule targets in fields such as food safety testing and environmental monitoring.

[0007] To address the core shortcomings of existing competitive fluorescence immunochromatographic detection technologies, such as a single signal output mode, limited improvement in ratiometric fluorescence sensitivity, and the C-line only serving a quality control function without participating in core quantitative calculations, this invention provides a highly sensitive competitive immunochromatographic detection method and device mediated by a hybrid labeled probe with dual specific recognition units. By constructing a competitive capture relationship between the T-line and C-line for the signal probes, and using the ratio of the two-line ratiometric fluorescence as a quantitative signal, this invention overcomes the sensitivity bottleneck at the detection mechanism level, achieving highly sensitive and stable detection of targets such as small molecules.

[0008] The technical solution of the present invention is as follows: A first aspect of the present invention provides a dual-line ratio fluorescence competitive immunochromatographic detection device, the dual-line ratio fluorescence competitive immunochromatographic detection device comprising a quenched fluorescent probe labeled with dual specific recognition units, a background fluorescent material and a solid substrate, wherein a T-line immobilized with a target binding ligand and a C-line immobilized with a quality control ligand are disposed on the solid substrate; The quenched fluorescent probe labeled with dual specific recognition units includes a quenched fluorescent material and a first specific recognition unit and a second specific recognition unit modified on the surface of the quenched fluorescent material. The first specific recognition unit can specifically bind to the target analyte and the target analyte binding ligand on the T line, and the second specific recognition unit can specifically bind to the quality control ligand on the C line. The background fluorescent material is immobilized on the T-line and the C-line; The background fluorescent material and the quenched fluorescent material exhibit different colors of fluorescence.

[0009] In a preferred embodiment of the present invention, the quenching fluorescent material is a material that has both fluorescence emission capability and background fluorescence quenching capability, and is selected from at least one of aggregation-induced emission microspheres, quantum dots and organic quenchers; The background fluorescent material is a nanomaterial with stable fluorescence emission capability, selected from at least one of quantum dots, fluorescent microspheres, and organic fluorescent dyes.

[0010] In a preferred embodiment of the present invention, the first specific recognition unit and the second specific recognition unit are each independently selected from at least one of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, nucleic acid aptamers, polypeptide aptamers, receptors, recombinant binding proteins, and antibody functional fragments; The target binding ligand on the T-line is at least one of the following: a natural ligand, a recombinant ligand, and a small molecule conjugated ligand, which can specifically bind to the first specific recognition unit. The quality control ligand on the C-line is an independent, specifically binding ligand that can specifically bind to the second specific recognition unit and has no cross-reaction with the target detection system.

[0011] In a preferred embodiment of the present invention, the target substance is a small molecule compound; the solid substrate is a nitrocellulose membrane, glass, or plastic substrate.

[0012] A second aspect of the present invention provides a method for preparing the aforementioned dual-line ratio fluorescence competitive immunochromatographic detection device, comprising the following steps: Quenched fluorescent material, first specific recognition unit and second specific recognition unit were added to buffer solution for reaction. After the reaction was completed, blocking solution was added for blocking. After centrifugation and reconstitution, quenched fluorescent probe with mixed labeling of dual specific recognition units was obtained. A quenched fluorescent probe labeled with a dual-specific recognition unit was sprayed onto the binding pad; Background fluorescent material was added to buffer solution, and EDC and NHSS were added for activation. After the reaction was completed, blocking solution was added for blocking, centrifugation and reconstitution were performed to obtain background fluorescent material solution. Background fluorescent material solution is added to buffer solution to obtain diluent; the diluent is mixed with target full antigen and quality control ligand respectively to obtain target full antigen solution and quality control ligand solution; the target full antigen solution and quality control ligand solution are coated onto nitrocellulose membrane to form T line and C line respectively; The sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper are assembled to obtain the dual-line ratio fluorescence competitive immunochromatographic detection device.

[0013] In a preferred embodiment of the present invention, the mass ratio of the first specific recognition unit or the second specific recognition unit to the quenching fluorescent material is 1~5:100; The mass ratio of quenched fluorescent material to background fluorescent material is 1~4:1; The mass ratio of EDC and NHSS to background fluorescent materials is 1~5:1~5:100.

[0014] In a preferred embodiment of the present invention, the concentration of background fluorescent material in the diluent is 0.4 mg / mL to 0.6 mg / mL; The concentration of the target antigen solution is 0.2 mg / mL to 0.8 mg / mL; The concentration of the quality control ligand solution is 0.2 mg / mL to 0.8 mg / mL.

[0015] A third aspect of the present invention provides the application of the aforementioned dual-line ratio fluorescence competitive immunochromatographic detection device in the detection of exogenous harmful substances, said exogenous harmful substances including at least one of pesticides, veterinary drugs and mycotoxins.

[0016] In a preferred embodiment of the present invention, the mycotoxin includes zearalenone and aflatoxin B1, and the veterinary drug includes chloramphenicol.

[0017] A fourth aspect of the present invention provides a method for detecting exogenous harmful substances in food, using the aforementioned dual-line ratio fluorescence competitive immunochromatographic detection device, wherein the exogenous harmful substances include at least one of pesticides, veterinary drugs, and mycotoxins, and includes the following steps: The target analyte is prepared into a solution, and after pretreatment, a sample solution is obtained; The sample solution was added to the dual-line ratio fluorescence competitive immunochromatographic detection device and incubated at 36℃~38℃ for 5min~15min. The detection was performed using a fluorescence immunoassay analyzer, with the detector's receiving wavelengths set to 510±25nm and 610±25nm. Qualitative detection can be performed by observing the colors of the T-line and C-line, or quantitative detection can be performed by collecting the background fluorescence intensity and quenched fluorescence intensity of the T-line and C-line.

[0018] A preferred embodiment of the present invention provides a highly sensitive competitive immunochromatographic detection method mediated by a hybrid labeled probe with dual specific recognition units, comprising the following steps: (1) Preparation of quenched fluorescent probes with dual specific recognition units: Construct quenched fluorescent probes with surfaces simultaneously modified with a first specific recognition unit and a second specific recognition unit; the first specific recognition unit can specifically bind to the target analyte and the target analyte binding ligand on the solid substrate; the second specific recognition unit can specifically bind to the quality control ligand on the solid substrate; (2) Constructing a competitive immunochromatographic detection system: The system comprises the bispecific recognition unit mixed-label quenched fluorescent probe prepared in step (1), background fluorescent material, and a solid substrate sequentially coated with a target binding ligand (T line) and an independent quality control ligand (C line) that does not cross-react with the first specific recognition unit and does not interfere with the detection of the target; the T line and the C line have a competitive capture relationship for the bispecific recognition unit mixed-label quenched fluorescent probe; (3) Perform an immune competitive reaction: After the sample to be tested is mixed with the detection system, the target analyte in the sample competes with the target analyte binding ligand on the T line to bind to the first specific recognition unit on the surface of the quenched fluorescent probe, while the second specific recognition unit on the surface of the quenched fluorescent probe binds to the quality control ligand on the C line; by competitive binding, the binding amount of the quenched fluorescent probe on the T line and the C line is controlled, thereby controlling the fluorescence quenching degree of the background fluorescent material. (4) Dual-line ratio fluorescence detection and analysis: After the detection system is excited, the ratio of quenched fluorescence to background fluorescence intensity of T line and C line is detected respectively; (T line quenched fluorescence / background fluorescence) / (C line quenched fluorescence / background fluorescence) is calculated, and this ratio is negatively correlated with the concentration of the target analyte; the dual-line synchronous ratio fluorescence change achieves highly sensitive detection of the target analyte.

[0019] In a preferred embodiment of the present invention, the quantitative detection method includes: Prepare a standard solution, and detect the standard solution using the described dual-line ratio fluorescence competitive immunochromatographic detection device. Calculate the (T) of the standard solution. R / T G ) / (C R / C G The ratio of (T) to the standard solution (T) R / T G ) / (C R / C G The ratio of the two values ​​is used as the ordinate and the concentration of the standard solution is used as the abscissa. A standard curve is plotted by fitting four parameters. The (T) of the sample to be tested R / T G ) / (C R / C G Substitute the ratio of the two values ​​into the standard curve to calculate the concentration of the target analyte. Among them, T R T represents the quenched fluorescence intensity of the T-line. G C represents the background fluorescence intensity of the T-line. R The quenched fluorescence intensity of the C-line, C G The background fluorescence intensity is represented by line C.

[0020] The fluorescence quenching mechanism of this invention is the internal filtering effect (IFE). The system controls the binding amount of the quenching fluorescent probe on the T-line and C-line, thereby quenching the fluorescence of the background fluorescent material and producing a change in the quenched fluorescence / background fluorescence ratio.

[0021] The ratio of (T-line quenched fluorescence / background fluorescence) / (C-line quenched fluorescence / background fluorescence) mentioned in step (4) is inversely proportional to the concentration of the target substance, and the detection sensitivity is significantly improved compared with the traditional detection modes of single T-line signal, T / C intensity value or single T-line ratio fluorescence.

[0022] This invention has at least one of the following beneficial effects: 1. This invention provides a highly sensitive competitive immunochromatographic detection device and method mediated by a hybrid labeled probe with dual specific recognition units. The invention uses a quenching fluorescent probe with hybrid labeled probes of dual specific recognition units as its core, constructing a competitive capture relationship between the T-line and C-line for the probe. The binding amount of the probe on the dual lines is controlled through the competitive reaction of the target analyte, thereby regulating the degree of quenching of background fluorescence based on the internal filtration effect. Finally, the quantitative signal is (T-line quenched fluorescence / background fluorescence) / (C-line quenched fluorescence / background fluorescence), which is negatively correlated with the target analyte concentration. The C-line participates in the ratio calculation to improve detection sensitivity.

[0023] 2. This invention is the first to combine a hybrid labeled probe with a dual-specific recognition unit with dual-line ratio fluorescence synergistic computation. By amplifying signal differences through a competitive capture mechanism, it breaks through the sensitivity bottleneck of existing ratio fluorescence technology at the detection mechanism level. It is suitable for highly sensitive qualitative and quantitative detection of targets such as small molecule compounds and has broad application prospects in the rapid detection of trace targets in fields such as food safety testing and environmental monitoring. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the principle of the bilinear ratio fluorescence competitive immunochromatographic detection method mediated by the hybrid labeled probes of the dual-specific recognition unit of the present invention.

[0025] Figure 2 The figures show the spectra and fluorescence intensities of the quenched fluorescent probe and the background fluorescent material. In the figure, (a) shows the absorption spectrum (red solid line) of the quenched fluorescent probe (AIEFM-R), the excitation spectrum (green dashed line) and the emission spectrum (green solid line) of the background fluorescent material (QBs-G). In the figure, (b) shows the fluorescence intensity changes of the background fluorescent material under the action of different concentrations of the quenched fluorescent probe (0 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.6 mg / mL).

[0026] Figure 3 Normalized fluorescence emission spectra of background fluorescent material (QBs-G) and quenched fluorescent probe (AIEFM-R).

[0027] Figure 4 The images show the detection results for three target objects; among them, Figure 4 (a)- Figure 4 (c) in the figure shows the fluorescence color development of the immunochromatographic test strips at different concentrations of the three target substances; Figure 4 (d)- Figure 4 In the diagram, (f) represents the IC used to detect three targets under different signal output modes. 50 Compare the bar charts.

[0028] Figure 5 When CAP is the target, the quenched fluorescence of the T and C lines (red, T) R C R ) and background fluorescence (green, T) G C G The relationship between signal intensity and CAP concentration is shown in the curve.

[0029] Figure 6 Comparison of CAP competition suppression curves under different signal output modes.

[0030] Figure 7 This is for the specificity verification of CAP detection; in the figure, (a) is the fluorescence color development diagram of test strips with different interfering substances and chloramphenicol; in the figure, (b) is the bar chart of the double-line ratio signal values ​​corresponding to each interfering substance, and the signal values ​​are expressed as (T... R / T G ) / (C R / C G )calculate.

[0031] Figure 8 Fluorescence color development images of test strips with different honey bases and different spiking concentrations. Detailed Implementation

[0032] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0033] Example 1 This embodiment provides a method for constructing a bilinear ratio fluorescence detection mode mediated by a hybrid labeled probe with dual specific recognition units and a verification method for enhancing sensitivity to small molecule targets. The specific method is as follows: (1) Selection and compatibility verification of quenched fluorescent probes and background fluorescent materials Red aggregation-induced emission microspheres (AIEFM-R) were selected as quenching fluorescent probes, and green quantum dot microspheres (QBs-G) were selected as background fluorescent materials. Both types of microspheres were prepared in-house, and the specific preparation methods are as follows: 1) Preparation of red aggregation-induced emission microspheres (AIEFM-R): Take 100 mg of PS (polystyrene microspheres), dilute with water to 10 mg / mL, and then add 4 mg of SDS (sodium dodecyl sulfate) and mix well. Weigh 10 mg of AIE dye, add it to 1 mL of tetrahydrofuran and sonicate to dissolve it. Transfer the resulting solution to a rotary evaporator flask, wash with 1 mL of tetrahydrofuran and combine the solution with the liquid in the flask, and record the weight change of the flask. Place the flask under sonication and slowly add 2.5 mL of the above-treated PS microsphere solution. Seal the flask and allow it to swell in a cool place for 2 h. Then, rotary evaporate under reduced pressure for about 25 min. Centrifuge the obtained product at 14000 g and 4 ℃ for 20 min. Redissolve the precipitate in 1 mL of alkaline water with pH 10 for 4 h. Wash three times with pure water, and finally resuspend in 2.5 mL of ultrapure water. Store at 4 ℃ for later use.

[0034] 2) Preparation of green quantum dot microspheres (QBs-G): 10 mg of quantum dots and 5 mg of PMAO (polymaleic anhydride octadecyl alcohol) were dissolved in 100 µL of chloroform and thoroughly mixed. Then, 400 µL of an aqueous solution with a concentration of 1 mg / mL SDS was added. The mixture was then sonicated in an ultrasonic cell disruptor for 2 min (96 W, 9.9 s operation, 5 s pause). The resulting emulsion was then transferred to a distillation flask and evaporated under reduced pressure for 30 min using a rotary evaporator. The product was centrifuged at 15000 rpm for 15 min, and the precipitate was redissolved in 1 mL of alkaline water with a pH of 10 and treated for 4 h. The precipitate was then washed three times with pure water and finally resuspended in 1 mL of ultrapure water and stored at 4 °C for later use.

[0035] After preparation, the spectral characteristics of both were measured using a fluorescence spectrometer to verify the feasibility of quenching. Specifically, Figure 2 Based on: Figure 2 In the image (a), the absorption spectrum of the quenching fluorescent probe (AIEFM-R), the excitation spectrum of the background fluorescent material (QBs-G), and the emission spectrum are shown. It can be seen that the two spectra have good overlap, which provides a spectroscopic basis for the quenching mechanism of the internal filter effect (IFE). Figure 2 (b) shows the fluorescence intensity changes of the background fluorescent material under the action of different concentrations of quenching fluorescent probes (0 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.6 mg / mL). As the concentration of the quenching fluorescent probe increases, the fluorescence intensity of the background fluorescent material gradually decreases, proving that the quenching fluorescent probe can effectively quench the background fluorescence, and the quenching effect is concentration-dependent, which meets the requirements of the detection system of this invention. Therefore, this set of probes is determined to be used in subsequent experiments.

[0036] (2) Preparation of immunochromatographic test strips and establishment of detection methods ① Preparation of dual-specific recognition unit hybrid labeled quenched fluorescent probe 1) Add 0.6 mL of 0.01 mol / L MOPS (3-(N-morpholino)propanesulfonic acid) (pH 7.0) buffer to 6 μL of AIEFM-R (10 mg / mL) dispersion; 2) Simultaneously add 1 / 10 volume of bispecific recognition unit to the reaction system, including the target monoclonal antibody and the quality control system digoxin monoclonal antibody. The final concentrations of each antibody in the reaction system are: chloramphenicol monoclonal antibody 20 μg / mg, zearalenone monoclonal antibody 15 μg / mg, aflatoxin B1 monoclonal antibody 30 μg / mg, and digoxin monoclonal antibody 7.5 μg / mg (based on AIEFM-R mass). The reaction is carried out at 37℃ and 150 r / min on a shaker for 20 min. 3) Blocking: Add 60 μL of blocking solution (10% BSA) dropwise and react in a shaker at 37℃ and 150 r / min for 40 min; 4) Centrifugation and reconstitution: Centrifuge at 14000 r / min for 15 min, and reconstitute with the reconstitution solution (0.01 M PB buffer (pH 6.0) containing 10% sucrose and 1% BSA) to 4 mL to prepare the labeled antibody complex; 5) The labeled antibody complex was sprayed onto the conjugate pad using a synovial gold spraying machine, dried at 50°C for 24 h, and then stored in a dry environment at room temperature for later use.

[0037] ② Preparation of background fluorescent materials 1) Add 5 mL of 0.01 mol / L MOPS (pH 7.0) buffer to 100 μL of QBs-G (10 mg / mL) dispersion; 2) Activation: Add a certain volume of EDC and NHSS to make the final concentration of EDC and NHSS in the reaction system 25 μg / mg (calculated based on QBs-G mass), and react in a shaker at 37℃ and 150 r / min for 15 min. 3) Blocking: Add 1 mL of blocking solution (10% BSA) dropwise and react in a shaker at 37℃ and 150 r / min for 40 min; 4) Centrifugation and reconstitution: Centrifuge at 14000 r / min for 15 min, reconstitute with 0.01 M PB buffer (pH 6.0) containing 10% sucrose and 1% BSA to 2 mL (0.5 mg / mL) and store at 4 ℃ for later use.

[0038] ③ Coating of nitrocellulose membrane The target antigen (chloramphenicol antigen, zearalenone antigen, or aflatoxin B1 antigen) and the independent control ligand (digoxigenin antigen) were coated onto nitrocellulose membranes to create the detection line and control line, respectively. The concentrations of the coatings were adjusted to 0.5 mg / mL for the detection line and 0.3 mg / mL for the control line using 0.01 mol / L phosphate buffered solution (PBS, pH 7.4, containing 0.05 mg / mL background fluorescent material), with a coating volume of 0.74 μL / cm and a 6 mm gap between the two zones. After drying at 50°C for 24 h, the membranes were stored at room temperature in an air-dry environment for later use.

[0039] ④ Assembly of test strips The following components are sequentially overlapped and glued onto the base plate: a sample pad, a binding pad sprayed with labeled antibody complex, a nitrocellulose membrane coated with the target antigen as the detection line and digoxin antigen as the control line, and absorbent paper to form the large plate of the immunochromatographic test strip. Then, it is cut into 3.0 mm wide pieces to assemble the immunochromatographic test strip for evaluation.

[0040] ⑤ Detection methods Add 90 μL of the test buffer solution to the sample well of the test card, incubate at 37°C for 10 min, and then perform detection using a fluorescence immunoassay analyzer. Figure 3 Explanation of the selection of the detector's receiving wavelength: Figure 3 The study specifies that the emission wavelengths of the two probes are 519 nm (background fluorescent material QBs-G, green fluorescence) and 627 nm (quenched fluorescent probe AIEFM-R, red fluorescence). Therefore, the detector receiving wavelengths are set to 510 ± 25 nm and 610 ± 25 nm, respectively, to collect the green background fluorescence intensity and red quenched fluorescence intensity of the T line and C line for subsequent ratio calculation.

[0041] Figure 1The diagram illustrates the principle of the dual-line ratio fluorescence competitive immunochromatographic detection method mediated by a hybrid labeled probe with a dual-specificity recognition unit according to the present invention. The principle is as follows: after mixing the sample solution with the labeled probe, the mixture is chromatographically analyzed along the test strip. The target analyte competes with the antigen coated on the T line for binding to the quenched fluorescent probe. For qualitative interpretation, the positive or negative result is determined by observing the fluorescence color changes of the T and C lines: when the sample does not contain the target analyte, the quenched probe binds extensively to the T line, resulting in strong quenching fluorescence and weak background fluorescence, appearing as red; while the quenched probe binds less to the C line, resulting in strong background fluorescence and weak quenching fluorescence, appearing as green. When the sample contains the target analyte, the binding of the quenched probe to the T line decreases, increasing the background fluorescence and appearing as green; the binding of the quenched probe to the C line increases, increasing the quenching fluorescence and appearing as red, thus achieving the positive or negative result. For quantitative detection, the doublet ratio (T0) is calculated by collecting the T-line quenched fluorescence and background fluorescence signals, and the C-line quenched fluorescence and background fluorescence signals. R / T G ) / (C R / C G The negative correlation between this ratio and the concentration of the target analyte is used to achieve accurate quantification by combining it with a standard curve.

[0042] (3) Comparison of sensitivity of different detection targets and mechanism analysis Standard solutions of zearalenone (ZEN), aflatoxin B1 (AFB1), and chloramphenicol (CAP) at different concentration gradients were prepared. The corresponding test strips and detection methods prepared above were used for detection. The results were obtained using traditional single-line fluorescent T-line assays. R Single T-line ratio fluorescence T R / C R Traditional T / C strength value T R / C G And the bilinear ratio fluorescence ratio (T) of the present invention R / T G ) / (C R / C G Four standard curves were plotted with the target analyte concentration on the x-axis and the target analyte concentration on the y-axis. These curves were used for quantitative detection of the target analyte and to compare the sensitivity of four signal output modes. Among them, T... R T represents the quenched fluorescence intensity of the T-line. G C represents the background fluorescence intensity of the T-line. R The quenched fluorescence intensity of the C-line, C G The background fluorescence intensity of the C line, combined with Figure 4 , Figure 5 and Figure 6 Analysis of detection sensitivity and improvement mechanism: ①Sensitivity verification Figure 4 (a)- Figure 4 (c) shows the fluorescence chromatograms of the immunochromatographic test strips with different concentrations of the target analytes (ZEN, AFB1, CAP). Figure 4 (d)- Figure 4 In the diagram, (f) represents the IC under different signal output modes for detecting three targets (ZEN, AFB1, CAP). 50 Compare the bar charts. (From...) Figure 4 (a)- Figure 4 As shown in (c), with increasing target concentration, the red quenched fluorescence intensity of line T gradually decreases while the fluorescence intensity against the green background gradually recovers; conversely, the red quenched fluorescence intensity of line C gradually increases while the fluorescence intensity against the green background gradually decreases, demonstrating a clear and distinguishable difference in fluorescence color development. Figure 4 (d)- Figure 4 As can be seen from (f) in the figure, the dual-line ratio fluorescence mode of the present invention is effective for the IC of ZEN, AFB1, and CAP. 50 The values ​​were 0.642 ng / mL, 0.046 ng / mL, and 0.014 ng / mL, respectively. The sensitivity was 1.476~2.223 times higher than that of the traditional single T-line ratio fluorescence mode (1.427 ng / mL, 0.068 ng / mL, 0.027 ng / mL), 2.405~2.693 times higher than that of the traditional T / C value mode (1.729 ng / mL, 0.111 ng / mL, 0.035 ng / mL), and 3.595~6.491 times higher than that of the traditional single T-line mode (4.167 ng / mL, 0.165 ng / mL, 0.082 ng / mL). This demonstrates that the detection mode of the present invention has significantly higher sensitivity than the traditional mode.

[0043] ②Analysis of the sensitivity enhancement mechanism Figure 5 When CAP is the target, the quenched fluorescence of the T and C lines (red, T) R C R ) and background fluorescence (green, T) G C G The curve showing the relationship between signal intensity and CAP concentration. Figure 5 It can be seen that as the CAP concentration increases, the background fluorescence signal of the T line (T) decreases. G ) and C-line quenched fluorescence signal (C R The fluorescence signal gradually increases, while the T-line quenches the fluorescence signal (T...). R ) and C-line background fluorescence signal (C G As the fluorescence intensity gradually decreases, the fluorescence signals of the T and C lines exhibit a significant inverse response characteristic.

[0044] To quantitatively elucidate the sensitivity differences among different signal output modes of this invention, the relative sensitivity of the signal is defined as the absolute value of the logarithmic derivative of the signal value with respect to the target concentration: in, d represents the relative sensitivity of signal S, d represents the differential operator (differentiation), S represents the signal value of any signal output mode, and C represents the concentration of the target substance.

[0045] based on Figure 5 The observed fluorescence signal variation patterns are used to theoretically derive the sensitivity of the four signal output modes, as detailed below: 1. Single T-line signal mode (T) R ): in, This indicates the relative sensitivity of a single T-line signal mode. Represents the differential operator (differentiation). The signal value represents any signal output mode. T represents the concentration of the target analyte. R This indicates the quenched fluorescence intensity of the T-line.

[0046] This mode relies on a single fluorescence signal response and has the lowest sensitivity.

[0047] 2. Single T-line ratio pattern (T) R / T G ): in, T represents the relative sensitivity of a single T-line ratio mode. R T represents the quenched fluorescence intensity of the T-line. G S(T) represents the background fluorescence intensity of the T line. R S(T) represents the relative sensitivity of a single T-line quenching fluorescence signal. G () indicates the relative sensitivity of the T-line background fluorescence signal.

[0048] This mode achieves a single-stage sensitivity amplification through the ratio effect of the two types of fluorescence signals in the T-line, resulting in a sensitivity superior to the single T-line signal mode.

[0049] 3. Traditional T / C fluorescence ratio mode (T R / C R ): in, This indicates the relative sensitivity of the traditional T / C fluorescence ratio mode; Indicates the quenched fluorescence intensity of the T-line; Indicates the quenched fluorescence intensity of the C-line; Indicates the relative sensitivity of T-line quenching of fluorescence signal; This indicates the relative sensitivity of the C-line to quench the fluorescence signal.

[0050] This mode achieves a single-step amplification of sensitivity through the ratio effect of T-line and C-line quenching fluorescence signals, with sensitivity comparable to the single T-line ratio mode.

[0051] 4. The bilinear ratio mode of this invention ((T) R / C R ) / (T G / C G ): Wherein, S represents the relative sensitivity of the bilinear ratio mode of the present invention; Indicates the quenched fluorescence intensity of the T-line; Indicates the background fluorescence intensity of the T-line; Indicates the quenched fluorescence intensity of the C-line; Indicates the background fluorescence intensity of the C line; , , , These represent the relative sensitivity of the corresponding signals.

[0052] This mode achieves linear superposition of the sensitivities of four fluorescence signals, and realizes secondary amplification of sensitivity through the dual ratio effect, resulting in the optimal theoretical sensitivity.

[0053] From the above mathematical derivation, a clear theoretical conclusion can be drawn: among the four signal output modes, the theoretical sensitivity of the dual-line ratio mode of this invention is significantly better than the other three modes, while the sensitivity of the single T-line ratio mode and the traditional T / C fluorescence ratio mode are on the same order of magnitude, both significantly higher than the single T-line signal mode. Based on the above fluorescence signal variation law and theoretical derivation results, this invention constructs (T... R / T G ) / (C R / C G The dual-line ratio signal output mode effectively increases the slope of the competitive inhibition curve by using the dual amplification effect of the T-line quenched fluorescence / background fluorescence ratio and the C-line quenched fluorescence / background fluorescence ratio, further amplifying the signal response difference and achieving the high-sensitivity detection effect expected by theoretical derivation.

[0054] To verify the scientific validity and accuracy of the above theoretical derivation conclusions, through... Figure 6 Experimental verification was performed using standard suppression curves of CAP under different signal output modes. Here, B / B0 is the standardized signal value, and the concentration corresponding to B / B0 = 0.5 is the half-inhibition concentration (IC50).50 IC 50 The smaller the value, the higher the detection sensitivity. Experimental results show that the actual sensitivity ranking of the four signal output modes is: dual-line ratio mode > single T-line ratio mode ≈ traditional T / C fluorescence ratio mode > single T-line signal mode, which is completely consistent with the sensitivity ranking derived from the aforementioned theoretical derivation. Furthermore, the dual-line ratio mode of this invention can achieve 50% signal suppression under lower CAP concentration conditions, with the largest slope of the fitting curve and the widest dynamic detection range. The above experimental results fully verify the correctness of the theoretical derivation conclusions, confirming that the dual-line ratio amplification mechanism constructed in this invention can effectively improve detection sensitivity, which is completely consistent with theoretical expectations.

[0055] Experimental results show that the dual-line ratio fluorescence detection mode of this invention is not limited by the type of target and can significantly improve the sensitivity of small molecule compounds (AFB1, ZEN, CAP) with different structures, demonstrating good versatility. As can be seen from the detection data and fluorescence colorimetric characteristics, this system has the potential for quantitative detection with low detection limits and the advantage of qualitative interpretation with clear differences in fluorescence colorimetry. It can meet the dual needs of ultra-trace precise quantification of small molecule targets and rapid on-site qualitative screening in the field of food safety.

[0056] Example 2 This embodiment provides a method for the specific detection and spiked recovery verification of chloramphenicol (CAP) in honey samples based on double-line ratio fluorescence immunochromatography. The specific method is as follows: (1) Specificity verification Figure 7 ) Common veterinary drug residues were selected as potential interfering substances, including thiamphenicol (TAP), florfenicol (FF), amantadine (AMD), metronidazole (MNZ), dimetridazole (DMZ), azithromycin (AZM), trimethoprim (TMP), tetracycline (TC), chlortetracycline (CTC), diazepam (DZP), enrofloxacin (ENR), clenbuterol (CLEN), ractopamine (RAC), salbutamol (SAL), and nicarbazine (NCB). Interfering substance solutions with a concentration of 0.25 ng / mL were prepared, and chloramphenicol standard solution with a concentration of 0.025 ng / mL was used as a positive control. Immunochromatographic detection was performed simultaneously with blank PBST buffer.

[0057] Figure 7(a) shows the fluorescence color development results of the test strips under different interfering agents: In the CAP positive sample, the competitive inhibition of the target analyte led to a reduction in the binding of the probe to the T-line antigen, resulting in a significant weakening of the quenched fluorescence signal, while the background fluorescence was not quenched, thus the T-line appeared green; simultaneously, a large number of probes flowed to the C-line and bound to the antibody, enhancing the quenched fluorescence signal and quenching the background fluorescence, thus the C-line appeared red. The results of all other interfering agent groups were consistent with the blank control group, with the probe mainly captured by the T-line antigen, resulting in a strong quenched fluorescence signal on the T-line and quenching of the background fluorescence, thus appearing red; the quenched fluorescence signal on the C-line was weak, while the background fluorescence was not quenched, thus appearing green. Figure 7 (b) in the figure represents the bilinear ratio signal value (T) corresponding to different interfering objects. R / T G ) / (C R / C G The bar chart shows that the signal values ​​of each interfering substance group are similar to those of the blank control group, with only the CAP group showing a significant decrease in signal value. These results indicate that under the experimental conditions, these interfering substances did not significantly affect the detection signal of chloramphenicol, and the method exhibits good specificity.

[0058] (2) Validation of spiked recovery of honey samples Figure 8 (and Table 1) Four honey samples with different matrices were selected from commercially available products: linden honey (Shanghai Guanshengyuan Food Co., Ltd., batch number: 090952F21), clover honey (Jiangxi Mufengyuan Biotechnology Co., Ltd., production date: 20260129), acacia honey (Jiangxi Wangshi Bee Garden Co., Ltd., production date: 20251014), and loquat honey (Chenzhou Fengshang Bee Industry Co., Ltd., production date: 20251018). Chloramphenicol standard at three concentration levels of 0.05 μg / kg, 0.1 μg / kg, and 0.2 μg / kg was added respectively. After pretreatment, the double-line ratio fluorescence immunochromatography method constructed in this study was used for detection. Three parallel experiments were set up for each concentration level. The spiked recovery rate was calculated according to formula (1), and the coefficient of variation (CV) was calculated simultaneously.

[0059] (1) The results are as follows Figure 8 As shown in Table 1.

[0060] Table 1 a The average of three measurements.

[0061] Note: Spike recovery rates between 70% and 130% are considered normal.

[0062] Figure 8Fluorescence results of test strips with different honey matrices and different spiking concentrations: As the spiking concentration increases, the T line changes from red to green and the C line changes from green to red, and the color change is consistent with the concentration gradient.

[0063] Table 1 shows the corresponding spiked recovery data. The results show that the spiked recoveries of chloramphenicol in the four honey matrices ranged from 75.74% to 126.25%, and the coefficients of variation (CV) ranged from 0.93% to 13.36%.

[0064] The above results indicate that this method can effectively detect chloramphenicol in different honey matrices, and the recovery rate and repeatability are within the acceptable range for the detection of veterinary drug residues in food. It can be used for the detection of chloramphenicol residues in actual samples of honey.

[0065] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of a dual-line ratio fluorescence competitive immunochromatographic detection device in the detection of exogenous harmful substances, characterized in that, The dual-line ratio fluorescence competitive immunochromatographic detection device includes a quenched fluorescent probe labeled with dual specific recognition units, a background fluorescent material, a solid substrate, and a binding pad. The solid substrate is provided with a T line immobilized with the full antigen of the target analyte and a C line immobilized with the quality control ligand. The quenched fluorescent probe, which is mixed-labeled with the dual-specific recognition unit, is disposed on the binding pad; The dual-specific recognition unit hybrid-labeled quenched fluorescent probe includes a quenched fluorescent material and a first specific recognition unit and a second specific recognition unit modified on the surface of the quenched fluorescent material. The first specific recognition unit can specifically bind to the target analyte and the target analyte full antigen on the T line, and the second specific recognition unit can specifically bind to the quality control ligand on the C line. The background fluorescent material and the target full antigen are mixed and then immobilized on the T line; the background fluorescent material and the quality control ligand are mixed and then immobilized on the C line. The quality control ligand on line C is an independent, specific binding ligand that can specifically bind to the second specific recognition unit and has no cross-reaction with the target detection system; The background fluorescent material and the quenched fluorescent material exhibit different colors of fluorescence; The method for quantitatively detecting exogenous harmful substances using the aforementioned dual-line ratio fluorescence competitive immunochromatographic detection device includes: Prepare a standard solution, and detect the standard solution using the described dual-line ratio fluorescence competitive immunochromatographic detection device. Calculate the (T) of the standard solution. R / T G ) / (C R / C G The ratio of (T) to the standard solution (T) R / T G ) / (C R / C G The ratio of the two values ​​is used as the ordinate and the concentration of the standard solution is used as the abscissa. A standard curve is plotted by fitting four parameters. The (T) of the sample to be tested R / T G ) / (C R / C G Substitute the ratio of the two values ​​into the standard curve to calculate the concentration of the target analyte. Among them, T R T represents the quenched fluorescence intensity of the T-line. G C represents the background fluorescence intensity of the T-line. R The quenched fluorescence intensity of the C-line, C G The background fluorescence intensity is represented by line C.

2. The application according to claim 1, characterized in that, The quenching fluorescent material is a material that combines fluorescence emission capability and background fluorescence quenching capability, and is selected from at least one of aggregation-induced emission microspheres and quantum dots; The background fluorescent material is a nanomaterial with stable fluorescence emission capability, selected from at least one of quantum dots and fluorescent microspheres.

3. The application according to claim 1, characterized in that, The first specific recognition unit and the second specific recognition unit are each independently selected from at least one of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, nucleic acid aptamers, polypeptide aptamers, receptors, recombinant binding proteins, and antibody functional fragments.

4. The application according to claim 1, characterized in that, The solid substrate is a nitrocellulose membrane; The preparation method of the dual-line ratio fluorescence competitive immunochromatographic detection device includes the following steps: Quenched fluorescent material, first specific recognition unit and second specific recognition unit were added to buffer solution for reaction. After the reaction was completed, blocking solution was added for blocking. After centrifugation and reconstitution, quenched fluorescent probe with mixed labeling of dual specific recognition units was obtained. A quenched fluorescent probe labeled with a dual-specific recognition unit was sprayed onto the binding pad; Background fluorescent material was added to buffer solution, and EDC and NHSS were added for activation. After the reaction was completed, blocking solution was added for blocking, centrifugation and reconstitution were performed to obtain background fluorescent material solution. Background fluorescent material solution is added to buffer solution to obtain diluent; the diluent is mixed with target full antigen and quality control ligand respectively to obtain target full antigen solution and quality control ligand solution; the target full antigen solution and quality control ligand solution are coated onto nitrocellulose membrane to form T line and C line respectively; The sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper are assembled to obtain the dual-line ratio fluorescence competitive immunochromatographic detection device.

5. The application according to claim 4, characterized in that, The mass ratio of the first or second specific recognition unit to the quenched fluorescent material is 1~5:100; The mass ratio of quenched fluorescent material to background fluorescent material is 1~4:1; The mass ratio of EDC, NHSS to background fluorescent material is 1~5:1~5:

100.

6. The application according to claim 4, characterized in that, The concentration of background fluorescent material in the diluent was 0.4 mg / mL to 0.6 mg / mL; The concentration of the target antigen solution is 0.2 mg / mL to 0.8 mg / mL; The concentration of the quality control ligand solution is 0.2 mg / mL to 0.8 mg / mL.

7. The application according to claim 1, characterized in that, The exogenous harmful substances include at least one of pesticides, veterinary drugs, and fungal toxins.

8. The application according to claim 7, characterized in that, The fungal toxins include zearalenone and aflatoxin B1, and the veterinary drug includes chloramphenicol.

9. The application according to claim 1, characterized in that, The method for detecting exogenous harmful substances in food using the aforementioned dual-line ratio fluorescence competitive immunochromatographic detection device includes the following steps: The target analyte is prepared into a solution, and after pretreatment, a sample solution is obtained; The sample solution was added to the dual-line ratio fluorescence competitive immunochromatographic detection device and incubated at 36℃~38℃ for 5min~15min. The detection was performed using a fluorescence immunoassay analyzer, with the detector's receiving wavelengths set to 510±25nm and 610±25nm. Qualitative detection can be performed by observing the colors of the T-line and C-line, or quantitative detection can be performed by collecting the background fluorescence intensity and quenched fluorescence intensity of the T-line and C-line.

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