Three-mode immunochromatography detection system based on bowl-shaped COF

Abstract

The invention relates to the technical field of immunodetection, and discloses a tri-modal immunochromatography detection system based on bowl-shaped COF, the tri-modal immunochromatography detection system comprises a signal marker ABCPP, an immune probe, an LFIA test strip and a signal detection module; the signal marker ABCPP is composed of a bowl-shaped covalent organic framework, an aggregation-induced luminophor and mesoporous palladium-platinum nanoparticles. According to the invention, fluorescence enhancement of AIEgens and colorimetric and photothermal effects of mPdPt NPs are creatively integrated on a bowl-shaped COF single platform; the bowl-shaped COF not only serves as an optical cage to remarkably enhance fluorescence emission of BTDTA through space limitation, but also serves as optical energy gathering to enhance interaction between light and substances. In addition, energy transfer between the internal AIGens and the external mPdPt NPs synergistically amplifies the photothermal effect, so that the photothermal conversion efficiency is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and more specifically, to a three-modal immunochromatographic assay system based on a bowl-shaped COF. Background Technology

[0002] Lateral flow immunoassay (LFIA) has been widely used in on-site diagnostics and food safety monitoring due to its advantages such as ease of operation, rapid detection, and low cost. Signal markers, as the core component of LFIA, directly determine the sensitivity, accuracy, and stability of the detection. Traditional signal markers, such as gold nanoparticles, can only achieve single colorimetric readings with limited sensitivity; while fluorescent quantum dots improve detection sensitivity, they suffer from aggregation-induced quenching problems; existing multifunctional nanolabeled materials generally face challenges such as signal crosstalk, matrix effect interference, and insufficient bioconjugation stability, making it difficult to meet the needs of accurate detection of low-concentration target analytes in complex samples.

[0003] Polymerization-induced emission (AIEgens) possess the unique characteristic of enhanced fluorescence during polymerization, overcoming the aggregation-quenching defects of traditional fluorophores and becoming highly promising candidate materials for signal labeling. Among them, BTDTA molecules exhibit both significant AIE effects and two-photon absorption capabilities, resulting in excellent light-harvesting performance. However, their optical behavior is highly dependent on the aggregated state and local environment; without appropriate host structure confinement, nonradiative decay severely weakens luminescence efficiency. Covalent organic frameworks (COFs) offer advantages such as crystal order and tunable porosity, but their spherical structure has limited effect on confinement and light enhancement of AIEgens. In contrast, the concave cavity of bowl-shaped COFs (BCs) can simultaneously achieve spatial confinement of AIEgens molecules and enhance photo-matter interactions.

[0004] Therefore, providing a three-modal immunochromatographic detection system based on a bowl-shaped COF has significant practical implications. Summary of the Invention

[0005] In view of this, the present invention proposes a three-modal immunochromatographic detection system based on a bowl-shaped COF, aiming to solve at least one of the problems in the current background art.

[0006] This invention proposes a three-modal immunochromatographic detection system based on a bowl-shaped COF, the three-modal immunochromatographic detection system comprising: Signal markers ABCPP, immune probes, LFIA test strips, and signal detection modules; The signal marker ABCPP is composed of a bowl-shaped covalent organic framework (BC), an aggregation-induced emission polymer (BTDTA), and mesoporous palladium-platinum nanoparticles (mPdPt NPs). Preferably, the aggregation-induced light-emitting polymer (BTDTA) is encapsulated in a concave cavity of a bowl-shaped covalent organic framework (BC), and mesoporous palladium-platinum nanoparticles (mPdPt NPs) are modified on the surface of the bowl-shaped covalent organic framework (BC).

[0007] Preferably, the preparation steps of the bowl-shaped covalent organic framework include: 1,3,5-Tris(4-aminophenyl)benzene (TAPB) and 2,5-dimethoxybenzene-1,4-dicarboxaldehyde (DMTP) were dissolved in acetonitrile and mixed to obtain a mixture; Add glacial acetic acid to the mixture, stir, and then collect the first product by centrifugation; The first product was dispersed in DMF, and HAc, ACN solution and 3% hydrogen peroxide aqueous solution were added sequentially. The mixture was placed at 60°C and allowed to stand for 10 minutes. After centrifugation, the bowl-shaped covalent organic framework was obtained.

[0008] Preferably, the preparation steps of the signal marker ABCPP include: Dissolve the bowl-shaped covalent organic framework in water to obtain an aqueous solution of the bowl-shaped covalent organic framework; The aggregation-induced emission factor (BTDTA) solution was mixed with the F-127 solution to obtain a mixture; The mixture was subjected to ultrasonic treatment, and the aqueous solution of the bowl-shaped covalent organic framework was added during the ultrasonic treatment. A bowl-shaped COF based on AIEgen was obtained under a nitrogen flow. Na2PdCl4, H2PtCl6, HCl and F-127 were added to the AIEgen-based bowl-shaped COF. After reacting for a period of time, ascorbic acid was added, and the mixture was continuously sonicated at 45°C for 3 hours. After cooling and washing, the signal marker ABCPP was obtained.

[0009] Preferably, the preparation steps of the immune probe include: The signal marker ABCPP was incubated with anti-NPAOZ antibody for 1 hour, then BSA solution was added and incubated for 30 minutes. The immune probe was then purified by centrifugation.

[0010] Preferably, the LFIA test strip comprises: a PVC board, a sample pad, a nitrocellulose membrane, and an absorbent pad; The sample pads were pretreated by soaking in a blocking buffer containing 2% BSA, 1% PVP K30 and 0.5% Tween-20 before assembly, and then dried for 6 hours. The nitrocellulose membrane forms a detection line (T line) by spotting CPAOZ-BSA and a control line (C line) by spotting anti-mouse IgG. The sample pad, nitrocellulose membrane, and absorbent pad are sequentially laminated onto a PVC board with a 2 mm overlap between adjacent layers. The assembled sheet is then cut into 3 mm wide strips to obtain the LFIA test strip.

[0011] Preferably, the signal detection module includes a smartphone for colorimetric signal detection, a 365nm ultraviolet lamp and ImageJ quantitative analysis software for fluorescence signal detection, and an 808nm near-infrared laser and a portable infrared thermal imager for photothermal signal detection.

[0012] The present invention also provides the application of the detection system described in any of the above technical solutions, for the rapid on-site detection of nitrofuran metabolites, antibiotic residues, biomarkers in biological samples, and 3-amino-2-oxazolidinone in food.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Trimodal signal output and synergistic enhancement: This invention creatively integrates the fluorescence enhancement of AIEgens, the colorimetric effect of mPdPtNPs, and the photothermal effect into a single platform called a bowl-shaped COF. The bowl-shaped COF not only acts as an optical cage to significantly enhance the fluorescence emission of BTDTA through spatial confinement, but also acts as an optical energy concentrator to enhance the interaction between light and matter. In addition, the energy transfer between the internal AIEgens and the external mPdPt NPs synergistically amplifies the photothermal effect, significantly improving its photothermal conversion efficiency.

[0014] (2) High sensitivity and ultra-low detection limit: Due to the multi-mode signal synergistic amplification, the immunochromatographic detection system constructed in this invention exhibits extremely high sensitivity to the target AOZ. Moreover, the three detection modes complement and verify each other, effectively avoiding false positive or false negative results caused by a single signal due to the complex sample matrix, and significantly improving the accuracy and reliability of detection.

[0015] (3) The preparation method is controllable and the structure is stable: The components of this invention, such as signal markers, have clear preparation steps and mild conditions. The resulting nanostructures are uniform and stable, and are easy to be efficiently coupled with antibodies, making them suitable for large-scale preparation and application. Attached Figure Description

[0016] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 SEM and TEM images of spherical covalent organic frameworks (COFs); Figure 2 XRD patterns of spherical covalent organic framework (COF) and bowl-shaped covalent organic framework (BC); Figure 3 FT-IR plots of TAPB and DMTP, spherical covalent organic framework (COF), and bowl-shaped covalent organic framework (BC); Figure 4 Zeta diagrams of spherical covalent organic frameworks (COF) and bowl-shaped covalent organic frameworks (BC); Figure 5 The UV / Vis absorption spectra of MB under different conditions; Figure 6 TEM image of mesoporous palladium-platinum nanoparticles mPdPt NPs; Figure 7 Characterization diagram of ABCPP-mAb; Figure 8 The optimization results of ABCPP-CM-LFIA are shown in the figure. Figure 9 The optimization results of ABCPP-FL-LFIA are shown in the figure. Figure 10 The diagram shows the preparation and characterization of ABCPP. Figure 11 The graph shows the fluorescence performance test results of ABC and ABCPP. Figure 12 The image shows the preliminary test results of the photothermal performance of ABCPP. Figure 13 The image shows the in-depth test results of the photothermal performance of ABCPP. Figure 14 The graph shows the analytical performance test results of ABCPP-LFIA. Detailed Implementation

[0017] 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. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0018] Furthermore, regarding the 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 within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] 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.

[0020] 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 apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] 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.

[0022] In this invention, the abbreviations of the various substances are as follows: the bowl-shaped covalent organic framework is abbreviated as BC, the aggregation-induced emission body is abbreviated as BTDTA, the mesoporous palladium-platinum nanoparticles are abbreviated as mPdPt NPs, and the spherical covalent organic framework is abbreviated as COF.

[0023] This invention proposes a three-modal immunochromatographic detection system based on a bowl-shaped COF, the three-modal immunochromatographic detection system comprising: Signal markers ABCPP, immune probes, LFIA test strips, and signal detection modules; The signal marker ABCPP is composed of a bowl-shaped covalent organic framework (BC), an aggregation-induced emission polymer (BTDTA), and mesoporous palladium-platinum nanoparticles (mPdPt NPs). In this invention, the aggregation-induced light-emitting polymer (BTDTA) is encapsulated in the concave cavity of a bowl-shaped covalent organic framework (BC), and mesoporous palladium-platinum nanoparticles (mPdPt NPs) are modified on the surface of the bowl-shaped covalent organic framework (BC).

[0024] In this invention, the preparation steps of the bowl-shaped covalent organic framework preferably include: 1,3,5-Tris(4-aminophenyl)benzene (TAPB) and 2,5-dimethoxybenzene-1,4-dicarboxaldehyde (DMTP) were dissolved in acetonitrile and mixed to obtain a mixture; Add glacial acetic acid to the mixture, stir, and then collect the first product by centrifugation; The first product was dispersed in DMF, and HAc, ACN solution and 3% hydrogen peroxide aqueous solution were added sequentially. The mixture was placed at 60°C and allowed to stand for 10 minutes. After centrifugation, the bowl-shaped covalent organic framework was obtained.

[0025] A further preferred method for preparing the bowl-shaped covalent organic framework is as follows: 0.02 mmol TAPB and 0.03 mmol DMTP are completely dissolved in 10 mL acetonitrile. Then, 0.25 mL glacial acetic acid is added to the mixture, and the mixture is stirred in a fume hood at room temperature until the solvent has largely evaporated. The product is collected by centrifugation and washed three times with deionized water. Subsequently, an oxidation strategy is used to convert the product into a nanobowl structure. The specific steps of this process are as follows: First, the product is dispersed in 10 mL DMF, and then 0.5 mL HAc and 0.2 mL ACN solution (containing 50 mg / mL) are added sequentially. -1 FeCl3) and 2 mL of 3% hydrogen peroxide aqueous solution were placed in a 60℃ incubator and allowed to stand for 10 minutes. The product was then collected by centrifugation and washed three times with ethanol.

[0026] In this invention, the preparation steps of the signal marker ABCPP preferably include: Dissolve the bowl-shaped covalent organic framework in water to obtain an aqueous solution of the bowl-shaped covalent organic framework; The BTDTA solution was mixed with the F-127 solution to obtain a mixture; The mixture was subjected to ultrasonic treatment, and the aqueous solution of the bowl-shaped covalent organic framework was added during the ultrasonic treatment. A bowl-shaped COF based on AIEgen was obtained under a nitrogen flow. Na2PdCl4, H2PtCl6, HCl and F-127 were added to the AIEgen-based bowl-shaped COF. After reacting for a period of time, ascorbic acid was added, and the mixture was continuously sonicated at 45°C for 3 hours. After cooling and washing, the signal marker ABCPP was obtained.

[0027] A further preferred method for preparing the signal marker ABCPP is as follows: BC is dispersed in 10 mL of deionized water. 200 µL of 1 mg / mL water is then added. -1 BTDTA solution with 240µL 10mg mL -1The F-127 solution was mixed in a 50 mL centrifuge tube. This mixture was rapidly added to 10 mL of BC aqueous solution while being subjected to vigorous sonication for 10 minutes. After evaporation of the organic solvent under a nitrogen stream, AIEgen-based bowl-shaped COF (ABC) was obtained. A surfactant-mediated synthesis method was used to load mPdPt nanoparticles onto the AIEgen-based bowl-shaped COF (ABC). The synthesis procedure was as follows: 0.6 mL of Na₂PdCl₄ (20 mM), 3 mL of H₂PtCl₆ (20 mM), 15 µL of HCl (6 M), and 60 mg of F-127 were added sequentially to the ABC solution under sonication. After 5 minutes, 3 mL of ascorbic acid (0.1 M) was added, and the mixture was continuously sonicated at 45 °C for 3 hours. After cooling to room temperature, the sample was washed with ethanol and water to remove unreacted substances and the F-127 surfactant, and the resulting signal marker ABCPP was finally collected.

[0028] In this invention, the preparation steps of the immune probe preferably include: The signal marker ABCPP was incubated with anti-NPAOZ antibody for 1 hour, then BSA solution was added and incubated for 30 minutes. The immune probe was then purified by centrifugation.

[0029] More preferably, the immune probe is an ABCPP-mAb immune probe, and the specific preparation steps of the immune probe are as follows: 1 mL of ABCPP solution (concentration 0.6 mg / mL) is prepared. -1 ) with 4 μL of anti-NPAOZ antibody (concentration 1 mg / mL) -1 Incubate for 1 hour. Then add 100 μL of 10% BSA (w / v) solution to block unbound sites. After incubation for 30 minutes, purify the ABCPP-mAb immunoprobe by centrifugation.

[0030] In this invention, the LFIA test strip preferably comprises: a PVC board, a sample pad, a nitrocellulose membrane, and an absorbent pad; The sample pads were pretreated by soaking in a blocking buffer containing 2% BSA, 1% PVP K30 and 0.5% Tween-20 before assembly, and then dried for 6 hours. The nitrocellulose membrane forms a detection line (T line) by spotting CPAOZ-BSA and a control line (C line) by spotting anti-mouse IgG. The sample pad, nitrocellulose membrane, and absorbent pad are sequentially laminated onto a PVC board with a 2 mm overlap between adjacent layers. The assembled sheet is then cut into 3 mm wide strips to obtain the LFIA test strip.

[0031] Further preferred, specifically: the LFIA test strip consists of four parts: a PVC plate, a sample pad, a nitrocellulose (NC) membrane, and an absorbent pad. The absorbent pad is used directly with the PVC plate. Before assembly, the sample pad is pretreated by soaking in a blocking buffer containing 2% BSA, 1% PVP K30, and 0.5% Tween-20, followed by drying for 6 hours. The nitrocellulose membrane is then sampled with CPAOZ-BSA (0.2 mg / mL). -1 A detection line (T line) is formed, and anti-mouse IgG (1 mg / mL) is spotted onto the sample. -1 A control line (C line) is formed. Subsequently, the sample pad, NC film, and absorbent pad are sequentially laminated onto a PVC board, with an overlap of 2 mm between adjacent layers. The assembled sheet is then cut into 3 mm wide strips for further application.

[0032] The application of LFIA test strips in AOZ detection is as follows: After incubating ABCPP-mAb probes with different concentrations of AOZ, the analytical performance of ABCPP-LFIA was systematically evaluated. First, the colorimetric reaction was assessed visually, followed by detection using fluorescence (365nm UV lamp) and photothermal (808nm near-infrared laser) imaging techniques. Colorimetric and fluorescence images were recorded using a smartphone and quantitatively analyzed using ImageJ software; the photothermal signal was measured using a portable infrared thermal imager. In qualitative detection, the cutoff value (COV) was defined as the lowest AOZ concentration at which the T line disappeared. The visual detection limit (vLOD) was defined as the lowest AOZ concentration that produced a T line significantly weaker than the negative control.

[0033] In this invention, the signal detection module includes a smartphone for colorimetric signal detection, a 365nm ultraviolet lamp and ImageJ quantitative analysis software for fluorescence signal detection, and an 808nm near-infrared laser and a portable infrared thermal imager for photothermal signal detection.

[0034] The present invention also provides the application of the detection system described in any of the above technical solutions, for the rapid on-site detection of nitrofuran metabolites, antibiotic residues, biomarkers in biological samples, and 3-amino-2-oxazolidinone in food.

[0035] Example (1) Synthesis of bowl-shaped covalent organic framework (BC) Preparation of spherical COF(C): 0.02 mmol TAPB and 0.03 mmol DMTP were completely dissolved in 10 mL acetonitrile, 0.25 mL glacial acetic acid was added, and the mixture was stirred in a fume hood at room temperature until the solvent was basically evaporated. The product was collected by centrifugation and washed three times with deionized water. Bowl-shaped structure transformation: Disperse spherical COF in 10 mL DMF, add 0.5 mL HAc, 0.2 mL ACN solution containing 50 mg / mL FeCl3 and 2 mL 3% hydrogen peroxide aqueous solution in sequence, incubate at 60℃ for 10 minutes, collect the product by centrifugation, wash 3 times with ethanol to obtain BC.

[0036] (2) Synthesis of signal markers ABCPP Preparation of bowl-shaped COF (ABC) based on AIEgen: BC was dispersed in 10 mL of deionized water; 200 μL of 1 mg / mL BTDTA solution and 240 μL of 10 mg / mL F-127 solution were mixed and sonicated vigorously for 10 minutes while adding the BC aqueous solution. The organic solvent was evaporated under a nitrogen flow to obtain ABC. mPdPt nanoparticle loading: Under ultrasonic conditions, 0.6 mL of 20 mM Na2PdCl4, 3 mL of 20 mM H2PtCl6, 15 μL of 6 M HCl and 60 mg of F-127 were added sequentially to the ABC solution. After 5 minutes, 3 mL of 0.1 M ascorbic acid was added, and the mixture was sonicated at 45 °C for 3 hours. After cooling, the mixture was washed with ethanol and water, and the ABCPP was collected.

[0037] (3) Preparation of immune probe (ABCPP-mAb) 1 mL of 0.6 mg / mL ABCPP solution was incubated with 4 μL of 1 mg / mL anti-NPAOZ antibody for 1 hour. 100 μL of 10% BSA solution was added to block the unbound sites. The incubation was continued for 30 minutes. The ABCPP-mAb immunoprobe was obtained by centrifugation and purification. The successful conjugation was verified by zeta potential, with a conjugation efficiency of 99.87%.

[0038] (4). Preparation of LFIA test strips Sample pad pretreatment: Immerse the sample pad in blocking buffer containing 2% BSA, 1% PVP K30 and 0.5% Tween-20, and dry for 6 hours; Nitrocellulose (NC) membrane modification: 0.2 mg / mL CPAOZ-BSA was spotted using a spotting apparatus to form a detection line (T line), and 1 mg / mL anti-mouse IgG was spotted to form a control line (C line). Assembly and cutting: Lay the sample pad, NC film, and absorbent pad onto the PVC board in sequence, with adjacent layers overlapping by 2 mm, and cut into 3 mm wide test strips.

[0039] (5) Optimization of the detection system Optimize key parameters through single-factor experiments to ensure optimal detection performance: ABCPP concentrations: Optimal concentration of CM-LFIA is 0.4 mg / mL, and optimal concentration of FL-LFIA is 0.3 mg / mL; Antibody consumption: 5 μg antibody consumption is optimal for both modes; The optimal specific binding effect of CPAOZ-BSA concentration in the T-line is 0.2 mg / mL. Probe volume: A 10 μL probe loading amount can achieve a clear signal response.

[0040] Application examples 1. Sample pretreatment Standard gradient preparation: Dilute AOZ standards to a series of concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, and 2.0 ng / mL using PBS buffer; Spiking of food samples: Add different concentrations of AOZ standard to honey and shrimp samples, vortex mix, extract with PBS buffer, centrifuge and take the supernatant as the sample to be tested.

[0041] 2. Trimodal Detection Reaction incubation: Mix 100 μL of the sample to be tested with 10 μL of ABCPP-mAb probe and incubate at room temperature for 10 minutes; Test strip detection: Add the mixture to the LFIA test strip sample pad, let it stand at room temperature for 15 minutes, and then read the three-modal signal. Colorimetric mode (CM): The test strip's T / C line is photographed by a smartphone, and ImageJ is used for quantitative analysis of the band intensity; Fluorescence mode (FL): Irradiated by a 365nm ultraviolet lamp, the fluorescence image is acquired and quantified by a smartphone; Photothermal mode (PT): The T-line region is irradiated by an 808nm near-infrared laser, and the temperature change is recorded by an infrared thermal imager.

[0042] 3. Results Limits of detection (LOD): CM-LFIA 0.07 ng / mL, FL-LFIA 0.92 ng / mL, PT-LFIA 0.18 ng / mL; Visual detection limit (vLOD): CM mode 0.2 ng / mL, FL mode 0.4 ng / mL, PT mode 0.1 ng / mL; Specificity: It shows no obvious response to interfering substances such as SEM, AHD, and tetracycline, but specifically identifies AOZ; Recovery rates: In honey samples, the recoveries were 82.42%–107.12% in CM mode, 95.41%–99.81% in FL mode, and 84.58%–102.62% in PT mode; in shrimp samples, the corresponding recoveries were 82.71%–101.81%, 98.62%–98.95%, and 87.63%–99.11%, respectively, with RSDs all less than 12%.

[0043] As described above, the three-modal immunochromatographic detection system based on bowl-shaped COF constructed in this embodiment of the invention achieves high sensitivity and high specificity for AOZ detection through the synergistic signal enhancement effect of ABCPP. The three detection modes mutually validate each other, effectively avoiding false positive / false negative results, and are suitable for rapid on-site detection of AOZ in complex food matrices such as honey and shrimp.

[0044] Performance testing 1. The synthesized substances were characterized, specifically as follows: Figure 1 Images A through B are SEM and TEM images of spherical covalent organic frameworks (COFs). Figure 2 XRD patterns of spherical covalent organic framework (COF) and bowl-shaped covalent organic framework (BC); Figure 3 A~B are FT-IR diagrams of TAPB and DMTP, spherical covalent organic framework COF and bowl-shaped covalent organic framework BC, respectively; Figure 4 Zeta diagrams of spherical covalent organic frameworks (COF) and bowl-shaped covalent organic frameworks (BC); Figure 5 The UV / Vis absorption spectra of MB under different conditions; Figure 6 TEM image of mesoporous palladium-platinum nanoparticles mPdPt NPs; Figure 7 This is a characterization diagram of ABCPP-mAb, specifically... Figure 7 A shows the zeta potential distribution of the ABCPP and ABCPP-mAb immune probes; Figure 7 B is the regression equation graph of OD450 nm versus the concentration of anti-CPAOZ monoclonal antibody; Figure 7 C is a graph showing the conjugation efficiency of ABCPP at different monoclonal antibody concentrations; Figure 8 The optimization results of ABCPP-CM-LFIA are shown in the figure. Figure 8 A~D represent the concentration of ABCPP, antibody consumption, CPAOZ-BSA concentration on the T-line, and probe volume, respectively. Figure 9The graph shows the optimization results of ABCPP-FL-LFIA. Specifically, Figure 9 A~D represent the concentration of ABCPP, antibody consumption, CPAOZ-BSA concentration on the T-line, and probe volume, respectively. Figure 10 The following are the preparation and characterization diagrams of ABCPP: Figure 10 A is a flowchart of the ABCPP preparation process; Figure 10 B is a transmission electron microscope image of BC; Figure 10 C is a transmission electron microscope image of ABCPP; Figure 10 D is the scanning electron microscope image of BC; Figure 10 E is a scanning electron microscope image of ABCPP; Figure 10 F is the energy spectrum distribution of ABCPP; Figure 10 G is the energy spectrum of ABCPP; Figure 10 H is the XPS energy spectrum of ABCPP; Based on the characterization diagrams above, specifically, imine covalent organic frameworks (COFs) are first synthesized using a solvent evaporation method. Figure 1 The image shows that the obtained product C has a highly monodisperse spherical morphology with a diameter of approximately 600 nm. Powder X-ray diffraction (PXRD) pattern ( Figure 2 The high crystallinity of C was confirmed. PXRD analysis showed significant peaks at 2.89°, 4.92°, 5.67°, 7.50°, and 9.85°, indicating the formation of a pure phase. In the second step, the ·OH radicals generated by the Fenton reaction oxidized the imine bonds in C, forming a BC structure. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images ( Figure 10 B and 10D spectra show a bowl-shaped morphology with a concave diameter of approximately 300 nm and a cavity depth of approximately 600 nm. Fourier transform infrared spectroscopy (FT-IR), Zeta potential analysis, and powder X-ray diffraction (PXRD) further confirmed the structural changes. According to the FT-IR spectrum of BC, a new peak corresponding to -OH appeared at 3437 cm⁻¹, confirming the formation of -COOH. Figure 3 The zeta potential changed from 4.7 eV to -18.8 eV, confirming the above results. Figure 4 Compared to the original spherical C, the crystal strength of BC is slightly reduced. Figure 2 These results indicate that the ·OH radical partially oxidizes the imine bonds in the ordered COF framework, generating amine and carboxyl groups, leading to partial structural collapse and the formation of BC ( ). Figure 5 Subsequently, BC acts as a nanocontainer to encapsulate BTDTA molecules and anchor mPdPt nanoparticles. (For example...) Figure 10 As shown in B and 10D, mPdPt nanoparticles ( Figure 6) are evenly distributed on the BC surface.

[0045] Energy spectral mapping ( Figure 1 F) and spectrum ( Figure 1 G) confirmed the uniform distribution of C, N, O, S, palladium, and platinum in ABCPP, with corresponding atomic percentages of 31.8%, 3.4%, 9.4%, 0.6%, 6.9%, and 47.8%, respectively. X-ray photoelectron spectroscopy (XPS) characterization ( Figure 10 H) further confirms the coexistence of these elements in ABCPP.

[0046] 2. The fluorescence properties of ABC and ABCPP were tested. Performance test results are as follows Figure 11 As shown, specifically, Figure 11 A is a schematic diagram of the fluorescence enhancement mechanism; Figure 11 B is a graph showing the relative emission intensity (I / I(0)) of BTDTA at the maximum emission wavelength as a function of different water contents; Figure 11 C represents the excitation and emission spectra of ABC, AC, BC, and C; Figure 11 D represents a comparison of the fluorescence intensities of ABC, AC, BC, and C; Figure 11 E represents the excitation and emission spectra of ABCPP; Figure 11 F is the excitation-emission matrix diagram of ABCPP; Figure 11 G is the CIE chromaticity coordinate diagram of ABC and ABCPP.

[0047] The luminescent properties of BTDTA were first investigated in THF / water mixed solvents with different water contents, and the results are as follows: Figure 11 As shown in Figure B, the fluorescence intensity of BTDTA first decreases and then increases with increasing water content. This phenomenon can be attributed to the synergistic effect of intramolecular charge transfer and aggregation-induced emission. Based on this, BTDTA molecules were encapsulated in nano-confined cavities of BC and COF to construct high-performance fluorescent materials. Fluorescence spectroscopy showed that the emission intensity of ABC was 1.52 times higher than that of AC (with BTDTA molecules encapsulated in COF). Figure 11 C and 11D). This enhancement can be attributed to the synergistic effect of geometric constraints, subject-object interactions, and the inherent AIE properties of BTDTA (C and 11D). Figure 11A) The bowl-shaped structure formed by the partial collapse of the spherical COF creates a concave cavity, which spatially confines the encapsulated BTDTA molecules, effectively suppressing intramolecular motion and non-radiative decay pathways. Simultaneously, the oxidative transformation of the imine bond introduces -COOH and -NH2 groups, further immobilizing the BTDTA molecules through hydrogen bonding and electrostatic interactions. The synergistic effect of spatial confinement and host-guest interactions significantly enhances the inherent AIE effect of BTDTA, achieving fluorescence enhancement. Based on this, ABC was selected as a fluorescent nanocontainer for loading mPdPt nanoparticles to prepare the multi-signal tracer ABCPP. The fluorescence properties of ABCPP were further investigated using fluorescence spectroscopy and excitation-emission matrix spectroscopy (EEM). Figure 11 As shown in E and 11F, ABCPP exhibits bright yellow fluorescence at 580 nm and a significant Stokes shift of 230 nm. Such a large Stokes shift helps reduce background fluorescence, minimize self-quenching effects, and avoid false positives caused by backscattering of excitation light. Furthermore, the CIE chromaticity coordinates of ABCPP were determined to be (0.48304, 0.50325), located in the yellow region and close to the edge of the chromaticity map. Figure 11 (G) indicates that it has high color purity.

[0048] The above results demonstrate that the synergistic effect of the AIE effect and the nano-confinement effect endows ABCPP with excellent fluorescence properties, providing a foundation for high-performance biosensing applications.

[0049] 3. Test the photothermal properties of ABCPP. Preliminary test results of the photothermal performance of ABCPP are as follows: Figure 12 As shown, specifically: (A) Photothermal model diagram of ABCPP. (B) and (C) Photothermal heating curves and thermal images of ABCPP with different concentrations. (D) Photothermal heating curves of ABCPP under different laser power densities. (E) Four heating-cooling photothermal cycles. (F) Comparison of temperature rise amplitudes of C, BC, ABC, PP, ABCPP, BCPP and ACPP. (G) Heating curve of ABCPP. (H) Linear fitting plot of cooling time versus the negative natural logarithm (-lnθ) of temperature driving force. This invention encapsulates BTDTA molecules within a BC structure and anchors mPdPt nanoparticles on its surface, thereby enabling it to exhibit superior photothermal properties. Figure 12 A), the following experimental results clearly confirm this. Under 808 nm laser irradiation, the ABCPP solution exhibits a significant concentration dependence ( Figure 12 B and 12C) and power dependence ( Figure 12D) The heating effect highlights its potential as a quantitative photothermal label. The system also exhibits excellent stability, maintaining a nearly constant peak temperature throughout four consecutive on / off cycles, indicating its structural robustness suitable for biosensing applications. Figure 12 E). Most importantly, after 10 minutes of irradiation, ABCPP achieved a temperature rise of 73.1°C, far exceeding all control samples: COF group (C group, 0.5°C), BC group (0.5°C), ABC group (1.1°C), mPdPt (52.1°C), ACPP (mPdPt nanoparticles modified on AC) (59.4°C), and BCPP (mPdPt nanoparticles modified on BC) (58.7°C). Figure 12 F). Furthermore, the photothermal conversion efficiency (η) measured through heating-cooling curve analysis was 47.35% (F). Figure 4 (G and 4H) fully verified the optimized structural design of ABCPP and its excellent photothermal conversion capability.

[0050] The photothermal performance test results of ABCPP are as follows: Figure 13 As shown, (A) is a schematic diagram of the photothermal synergistic enhancement mechanism. (B) is a simulation of the electromagnetic distribution of ABCPP, BCPP, and ACPP under 808 nm laser irradiation. (C) is a simulation of the power absorption density of ABCPP, BCPP, and ACPP under 808 nm laser irradiation. Specifically, to gain a deeper understanding of the superior photothermal performance of ABCPP, a finite-difference time-domain (FDTD) simulation study was conducted. For example... Figure 13 As shown in Figure B, the near-field electromagnetic field of ABCPP (|E / E(0)|=16) is significantly higher than that of BCPP (|E / E(0)|=5) and ACPP (|E / E(0)|=8). Furthermore, the optical absorption power of ABCPP reaches 15 × 10⁻⁶. 12 Joules, approximately 5 times and 2 times that of BCPP and ACPP, respectively. Figure 13 C). These results are consistent with experimental observations, indicating that the superior photothermal effect of ABCPP originates from its rationally designed nanobowl-shaped structure. Specifically, the bowl-shaped COF structure acts as a highly efficient optical concentrator, trapping incident light within the cavity and extending the photon path length. This greatly enhances the light-harvesting ability of the densely packed BTDTA molecules (acting as highly efficient primary energy absorbers) within the bowl. More importantly, the nanoscale proximity between the internal BTDTA molecules and the externally deposited mPdPt nanoparticles promotes efficient energy transfer. The photon energy absorbed by AIEgens is rapidly transferred to the mPdPt alloy, which, with its strong localized surface plasmon resonance effect and efficient non-radiative attenuation characteristics, becomes an excellent heat exchanger (…). Figure 13A). This demonstrates that the synergistic effect of optical confinement, energy transfer, and plasma conversion forms the basis for the excellent photothermal performance of ABCPP.

[0051] 4. The analytical performance of ABCPP-LFIA was tested, and the results are as follows: Figure 14 As shown. Specifically, Figure 14 (A) is a schematic diagram of ABCPP-LFIA detection. Figure 14 (B) Photograph of the prototype reagent strip. Based on Figure 14 (C)CM-LFIA, Figure 14 (D) FL-LFIA and Figure 14 (E) Regression analysis and linear relationship of AOZ detection in PT-LFIA. Based on Figure 14 (F)CM-LFIA, Figure 14 (G)FL-LFIA and Figure 14 Specificity assessment of (H)PT-LFIA Immunoplasmic probes are the core component of the LFIA system and play a crucial role in the detection performance of the test strip. The ABCPP-mAb probe has been successfully synthesized and validated by measuring the zeta potential before and after antibody labeling. Furthermore, the conjugation efficiency of ABCPP was calculated to be 99.87% after adding antibody (5 μg). Leveraging the excellent signal transduction and antibody conjugation capabilities of ABCPP, an ABCPP-LFIA test strip for detecting AOZ was successfully developed. To obtain better analytical performance, key experimental parameters were first optimized. Under optimal conditions, different concentrations of AOZ (…) were analyzed using colorimetric, fluorescence, and photothermal triple readout signals via ABCPP-LFIA. Figure 14 A). In LFIA detection, vLOD, COV, and LOD are used as indicators to evaluate the detection capability of ABCPP-LFIA. Figure 14 B shows that on the ABCPP-LFIA test strip, the signal intensity of the T line gradually decreases with increasing AOZ concentration under different detection modes. The vLOD values ​​of CM-LFIA, FL-LFIA, and PT-LFIA are 0.2, 0.4, and 0.1 ng / mL, respectively. -1 The COV values ​​of CM-LFIA, FL-LFIA, and PT-LFIA were 2, 8, and 2 ng mL⁻¹, respectively. Standard curves showing good correlation between the signal intensity of the T-line and the concentration of AOZ were plotted. Figure 14(C, 13D, and 14E). The linear regression equations for CM-LFIA, FL-LFIA, and PT-LFIA on AOZ were y=17758.81-29708.02x (R2=0.99), y=21843.25-5194.69x (R2=0.99), and y=34.69-13.68x (R2=0.94), respectively. Based on these results, the detection limits for the three methods were 0.07 ng / mL (CM), 0.92 ng / mL (FL), and 0.18 ng / mL (PT), respectively. These results demonstrate that the developed ABCPP-LFIA can indeed significantly improve the performance of lateral flow immunoassay for AOZ detection.

[0052] Evaluation was conducted against common interfering agents such as aminourea (SEM), 1-aminohydantoin (AHD), and 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), as well as tetracycline (TC), neomycin (NEO), and kanamycin (Kana). Figure 14 As shown in F, 14G, and 14H, except for AOZ, the CM, FL, and PT detection signals did not produce significant responses to other interfering objects.

[0053] These results demonstrate that the ABCPP-LFIA platform achieves excellent selectivity for AOZ in all detection modes. To evaluate the practicality of ABCPP-LFIA in real food matrices, different concentrations of AOZ were added to honey and shrimp samples, and analyzed using this method. Based on colorimetric, fluorescence, and photothermal signals measured at the T-line, the average recoveries of CM in honey samples were 82.42%–107.12%, FL was 95.41%–99.81%, and PT was 84.58%–102.62%. The corresponding recoveries in shrimp samples were: CM method 82.71%–101.81%, FL method 98.62%–98.95%, and PT method 87.63%–99.11% (Table S1). These results indicate that ABCPP-LFIA has satisfactory accuracy and reliability in analyzing complex food matrices, highlighting its practicality in field and bedside detection.

[0054] In summary, this invention integrates BTDTA molecules into a bowl-shaped covalent organic framework and modifies its surface with mPdPt nanoparticles, resulting in a nanobowl-confined optical energy cascade platform. This structure transcends the functions of traditional nanocarriers, acting as an active platform that coordinates light capture, energy conversion, and signal amplification. The COF nanobowl not only acts as an "optical cage" to confine BTDTA molecules (increasing its fluorescence intensity by 1.52 times compared to spherical COF analogs), but also serves as a highly efficient optical concentrator to enhance light-matter interactions. Furthermore, the nanoscale spatial arrangement of the internal BTDTA and external mPdPt nanoparticles achieves highly efficient optical energy cascades. The photon energy captured by BTDTA is transferred to the mPdPt nanoparticles, which, combined with the absorption effect of mPdPt itself, amplifies the photothermal effect. Both experimental and theoretical analyses confirm that ABCPP achieves a photothermal conversion efficiency as high as 47.35%. This synergistic design ultimately forms a multifunctional nanolabel integrating colorimetry, fluorescence, and photothermal output. When applied to lateral flow immunoassay for AOZ detection, ABCPP-LFIA exhibits an ultra-low limit of detection of 0.07 ng mL⁻¹. Beyond food safety testing, this energy-driven nanolabel provides a new paradigm for developing high-performance multimodal biosensors, with potential applications in biomedical diagnostics and portable point-of-care testing.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A three-modal immunochromatographic test system based on bowl-shaped COF, characterized in that, The three-mode immunochromatographic detection system comprises: a signal marker ABCPP, an immunological probe, an LFIA test strip and a signal detection module; The signal marker ABCPP is composed of a bowl-shaped covalent organic framework, an aggregation-induced emission body and a mesoporous palladium-platinum nanoparticle.

2. The detection system of claim 1, wherein, The aggregation-induced emission body is encapsulated in the concave cavity of the bowl-shaped covalent organic framework, and the mesoporous palladium-platinum nanoparticle is modified on the surface of the bowl-shaped covalent organic framework.

3. The detection system of claim 1, wherein, The preparation steps of the bowl-shaped covalent organic framework comprise: 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxybenzene-1,4-diformaldehyde are dissolved in acetonitrile to obtain a mixture; glacial acetic acid is added to the mixture, and stirring is performed, and then the first product is collected by centrifugation; the first product is dispersed in DMF, HAc, ACN solution and 3% hydrogen peroxide solution are sequentially added, and the mixture is placed at a temperature of 60 DEG C for 10 minutes, and then the bowl-shaped covalent organic framework is obtained after centrifugation.

4. The detection system of claim 3, wherein, The preparation steps of the signal marker ABCPP comprise: the bowl-shaped covalent organic framework is dissolved in water to obtain a bowl-shaped covalent organic framework aqueous solution; the AIEgen solution and the F-127 solution are mixed to obtain a mixed solution; the mixed solution is subjected to ultrasonic treatment, and the bowl-shaped covalent organic framework aqueous solution is added during the ultrasonic treatment, and the AIEgen-based bowl-shaped COF is obtained under a nitrogen flow; Na2PdCl4, H2PtCl6, HCl and F-127 are added to the AIEgen-based bowl-shaped COF, ascorbic acid is added after a period of reaction, and the mixture is continuously subjected to ultrasonic treatment at a temperature of 45 DEG C for 3 hours, and then the signal marker ABCPP is obtained after cooling and washing.

5. The detection system of claim 1, wherein, The preparation steps of the immunological probe comprise: the signal marker ABCPP is incubated with anti-NPAOZ antibody for 1 hour, BSA solution is then added, and the mixture is incubated for 30 minutes, and then the immunological probe is obtained by centrifugal purification.

6. The detection system of claim 1, wherein, The LFIA test strip comprises a PVC plate, a sample pad, a nitrocellulose membrane and a water absorption pad; the sample pad is immersed in a blocking buffer containing 2% BSA, 1% PVP K30 and 0.5% Tween-20 for pretreatment before assembly, and then dried for 6 hours; the nitrocellulose membrane is formed by spotting CPAOZ-BSA to form a test line (T line) and spotting anti-mouse IgG to form a control line (C line); the sample pad, the nitrocellulose membrane and the absorption pad are laminated on the PVC plate in sequence, and the adjacent layers are overlapped by 2 mm, and then the assembled sheet is cut into a 3 mm wide strip to obtain the LFIA test strip.

7. The detection system of claim 1, wherein, The signal detection module comprises a smartphone for colorimetric signal detection, a 365 nm ultraviolet lamp for fluorescent signal detection and ImageJ quantitative analysis software, an 808 nm near-infrared laser for photothermal signal detection and a portable infrared thermal imager.

8. Use of a detection system according to any one of claims 1 to 7, characterized in that It is used for on-site rapid detection of nitrofuran metabolites in food, antibiotic residues, biomarkers in biological samples and 3-amino-2-oxazolidinone.

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