An aptamer sensor and applications thereof

Abstract

The application belongs to the technical field of biosensors for food detection, and particularly relates to an aptamer sensor and application thereof. The aptamer sensor comprises an electrode matrix and a covalent organic framework material modified on the surface of the electrode matrix; the covalent organic framework material takes 5,10,15,20-tetra(4-carboxyphenyl)porphyrin and 4',4''-(1,3,5-triazine-2,4,6-triazyl)triphenylamine as a building unit, and is prepared by a solvothermal method through a condensation reaction; the covalent organic framework material surface is combined with aptamers for specific recognition of mycotoxins; and the electrode matrix is an SPR sensor matrix and / or an ECL sensor matrix. The SPR-ECL dual-mode aptamer sensor developed by the application shows a wide linear range and an ultra-low detection limit when detecting FB1, and can realize high-sensitivity and high-selectivity detection of mycotoxins in food.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology for food testing, specifically relating to an aptamer sensor and its application. Background Technology

[0002] Fumonisins are among the most harmful mycotoxins produced by *Fusarium verticillatum* and *Fusarium oxysporum*, damaging liver and kidney function and causing equine leukomalacia and pulmonary edema in pigs. Fumonisin B1 (FB1) is the most toxic and prevalent of these mycotoxins, exhibiting immunotoxicity, neurotoxicity, hepatic toxicity, and nephrotoxicity. Therefore, accurate and sensitive detection of FB1 is crucial to mitigating potential health hazards in livestock and humans. To date, various methods have been developed to detect FB1 in grains and foods. Compared to chromatographic and spectroscopic methods, which often suffer from drawbacks such as cumbersome sample preparation, time-consuming processes, high costs, poor selectivity, low sensitivity, and the need for expensive instruments, biosensing strategies offer advantages such as rapid response, low cost, feasibility, on-site operation, and high selectivity and sensitivity. Therefore, various biosensors have been constructed to detect FB1 by coupling with different technologies, including ECL, optical techniques, flumetochemical immunoassay, electrochemical methods, photoelectrochemical methods, colorimetry, and SPR. While extensive research has been conducted on developing biosensors for the sensitive determination of FB1 in foods or grains, most studies have focused on developing single-mode biosensing strategies. Fumonisin contamination in corn and grains has very low permissible limits, and the food matrix is ​​complex and may coexist with various mycotoxins. A single signal may result in false positives due to the complex matrix, thus limiting the accuracy of target analyte analysis. Dual-mode biosensors, which cross-verify through two signals, have the potential for accurate FB1 detection, but have not yet been reported.

[0003] As early as 1998, Liu et al. used SPR gold chips as a platform for detecting FB1 by adsorbing polyclonal FB1 antibodies, achieving a detection limit of 50 ng / mL. Subsequently, researchers developed various SPR biosensors, including immunosensors and aptamer sensors, for sensitive analysis of mycotoxins. Compared with SPR immunosensors constructed using polyclonal or monoclonal antibodies as probes, SPR aptamer sensors have advantages such as low cost, high selectivity, and structural feasibility, thus showing great potential for mycotoxin detection. However, the detection limits of current SPR aptamer sensors used for mycotoxin analysis are typically at the pg / mL level. Even though researchers have explored some signal amplification methods, it is still difficult to detect ultra-low levels of mycotoxins in food. Therefore, there is an urgent need to find a novel SPR biological platform that can not only transduce the SPR response but also significantly improve the sensitivity of constructed SPR aptamer sensors.

[0004] Furthermore, the construction of ECL aptamer sensors for the detection of mycotoxins has also attracted researchers' interest. ECL biosensing technology combines chemiluminescence and electrochemistry, offering advantages such as rapid response, high sensitivity, wide dynamic range, low background, and simple equipment. However, research on the detection of FB1 using ECL biosensors is still scarce. To construct advanced ECL aptamer sensors, ECL luminescent materials with high luminescence capabilities are crucial for enhancing the sensor's ability to detect analytes. External radiation (such as light) can induce collective oscillations (SPR) of conduction electrons on the surface of noble metal nanoparticles (especially gold or silver nanoparticles), significantly enhancing the electromagnetic field in their vicinity under appropriate stimulation. Based on this surface plasmon effect, the performance of ECL biosensors can also be improved [LIANG Z, ZHANG Q, NIE Y, et al. Polarized-Electrochemiluminescence Biosensor Based on Surface PlasmonCoupling Strategy and Fluorine-Doped BN Quantum Dots [J]. Analytical Chemistry, 2020]. Due to their superior performance, SPR-based biological platforms can simultaneously function as ECL luminescent materials to construct ECL aptamer sensors, thereby significantly improving the detection sensitivity and response performance for FB1. Therefore, developing an SPR-ECL dual-mode aptamer sensing platform can provide a solution for the efficient detection of FB1, demonstrating significant application potential in the field of rapid food safety detection.

[0005] Due to their high specific surface area, unique adsorption properties, ordered channel structure, inherent chemical and thermal stability, and strong affinity for probes, covalent organic frameworks (COFs) have been used to construct sensing platforms. Researchers have developed various COF-based sensors for detecting food contaminants, such as antibiotics, food additives, and mycotoxins. Among numerous COFs, porphyrin-based COFs exhibit enhanced light absorption and promoted charge transfer, thus demonstrating high ECL emission capabilities [WANG C, XIONG Z, FENG X, et al. Novel pore confinement enhancedeuropium functionalized porphyrin covalent organic framework electrochemiluminescence microreactor and its application to microRNA-21 detection [J]. Sensors and Actuators B: Chemical, 2024]. Simultaneously, π-π stacked electroactive porphyrin molecules possess excellent light absorption and can couple with SPR [WANG Y, MAO Z, CHEN Q, et al. Rapid and sensitive detection of PD-L1 exosomes using Cu-TCPP 2D MOF as a SPRsensitizer [J]. Biosensors and Bioelectronics, 2022]. Therefore, it is promising to utilize porphyrin-based COFs as a bifunctional biological platform to construct SPR-ECL aptamer sensors, achieving highly sensitive and selective detection of FB1. Summary of the Invention

[0006] The first objective of this invention is to provide an aptamer sensor to address the problems of low sensitivity and poor selectivity in existing biosensors for detecting mycotoxins.

[0007] A second objective of this invention is to provide the application of the aforementioned aptamer sensor in single-mode or dual-mode detection of mycotoxins using SPR / ECL.

[0008] To achieve the above objectives, the technical solution adopted by the aptamer sensor in this invention is as follows:

[0009] An aptamer sensor includes an electrode substrate and a covalent organic framework material modified on the surface of the electrode substrate; the covalent organic framework material is prepared by a solvothermal method via a condensation reaction using 5,10,15,20-tetra(4-carboxyphenyl)porphyrin and 4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine as building blocks; the surface of the covalent organic framework material is bound with aptamers for the specific recognition of mycotoxins; the electrode substrate is an SPR sensor substrate and / or an ECL sensor substrate, thereby forming a single-mode aptamer sensor or a dual-mode aptamer sensor.

[0010] By integrating electron donor (D) and electron acceptor (A) units into the structure of COFs, their structures can be made more regular, effectively enhancing intermolecular π-π interactions, regulating the optical band gap of COFs, and improving carrier mobility. This invention uses a photoelectro-active porphyrin-based COF with a DA conjugated structure (TCPP-TPT-COF) as a biological platform to develop a bifunctional SPR-ECL aptamer biosensing strategy for the first time. This opens a new path for developing COF-based SPR and its auxiliary enhancement of ECL biosensing technologies, enabling highly sensitive and selective detection of mycotoxins in food.

[0011] Preferably, the molar ratio of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin and 4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine is (1.8~2.2):1.

[0012] The photoelectro-active porphyrin-based COF (TCPP-TPT-COF) with a DA conjugated structure, developed in this invention using 5,10,15,20-tetra(4-carboxyphenyl)porphyrin (TCPP) and 4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine (TPT) as raw materials, exhibits excellent light absorption efficiency and photogenerated carrier mobility. It can couple with a gold film to generate an enhanced SPR response and is also available in S2O8. 2- The presence of porphyrin-based COFs enhances the luminescence ability of ECL. Furthermore, the versatility, high porosity, large specific surface area, and regular pore structure of porphyrin-based COFs endow them with strong affinity and the ability to immobilize a large number of aptamers, which helps to enhance the sensitivity and selectivity of SPR-ECL aptamer sensors.

[0013] Preferably, the holding temperature of the solvothermal method is 140~180 ℃, and the holding time is 60~96 hours.

[0014] Preferably, the solvent used in the solvothermal method is a mixed solvent of m-xylene and 1,4-dioxane, and the catalyst used is glacial acetic acid.

[0015] Specifically, the volume ratio of intermediate xylene to 1,4-dioxane in the mixed solvent is 6:(9~10), and the glacial acetic acid is added to the mixed solvent in the form of a glacial acetic acid solution with a concentration of 6 M, and the amount added is 10~12% of the volume of the mixed solvent.

[0016] Preferably, the surface bonding is achieved by attaching an amino-modified aptamer and a carboxyl group on a covalent organic framework material to the electrode substrate via a condensation reaction.

[0017] The TCPP-TPT-COF developed in this invention has the characteristics of multifunctionality, high porosity, large specific surface area and regular channels. These characteristics endow it with strong affinity and the ability to fix a large number of aptamers, which helps to enhance the sensitivity and selectivity of SPR-ECL aptamer sensors.

[0018] Preferably, the nucleotide sequence of the aptamer is shown in SEQ ID NO.1; the 5' end of the aptamer is attached with an NH2-(CH2)6- group.

[0019] The TCPP-TPT-COF developed in this invention has -COOH attached to its surface, which can be linked to amino-modified aptamers.

[0020] Specifically, the mycotoxin is fumonisin B1.

[0021] The aptamer sensor of the present invention has an aptamer attached to its surface that can specifically recognize fumonisin B1. The aptamer specifically binds to fumonisin B1 and has good selectivity.

[0022] To achieve the above objectives, the technical solution adopted by the aptamer sensor in the application of SPR / ECL single-mode or dual-mode detection of mycotoxins in this invention is as follows:

[0023] The above-mentioned aptamer sensor is used in SPR / ECL single-mode or dual-mode detection of mycotoxins.

[0024] Specifically, the mycotoxin is fumonisin B1.

[0025] The SPR-ECL dual-mode aptamer sensor developed based on TCPP-TPT-COF exhibits a wide linear range and an ultra-low detection limit when detecting FB1, with a LOD as low as fg / mL. Attached Figure Description

[0026] Figure 1 The image shows the surface morphology analysis results of TCPP-TPT-COF in Embodiment 1 of the present invention, where a is a low-magnification SEM image, b is a high-magnification SEM image, c, d, and e are TEM images at different magnifications, and f is an EDS elemental mapping image and an EDS spectrum.

[0027] Figure 2 The figure shows the structural characterization results of TCPP-TPT-COF in Embodiment 1 of the present invention, where a is the FT-IR spectrum and b is the XRD spectrum;

[0028] Figure 3 The X-ray photoelectron spectrum of TCPP-TPT-COF in Embodiment 1 of the present invention is shown in the figure, where a is the XPS full spectrum, b is the high-resolution C 1s spectrum, c is the high-resolution N 1s spectrum, and d is the high-resolution O 1s spectrum.

[0029] Figure 4 The graph shows the electrochemical performance analysis results of TCPP-TPT-COF in Example 1 of this invention, where a is the CV curve and b is the EIS spectrum.

[0030] Figure 5 The following are test results of the aptamer sensor in Embodiment 2 of the present invention: a is the result of detecting the RU value during the construction of the SPR sensor, b is the result of detecting the ECL intensity during the construction of the ECL sensor, c is the result of ECL intensity of different concentrations of TCPP-TPT-COF, d is the result of ECL intensity of different concentrations of aptamer, and e is the result of ECL intensity of aptamer at different binding times.

[0031] Figure 6 The graph shows the sensitivity test results of the aptamer sensor in Embodiment 3 of the present invention. Figure a shows the SPR response of the SPR aptamer sensor to different concentrations of FB1. Figure b shows the curve of the SPR response signal change (ΔRU) as a function of FB1 concentration. The inset in b is a linear fit graph of ΔRU and the logarithm of FB1 concentration, where the error bars represent the standard deviation of n=3. Figure c shows the response curve of the ECL aptamer sensor to different concentrations of FB1. Figure d shows the ECL intensity change ΔI. ECL The curve showing the change in FB1 concentration, with the inset in d representing ΔI. ECL Linear fit plot of FB1 concentration versus logarithm, where the error bars are the standard deviations of n=3;

[0032] Figure 7 The figures shown are the selective test results of the aptamer sensor in Embodiment 3 of the present invention, where a is the selective test result of the SPR aptamer sensor and b is the selective test result of the ECL aptamer sensor.

[0033] Figure 8 The figures shown are the reproducibility, stability, and regenerability test results of the aptamer sensor in Embodiment 3 of the present invention, where a is the reproducibility test result of the ECL aptamer sensor, b is the stability test result of the ECL aptamer sensor, and c is the regenerability test result of the SPR aptamer sensor. Detailed Implementation

[0034] The 5,10,15,20-tetra(4-carboxyphenyl)porphyrin (TCPP) and 4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine (TPT) used in this invention were purchased from Shanghai E. En Chemical Technology Co., Ltd. The FB1 aptamer sequence used, 5'-NH2-(CH2)6-TCTAACGTGAATGATAGATGAACTTATTCGACCATACACGTCTGCATTAACTTATTCGACCATATTCCATTACGCTAATTAACTTATTCGACCATA-3', was provided by Shanghai Sangon Biotech Co., Ltd.

[0035] The PBS buffer solution used in this invention is prepared as follows: 0.242 g potassium dihydrogen phosphate, 2.951 g disodium hydrogen phosphate, 0.2 g potassium chloride and 8.003 g sodium chloride are dissolved in deionized water, and the volume is adjusted to 1.0 L. The pH of the solution is adjusted to 7.4 with 0.1 M hydrochloric acid solution, filtered using a 0.22 μm pore size filter membrane, and stored in a refrigerator at 4°C for testing.

[0036] The preparation process of the EDC / NHS reagent used in this invention is as follows: Take 9.6 mg of EDC and 5.8 mg of NHS, disperse them in 50 mL of deionized water, stir magnetically for 3 h, and then place them in a refrigerator at 4°C for use.

[0037] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art, and the equipment and raw materials used are commercially available or commonly used in the art.

[0038] Example 1: Covalent organic framework material TCPP-TPT-COF

[0039] This embodiment provides a covalent organic framework material TCPP-TPT-COF and its preparation method, as detailed below:

[0040] Porphyrin-based COF materials were prepared via a solvothermal method using TCPP and TPT as building blocks through a condensation reaction. The molar ratio of TCPP to TPT was (1.8~2.2):1. The solvothermal method was carried out at a temperature of 140~180 °C for 60~96 hours. The solvent was a mixture of m-xylene and 1,4-dioxane (volume ratio 6:(9~10)), and the catalyst was a glacial acetic acid solution (6 M concentration), added at 10~12% of the volume of the mixed solvent. One specific preparation method is as follows:

[0041] 23.6 mg TCPP (0.04 mmol) and 7.8 mg TPT (0.02 mmol) were simultaneously dissolved in a mixed solvent of 0.8 mL m-xylene and 1.2 mL 1,4-dioxane. After sonication for 10 min until the solution was uniformly dispersed, 0.2 mL of glacial acetic acid solution (6 M) was added dropwise to the mixture. The mixture was then transferred to a pressure-resistant tube and degassed three times using a freeze-pump-thaw cycle, followed by argon gas purging and sealing. The tube was heated at 150 °C for 72 hours. After cooling to room temperature, a dark brown precipitate was obtained by centrifugation, washed several times with dichloromethane, washed three times with anhydrous ethanol, and dried overnight at 60 °C. The product was named TCPP-TPT-COF.

[0042] The surface morphology, structure, and electrochemical performance of TCPP-TPT-COF were analyzed, as follows:

[0043] (1) Surface morphology analysis of TCPP-TPT-COF

[0044] SEM images were generated by analyzing the surface morphology of TCPP-TPT-COF using scanning electron microscopy and transmission electron microscopy (e.g., Figure 1 (as shown in a, b), TEM images (such as...) Figure 1 (as shown in c, d, and e), and based on the energy and intensity of the characteristic X-rays generated by the electron beam excitation of the sample, EDS elemental mapping images and EDS spectra (as shown in c, d, and e) are generated to visualize the elemental distribution. Figure 1 (as shown in f), the results are as follows Figure 1 As shown.

[0045] SEM images clearly show that TCPP-TPT-COF is composed of stacked two-dimensional nanosheets with a size of approximately 300–800 nm. TEM images further indicate that it has numerous pores and exhibits a typical nanostructure. HR-TEM elemental mapping of TCPP-TPT-COF shows that C, N, and O elements are uniformly distributed within the nanosheets.

[0046] (2) Characterization of TCPP-TPT-COF structure

[0047] The synthesized TCPP-TPT-COF was characterized by Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD), and the results are as follows: Figure 2 As shown.

[0048] FT-IR spectrum ( Figure 2 a) at 1514 cm -1 and 1340 cm -1 The absorption peak appearing at 1600 cm⁻¹ is mainly due to the C=N stretching vibration and CN-ring stretching vibration of the triazine ring.-1 and 1450 cm -1 The absorption peak at 1627 cm⁻¹ originates from the stretching vibration absorption of the C=C skeleton of the porphyrin aromatic ring. Notably, the peak at 1627 cm⁻¹... -1 A distinct absorption peak appears at [location], corresponding to the stretching vibration peak of C=N, indicating the formation of imine bonds, thus demonstrating the successful preparation of TCPP-TPT-COF material. XRD pattern ( Figure 2 b) A weak diffraction peak appears near 2θ of 4°, while there are no obvious characteristic diffraction peaks in the range of 10~30°, which indicates that the synthesized TCPP-TPT-COF material has low crystallinity.

[0049] The chemical structure and composition of TCPP-TPT-COF were investigated using X-ray photoelectron spectroscopy (XPS), and a full XPS spectrum was generated (e.g., Figure 3 (as shown in a) and high-resolution fine spectra (as shown in a) Figure 3 (as shown in b, c, and d), the results are as follows Figure 3 As shown.

[0050] The XPS full spectrum of TCPP-TPT-COF shows binding energies (BEs) of 284.6 eV, 399.3 eV, and 532.4 eV for C 1s, N 1s, and O 1s signals, respectively. The high-resolution C 1s spectrum of TCPP-TPT-COF can be divided into five peaks located at binding energies (BEs) of 284.6, 285.1, 286.4, 288.7, and 291.1 eV, which are attributed to C–C / C=C, C–N, C=N, C=O groups, and... The high-resolution N 1s spectrum of TCPP-TPT-COF yielded three peaks: triazine N (398.4 eV), pyrrole N (399.9 eV), and oxide N (401.6 eV). The high-resolution O 1s spectrum of TCPP-TPT-COF included C=O (531.8 eV) and –OH groups (533.2 eV). These C, N, and O bonding states indicate the synthesis of TCPP-TPT-COF.

[0051] (3) Electrochemical performance analysis of TCPP-TPT-COF

[0052] The electrochemical performance of TCPP-TPT-COF was characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). In the CV experiments, TCPP-TPT-COF / AE was used as the working electrode, platinum as the counter electrode, and Ag / AgCl (saturated potassium chloride) as the reference electrode. CV curves were generated (e.g., ...). Figure 4 (As shown in a); In the EIS measurement experiment, 5 mM [Fe(CN)6] was used. 3- / 4-The experiment was performed in a PBS buffer containing 0.14 M sodium chloride and 0.1 M potassium chloride, at a potential of 0.21 V within a frequency range of 100 kHz to 0.1 Hz, to generate an EIS spectrum (e.g., Figure 4 (as shown in b), the results are as follows Figure 4 As shown.

[0053] The CV curves showed a significant decrease in the redox peak current of TCPP-TPT-COF, and the EIS spectrum showed that the electron transfer resistance (Rct) of TCPP-TPT-COF was 956 Ω. This indicates that TCPP-TPT-COF can significantly reduce the transfer of surface electrons and has relatively low electrochemical activity.

[0054] Example 2: Adaptor Sensor

[0055] This embodiment provides two aptamer sensors constructed using TCPP-TPT-COF: an SPR sensor and an ECL sensor. The successful construction of the sensors was tested, and the sensor fabrication method was optimized. The test results and optimization results are as follows: Figure 5 As shown.

[0056] The method for preparing aptamer sensors is as follows:

[0057] ① Preparation: The SPR chip used was a 15×10×0.5 mm glass chip with a ~50 nm thick gold film (Au chip, denoted as AuC) covering its surface. Before preparation, it was cleaned with cooled piranha solution (H2SO4 and H2O2, volume ratio 7:3). To protect the integrity of the gold film, 60-80 μL of the cooled piranha solution was completely applied to the surface of the gold film. After waiting for 10 seconds, the surface was immediately rinsed with deionized water to remove any remaining organic matter. This process was then repeated several times, and the surface was dried with a stream of N2 gas for further use.

[0058] ② Weigh 1 mg of TCPP-TPT-COF and disperse it in 1 mL of DMF to prepare a TCPP-TPT-COF dispersion with a concentration of 1 mg / mL. Use a pipette to add 10 μL of the TCPP-TPT-COF dispersion onto the SPR chip, and prepare the sensitive layer by spin coating to form TCPP-TPT-COF / AuC.

[0059] ③ Add 100 μL of EDC / NHS reagent to TCPP-TPT-COF / AuC and incubate statically at 4℃ for 3 h to fully activate the -COOH on the material. Then rinse the sensitive membrane surface with deionized water. After the SPR signal stabilizes, add 100 μL (10 nM) of FB1 aptamer to the -COOH activated TCPP-TPT-COF / AuC and incubate at 4℃ for 1 h. Then rinse with PBS buffer to remove unbound aptamers to obtain Apt / TCPP-TPT-COF / AuC.

[0060] ④ Incubate the obtained Apt / TCPP-TPT-COF / AuC with 10 μL of 6-mercapto-1-hexanol solution (MCH, 1.0 mM) for 10 minutes to eliminate the non-specific binding of the target to the SPR chip. Then rinse with PBS buffer. The SPR sensor is now constructed and is named MCH / Apt / TCPP-TPT-COF / AuC.

[0061] The construction steps of ECL sensors are similar, except that the SPR chip is replaced with an AE chip (gold electrode, 0.3 mm in diameter). The ECL sensor constructed using bare gold electrodes is denoted as MCH / Apt / TCPP-TPT-COF / AE.

[0062] Both the SPR and ECL sensors mentioned above require testing during fabrication to prove successful construction and normal operation. Specific testing methods are as follows:

[0063] (1) SPR sensor test

[0064] During the construction of the SPR sensor, the RU value was tested at each stage (e.g., Figure 5 (as shown in a).

[0065] This test was conducted at 25°C using PBS buffer. A baseline equilibrium was established by continuously passing PBS buffer through the SPR chip at a flow rate of 5 μL / min for 2 h. After the aptamer solution was continuously flowed through the TCPP-TPT-COF / AuC surface at a flow rate of 5 μL / min for 1080 s, the SPR signal changed by 68 RU after signal stabilization. Continued injection of MCH to block excess active sites resulted in almost no change in the SPR signal after signal stabilization. Finally, when 1 fg / mL of FB1 solution was slowly injected onto the chip surface, the RU value gradually increased. When unbound FB1 was washed away with PBS buffer, a decrease in the RU value was observed, followed by another equilibrium at 62 RU.

[0066] (2) ECL sensor testing

[0067] During the construction of the ECL sensor, the ECL strength at each stage (such as...) was tested. Figure 5 (as shown in b).

[0068] The ECL intensity of Apt / TCPP-TPT-COF / AE decreased from 20267 au to 19356 au compared to TCPP-TPT-COF / AE. This is because TCPP-TPT-COF has a nanosheet-like structure and a porous network structure, allowing a large number of aptamer chains to pass through. Adsorption and fixation occur on the surface and internal pores of TCPP-TPT-COF through stacking, electrostatic interactions, van der Waals forces, and hydrogen bonding. The active sites in TCPP-TPT-COF are occupied by aptamer chains, resulting in a significant reduction in electron transfer and luminescence efficiency of Apt / TCPP-TPT-COF / AE, which in turn leads to a decrease in ECL conversion efficiency.

[0069] Subsequently, when blocked by MCH, the ECL intensity of MCH / Apt / TCPP-TPT-COF / AE further decreased, indicating that MCH blocked excess active sites on the TCPP-TPT-COF sensitive layer. Since most sites on TCPP-TPT-COF / AE are bound to the aptamer chain, only a small amount of MCH was adsorbed by TCPP-TPT-COF.

[0070] When FB1 is detected, the Aptamer-FB1 complex is formed between the aptamer chains of FB1 and FB1, which further hinders the transfer of electrons at the electrode / electrolyte interface, resulting in a further decrease in the ECL response signal of FB1 / MCH / Apt / TCPP-TPT-COF / AE.

[0071] The above results show that both the SPR aptamer sensor and the ECL aptamer sensor were successfully constructed, and TCPP-TPT-COF can be used as a sensitive layer for constructing both the SPR aptamer sensor and the ECL aptamer sensor.

[0072] The fabrication methods for the SPR and ECL sensors described above have been optimized. Taking the ECL aptamer sensor as an example, the optimization process is as follows:

[0073] (1) TCPP-TPT-COF concentration

[0074] Under magnetic stirring, 2 mg of TCPP-TPT-COF was added to 1 mL of deionized water to prepare a TCPP-TPT-COF suspension with a concentration of 2 mg / mL. Then, the TCPP-TPT-COF suspension was diluted to different concentrations (0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL), and its ECL signal was tested (e.g., ...). Figure 5 (As shown in c). As the concentration of TCPP-TPT-COF suspension increased from 0.2 mg / mL to 1 mg / mL, its ECL signal response increased. When the concentration of TCPP-TPT-COF exceeded 1 mg / mL, the ECL signal decreased slightly.

[0075] (2) Aptamer concentration

[0076] TCPP-TPT-COF / AE was incubated with FB1 aptamer solutions (1, 5, 10, 20, and 50 nM) at 4 °C for 1 h to ensure the probe molecules were adsorbed and immobilized on TCPP-TPT-COF. Then, non-specific adsorption was eliminated using the immobilization blocking agent MCH to construct an ECL sensor, which was used to detect FB1. The response of the ECL sensor to FB1 was tested (e.g., ...). Figure 5 (As shown in d). The response to FB1 is greatest at an aptamer solution concentration of 10 nM, indicating that the number of aptamer chains is most immobilized at a concentration of 10 nM. Therefore, the optimal concentration of the aptamer is 10 nM.

[0077] (3) aptamer binding time

[0078] FB1 was detected using an ECL sensor constructed after optimization of TCPP-TPT-COF dosage and aptamer concentration. Figure 5 As shown in e), when the binding time is in the range of 15 to 60 min, its response value to FB1 becomes smaller and smaller. When the binding time is extended to 75 min, its response to FB1 is almost equivalent to the ECL signal intensity at 60 min, indicating that FB1 binding has reached saturation at 60 min.

[0079] In summary, the optimized experimental conditions are as follows: TCPP-TPT-COF material concentration of 1 mg / mL, aptamer concentration of 10 nM, and FB1 incubation time of 60 min.

[0080] Example 3: Performance Testing of the Aptamer Sensor

[0081] This embodiment tests the sensitivity, specificity, reproducibility, stability, and reproducibility of the aptamer sensor constructed in Example 2, as detailed below:

[0082] (1) SPR sensor sensitivity

[0083] The sensitivity of the SPR sensor constructed under optimized conditions was analyzed (e.g., Figure 6 (As shown in a and b). A single-cycle kinetic analysis was used to study the SPR aptamer sensor, detecting the SPR signal response of different concentrations of FB1 (1 fg / mL to 10 ng / mL). After the baseline signal stabilized, the target FB1 was injected onto the chip surface, and then unbound target FB1 was washed away with PBS buffer. Figure 6 As shown in Figure a, as different concentrations of FB1 solution are injected onto the surface of the SPR chip, the SPR signal response gradually increases, and the RU value increases from 71 to 271. Figure 6 b indicates that within the range of 1 fg / mL to 1 ng / mL, the value of ΔRU increases significantly with the increase of FB1 concentration, and the signal response of SPR gradually stabilizes when the concentration exceeds 1 ng / mL. Figure 6 The illustration shows the relationship between ΔRU and the logarithm of FB1 concentration (lgC). FB1 The relationship between ΔRU and 1gC can be seen from this. FB1 A linear relationship was observed within the range of 1 fg / mL to 10 ng / mL, and the linear regression equation was ΔRU = 30.39 lgC. AFB1 (pg / mL) +162.33, correlation coefficient (R²) 2 The limit of detection (LOD) for FB1 is 0.9839. Based on LOD = 3 SD / k (SD is the standard deviation of the blank sample, and k is the slope of the standard curve), the constructed TCPP-TPT-COF-based SPR aptamer sensor has a detection limit (LOD) of 0.28 fg / mL for FB1.

[0084] (2) ECL sensor sensitivity

[0085] The sensitivity of the ECL sensor constructed under optimized conditions was analyzed (e.g.) Figure 6 (As shown in c and d). It was used to detect different concentrations of FB1 (1 fg / mL to 10 ng / mL), and the sensitivity of FB1 detection was evaluated. Figure 6 As can be seen from c, within the range of 1 fg / mL to 10 ng / mL, the ECL response of the ECL sensor gradually decreases with increasing FB1 concentration. Analysis reveals the change in ECL intensity (ΔI). ECL The logarithm of FB1 concentration (lgC) FB1 ) exhibits a good linear relationship ( Figure 6 d). Therefore, using the linear regression equation ΔI ECL (au) = 1439.36 lgC FB1(pg / mL) + 7037.98 can be used for the quantitative detection of FB1, with a correlation coefficient (R). 2 The LOD value for FB1 detection by the ECL sensor was calculated to be 0.9827. Similarly, the LOD value for FB1 detection by the ECL sensor was calculated to be 0.32 fg / mL using the three-fold signal-to-noise ratio method.

[0086] Compared to previously reported biosensors for FB1 detection (Table 1), the SPR-ECL dual-mode aptamer sensor based on TCPP-TPT-COF in this invention exhibits a lower limit of detection (LOD) and a wider dynamic linear range. This is mainly attributed to the high specific surface area, large pore size, regular channels, and functionalization properties of TCPP-TPT-COF, which provides abundant binding sites for aptamer chain anchoring through the COF surface and internal channels. Furthermore, the porphyrin unit and DA conjugated structure in TCPP-TPT-COF significantly enhance light-harvesting ability and accelerate photogenerated carrier transport. This synergistic effect strengthens the coupling with the plasmon polarization of the gold chip surface, while simultaneously improving the self-enhanced ECL performance. Based on the multiple advantages of TCPP-TPT-COF, the SPR-ECL dual-mode aptamer sensor developed in this invention shows significant application potential in the accurate detection of mycotoxin FB1.

[0087] Table 1. Performance comparison of different sensors for detecting FB1.

[0088]

[0089] Literature 1: WEI JJ, WANG GQ, ZHENG JY, et al. Z-scheme Cu2MoS4 / CdS / In2S3nanocages heterojunctions-based PEC aptasensor for ultrasensitive assay offumonisin B1 via signal amplification with hollow PtPd–CoSnO3 nanozyme [J]. Biosensors and Bioelectronics, 2023.

[0090] Literature 2: MAO L, JI K, YAO L, et al. Molecularly imprinted photoelectrochemical sensor for fumonisin B1 based on GO-CdS heterojunction[J]. Biosensors and Bioelectronics, 2018.

[0091] Reference 3: ZHANG W, XIONG H, CHEN M, et al. Surface-enhanced molecularly imprinted electrochemiluminescence sensor based on Ru@SiO2 for ultrasensitive detection of fumonisin B1 [J]. Biosensors and Bioelectronics, 2017.

[0092] Reference 4: DONG N, LIU S, LI Y, et al. All-in-one fabrication of a ratiometric electrochemical aptasensor with tetrahedral DNA nanostructure for fumonisin B1 detection [J]. Chemical Communications, 2023.

[0093] Reference 5: SHI Z-Y, ZHENG Y-T, ZHANG H-B, et al. DNA Electrochemical Aptasensor for Detecting Fumonisins B1 Based on Graphene and Thionine Nanocomposite [J]. Electroanalysis, 2015.

[0094] Reference 6: TARUN KUMAR D, LAKSHMI G B V S, KASHYAP D, et al. Rapid and Label-Free Electrochemical Detection of Fumonisin-B1 Using Microfluidic Biosensing Platform Based on Ag-CeO2 Nanocomposite [J]. Journal of The Electrochemical Society, 2021.

[0095] Document 7: YANG X, ZHOU X, ZHANG

[0096] Literature 8: MUNAWAR H, SMOLINSKA-KEMPISTY K, CRUZ AG, et al. Molecularly imprinted polymer nanoparticle-based assay (MINA): application for fumonisinB1 determination [J]. The Analyst, 2018.

[0097] (3) Selectivity of FB1 detection by aptamer sensor

[0098] To evaluate the selectivity of the aptamer sensor, other biomolecules, such as LCM, ZEN, GEN, OTA, OTC, and TC, were used as interfering agents in this experiment. The concentration of the interfering agents was set at 100 fg / mL, which is 100 times the concentration of FB1 (1 fg / mL). The aptamer sensor was then used to detect different interfering agents and FB1, and the results are as follows: Figure 7 As shown.

[0099] Figure 7 The results show that the SPR response signal does not change significantly when detecting interfering substances, but causes a large change when detecting FB1. Comparing the SPR signal changes, it can be seen that the SPR signal change is less than 6 RU when detecting interfering substances, while it reaches 54 RU when detecting FB1. A comparable SPR response is obtained when detecting a mixture of FB1 and interfering substances. This is due to the specific recognition of the aptamer and FB1, as well as the small amount of non-specific adsorption between the target and the sensitive layer. These results indicate that the SPR aptamer sensor has good selectivity for FB1 even in complex environments.

[0100] Figure 7 b shows that the ECL response does not change significantly when detecting interfering substances, but it can induce a very high ECL response when detecting FB1. Furthermore, the ECL intensity for detecting mixtures is comparable to that for detecting FB1. These results demonstrate that the ECL aptamer sensor constructed in this experiment exhibits excellent selectivity.

[0101] (4) The reproducibility, stability and regenerability of FB1 detected by aptamer sensor

[0102] The reproducibility and stability evaluation results of the ECL aptamer sensor are similar to those of the SPR aptamer sensor regenerative performance evaluation results. Figure 8 As shown.

[0103] Five ECL aptamer sensors were constructed in parallel to detect FB1 (concentration of 1 fg / mL). The detection results are as follows: Figure 8 As shown in figure a, the ECL intensities of the five ECL aptamer sensors are not significantly different, and the relative standard deviation (RSD) is low, at only 1.94%. This indicates that the ECL aptamer sensors have good reproducibility.

[0104] Select one of the ECL aptamer sensors, record the sensor's ECL response to FB1 detection daily, and measure continuously for 7 days. The results are as follows: Figure 8 As shown in b, the ECL intensity of the ECL aptamer sensor was quite similar throughout the entire detection period, and the response value (ΔI) after 7 days was similar. ECL The value is 2738 au, which is 98.6% of the initial value, indicating its good time stability.

[0105] The SPR aptamer sensor underwent 8 regenerative tests. Figure 8 c) The specific implementation steps are as follows: After the SPR detection of the FB1 signal stabilizes, a 0.1 M PBS solution containing 50 nM NaOH is injected onto the chip surface for 5 min, followed by rinsing with PBS buffer (0.1 M, pH 7.4) to regenerate the chip for the next FB1 detection. The results show that the RSD for the FB1 response signal after 8 cycles is 1.16%, indicating its excellent regenerative properties.

[0106] Based on the above analysis, it can be seen that the SPR-ECL dual-mode aptamer sensor developed by this invention based on TCPP-TPT-COF has low detection limit, high selectivity, good reproducibility, stability and regenerability.

[0107] Application Example 1

[0108] To verify the practicality of the aptamer sensor of the present invention, a real sample study was conducted using the standard addition method, and the results are shown in Table 2 below.

[0109] Sample preparation: Grind corn and peanut samples into powder using PBS buffer, then soak them in methanol at 2 / 3 volume of PBS buffer. After 30 min, centrifuge the mixture for 10 min, collect the supernatant, and use it to prepare different concentrations of FB1 (1 fg / mL~100 pg / mL) for actual testing.

[0110] SPR aptamer sensor testing: PBS buffer was flowed through the chip to equilibrate the SPR aptamer sensor. Then, actual samples of different concentrations were sequentially passed through the SPR aptamer sensor, and the difference in SPR response signal ΔRU between the sensor and the blank sample was obtained. The corresponding FB1 concentration was calculated based on the calibration curve. Table 2 shows that the apparent recovery rate of the SPR aptamer sensor for detecting different concentrations of FB1 in corn samples was 95.4%–109.0%, with an RSD of 1.81%–3.64%. In peanut samples, the apparent recovery rate for FB1 detection was 98.4%–112.0%, with an RSD of 1.72%–3.26%.

[0111] ECL aptamer sensor testing: The apparent recovery rate of FB1 detection in corn samples ranged from 96.3% to 100.4%, with an RSD of 1.21% to 3.23%. The apparent recovery rate of FB1 detection in peanut samples ranged from 97.5% to 102%, with an RSD of 1.27% to 4.07%.

[0112] In summary, the TCPP-TPT-COF-based SPR-ECL dual-mode aptamer sensor demonstrates good applicability for FB1 testing in actual peanut and corn samples.

[0113] Table 2. Aptamer sensors used for FB1 detection in actual samples of corn and peanut (n = 3).

[0114]

[0115]

[0116] Leveraging the excellent photoelectric activity, self-enhanced ECL response, and high affinity for aptamers, TCPP-TPT-COF was used in this invention as both an SPR and ECL signal amplifier and a biosensing platform to successfully construct a dual-mode aptamer sensor for the sensitive detection of the mycotoxin FB1 in food samples. This DA-conjugated TCPP-TPT-COF material possesses high porosity, a regular pore structure, and abundant functional groups, which can promote photogenerated carrier migration, accelerate electron transport, and exhibit strong affinity to immobilize a large number of aptamer chains, achieving a kd value of 0.064 pM for the aptamers. These characteristics significantly enhance the detection sensitivity for FB1. The SPR-ECL dual-mode aptamer sensor exhibits ultrasensitivity in detecting FB1, with LODs of 0.28 fg / mL (SPR mode) and 0.32 fg / mL (ECL mode) over a wide linear range of 1 fg / mL to 10 ng / mL, outperforming previously reported biosensors for FB1 detection. Furthermore, the aptamer sensor of this invention exhibits high selectivity in the presence of various interfering agents (LCM, ZEN, GEN, OTA, OTC, TC), and also possesses excellent reproducibility and stability. Simultaneously, the SPR-ECL dual-mode aptamer sensor constructed in this invention can effectively detect FB1 in corn and peanut samples. This invention provides a novel strategy for developing COFs-based SPR-ECL dual-mode aptamer sensors, demonstrating significant potential in the detection of trace toxins in food.

[0117] Application Example 2

[0118] Mycotoxins in food are typically present in micrograms per kilogram or even nanograms per kilogram, which places high demands on the sensitivity of detection methods. Food contains large amounts of protein, fat, carbohydrates, pigments, etc., which can severely interfere with the detection process, potentially masking toxin signals and producing false positive results, leading to misjudgment. Mycotoxins are generally not present in isolation; multiple toxins often contaminate the food simultaneously, which places even higher demands on the selectivity of sensors.

[0119] This invention develops an SPR aptamer sensor and an ECL aptamer sensor based on TCPP-TPT-COF material, and further develops an SPR-ECL dual-mode aptamer sensor. This SPR-ECL dual-mode aptamer sensor detects FB1 in the sample and simultaneously records both SPR and ECL signals. The ratio of the two signals is used for mutual correction to offset interference from environmental factors on a single signal. Through cross-validation of the SPR and ECL signals, false alarms caused by a single signal anomaly and potential false positives or false negatives are effectively avoided, resulting in higher reliability of the final detection results.

[0120] 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 them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An aptamer sensor, characterized in that, The device includes an electrode substrate and a covalent organic framework material modified on the surface of the electrode substrate. The covalent organic framework material is prepared by a solvothermal method via a condensation reaction using 5,10,15,20-tetra(4-carboxyphenyl)porphyrin and 4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine as building blocks. The surface of the covalent organic framework material is bound with aptamers for the specific recognition of mycotoxins. The electrode substrate is an SPR sensor substrate and / or an ECL sensor substrate, thereby forming a single-mode aptamer sensor or a dual-mode aptamer sensor.

2. The aptamer sensor as described in claim 1, characterized in that, The molar ratio of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin to 4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine is (1.8~2.2):

1.

3. The aptamer sensor as described in claim 1, characterized in that, The solvothermal method involves holding the material at a temperature of 140~180 ℃ for 60~96 hours.

4. The aptamer sensor as described in claim 3, characterized in that, The solvent used in the solvothermal method is a mixture of m-xylene and 1,4-dioxane, and the catalyst used is glacial acetic acid.

5. The aptamer sensor as described in claim 4, characterized in that, The volume ratio of intermediate xylene to 1,4-dioxane in the mixed solvent is 6:(9~10). The glacial acetic acid is added to the mixed solvent in the form of a glacial acetic acid solution with a concentration of 6 M and an addition amount of 10~12% of the volume of the mixed solvent.

6. The aptamer sensor as described in claim 1, characterized in that, The surface bonding is achieved by bonding the amino-modified aptamer and the carboxyl group on the covalent organic framework material to the electrode substrate via a condensation reaction.

7. The aptamer sensor as described in claim 1, characterized in that, The nucleotide sequence of the aptamer is shown in SEQ ID NO.1; the 5' end of the aptamer is attached with an NH2-(CH2)6- group.

8. The aptamer sensor as described in claim 7, characterized in that, The mycotoxin in question is fumonisin B1.

9. The application of the aptamer sensor as described in any one of claims 1 to 8 in single-mode or dual-mode detection of mycotoxins in SPR / ECL.

10. The application of the aptamer sensor as described in claim 9 in single-mode or dual-mode detection of mycotoxins in SPR / ECL, characterized in that, The mycotoxin in question is fumonisin B1.

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