A competition chemiluminescence sensor based on quartz optical fiber-ZIF-8-aptamer interface and a preparation method and application thereof

Abstract

This invention discloses a competitive chemiluminescence sensor based on a quartz fiber-ZIF-8-aptamer interface, its preparation method, and its application in the detection of aflatoxin B1, belonging to the field of food safety detection and chemiluminescence sensing technology. The invention first involves ultrasonic cleaning of the quartz fiber with deionized water, oxygen plasma activation, and APTES silanization treatment to introduce amino groups onto the fiber surface. Then, carboxylated ZIF-8 is chemically covalently fixed using EDC / NHS to form a high specific surface area three-dimensional micro / nano interface. Next, the remaining carboxyl groups on the ZIF-8 surface are reactivated, and the AFB1 amino aptamer is covalently coupled to the ZIF-8-fiber interface. After BSA sealing, a competitive chemiluminescence sensing probe is constructed. This invention has advantages such as high sensitivity, strong specificity, low cost, and simple operation, and can be widely used for rapid screening of aflatoxin B1 and other mycotoxins in grains and foods.

Description

Technical Field

[0001] This invention belongs to the fields of food safety detection, chemiluminescence sensing and interface materials technology, specifically relating to a competitive chemiluminescence sensor based on a quartz fiber-ZIF-8-aptamer interface, its preparation method and application. Background Technology

[0002] Fungal toxins (such as aflatoxin B1 and AFB1) are widely found in grains and food, and are highly carcinogenic, making their highly sensitive and rapid detection crucial. Existing detection methods, such as high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA), suffer from problems such as expensive instruments and complex operation. Fiber optic chemiluminescence sensors have attracted attention due to their high sensitivity, fast response, and ease of miniaturization. However, the low amount of biomolecules immobilized on the surface of traditional optical fibers and their poor stability limit sensing performance. Metal-organic architectures (MOFs) possess high specific surface area, tunable channels, and abundant functional groups, enabling efficient loading of biorecognition molecules; aptamers, as artificial nucleic acid probes, exhibit high specificity and good stability. However, there is currently no systematic technical solution for controllable interfacial modulation of MOFs and aptamers on the surface of optical fibers, and for utilizing their synergistic enhancement of chemiluminescence signals. Summary of the Invention

[0003] The purpose of this invention is to provide a competitive chemiluminescence sensor based on a quartz optical fiber-ZIF-8 aptamer interface, its fabrication method, and its applications, to address the problems of complex operation, expensive instruments, insufficient sensitivity, and low biomolecule immobilization and poor stability on the optical fiber surface in existing fungal toxin detection methods. This invention achieves low-cost, high-sensitivity, and high-specificity detection of aflatoxin B1 (AFB1) by constructing a covalently immobilized ZIF-8 aptamer three-dimensional micro / nano interface.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a competitive chemiluminescence sensor based on a quartz fiber-ZIF-8-aptamer interface. The sensor comprises: (1) a quartz fiber substrate: serving as a carrier for optical signal transmission and sensing interface; (2) carboxylated ZIF-8 (ZIF-8-COOH): covalently fixed to the surface of the quartz fiber via amide bonds to form a three-dimensional micro / nano structure with a high specific surface area; and (3) an AFB1 amino aptamer: covalently coupled to the ZIF-8 surface via amide bonds as a specific recognition element.

[0005] Furthermore, the surface of the quartz optical fiber substrate is activated by oxygen plasma and treated with APTES silanization to introduce amino functional groups. The ZIF-8-COOH is activated by EDC / NHS and then fixed by forming amide bonds with the amino groups on the optical fiber surface; the AFB1 amino aptamer is covalently coupled by amide bonds after reactivating the remaining carboxyl groups on the ZIF-8 surface.

[0006] The method for preparing the sensor includes the following steps: S1: Surface pretreatment and amination modification of quartz optical fiber: S1.1: Fix the quartz optical fiber, remove the plastic coating of the sensing area using wire strippers, ultrasonically clean it in deionized water for 2 minutes, and dry it with nitrogen.

[0007] S1.2: Place the cleaned and dried optical fiber in a plasma cleaner and treat it for 5 minutes with oxygen as the working gas (flow rate 30 sccm, pressure 0.3 mbar, power 100 W).

[0008] S1.3: The optical fiber treated with oxygen plasma was immediately immersed in an APTES silanization solution, which was prepared by mixing 2 mL of APTES, 98 mL of anhydrous ethanol and 200 μL of glacial acetic acid, and incubated at 25°C in the dark for 2 h.

[0009] S1.4: After the reaction is complete, the optical fiber is taken out and washed three times with anhydrous ethanol and twice with ultrapure water. It is then dried at 25°C overnight for 12-16 hours to obtain surface-amino-aluminized silica optical fiber.

[0010] S2: Pretreatment, activation, and covalent fixation of carboxylated ZIF-8: S2.1: Take 200 μL of ZIF-8-COOH dispersion with a concentration of 2 mg / mL, vortex for 15 s; add 1 mL of MES buffer (pH 5.0), vortex to mix, centrifuge in a high-speed centrifuge for 10 min and discard the supernatant; wash 3 times with MES buffer.

[0011] S2.2: Resuspend the washed ZIF-8-COOH precipitate in 100 μL MES buffer (pH 5.0), add 100 μL EDC solution (10 mg / mL, dissolved in MES) and 100 μL NHS solution (10 mg / mL, dissolved in MES), and activate by shaking at 25°C in the dark for 30 min.

[0012] S2.3: Vortex the activated ZIF-8 mixture for 10 s, then completely immerse the aminated silica optical fiber in it and let it stand for 3 h at 25°C in the dark.

[0013] S2.4: Remove the optical fiber and rinse it three times with 200 μL PBST washing solution (PBS buffer containing 0.05% Tween-20); immerse the optical fiber in 1% BSA blocking solution and block it at 25℃ for 1 h; wash it three times again with PBST and dry it with nitrogen to obtain the optical fiber-ZIF-8 sensing probe.

[0014] S3: Reactivation of carboxyl groups on the surface of optical fiber-ZIF-8 and covalent fixation with aptamers.

[0015] S3.1: Mix 100 μL of MES buffer (pH 5.0), 100 μL of EDC solution (10 mg / mL) and 100 μL of NHS solution (10 mg / mL), immerse the fiber-ZIF-8 probe in the mixture, and incubate at 25°C in the dark for 30 min; after removal, wash three times with 200 μL of PBST.

[0016] S3.2: Immerse the activated optical fiber in 200 μL of AFB1 amino aptamer solution (50 nM, dissolved in PBS, pH 7.4) and coat it at 4℃ for 12-16 h.

[0017] S3.3: Remove the optical fiber and wash it three times with 200 μL PBST; then immerse it in 200 μL 1% BSA blocking solution and incubate it at 37℃ for 30 min; after removing it, wash it three times with PBST and dry it with nitrogen to obtain the optical fiber-ZIF-8-aptamer sensor.

[0018] The method for using the sensor in detecting aflatoxin B1 includes the following steps: (1) Indirect competitive immune reaction: AFB1 standard solutions of different concentrations (0, 0.25, 0.5, 1, 5, 10 ng / mL) and AFB1-OVA complete antigen solution with a fixed concentration of 1 μg / mL were prepared using PBS buffer. 100 μL of AFB1 solution and 100 μL of AFB1-OVA solution were mixed and the fiber-ZIF-8-aptamer sensor was completely immersed in the mixture and incubated in a constant temperature water bath at 30℃ for 30 min, with gentle shaking every 10 min. After the competition, the fiber was removed and rinsed 5 times with 200 μL of PBST washing solution.

[0019] (2) Antibody cascade amplification: The optical fiber after the competitive reaction was immersed in 100 μL of rabbit anti-OVA polyclonal antibody solution (1 μg / mL, diluted with PBS) and incubated at 30℃ for 30 min; after removal, it was washed 5 times with 200 μL PBST. Then the optical fiber was immersed in 100 μL of HRP-labeled goat anti-rabbit IgG solution (400 ng / mL, diluted with PBS) and incubated at 37℃ for 30 min; after removal, it was washed 5 times with 200 μL PBST.

[0020] (3) Chemiluminescence detection and quantification: 200 μL of a chemiluminescence substrate mixture was added to a centrifuge tube. The substrate was a Tris-HCl buffer solution (pH 8.5) containing 2 mM luminol, 0.5 mM p-iodophenol, and 2 mM H2O2. An optical fiber was immersed in the substrate solution, and the chemiluminescence signal was acquired using a photomultiplier tube or fiber optic spectrometer under completely dark conditions. The luminescence intensity was recorded. The luminescence intensity was negatively correlated with the AFB1 concentration. A standard curve was plotted, and the AFB1 content in the sample was calculated.

[0021] The performance evaluation and optimization include: conducting specificity evaluation, repeatability experiments, stability experiments, and spike recovery experiments on actual stored grain samples to evaluate the specificity of the constructed sensor. Furthermore, the effects of different MOF materials (such as MIL-101, UiO-66), different aptamer sequences, and different fiber types (plastic fiber, hollow fiber) on sensor performance can be investigated to determine the optimal combination of material parameters.

[0022] Compared with the prior art, the beneficial effects of the present invention are: (1) The interface is firmly fixed and highly stable: Through oxygen plasma activation, APTES silanization and EDC / NHS-mediated amide reaction, ZIF-8 and aptamer are covalently fixed on the surface of the optical fiber, which significantly improves the stability and service life of the sensing interface.

[0023] (2) Significantly improved sensitivity: ZIF-8 has a high specific surface area and abundant carboxyl functional groups, which can greatly increase the fixation density of aptamers. At the same time, the ZIF-8-aptamer interface has a synergistic enhancement effect on chemiluminescence reaction, and the detection limit can reach below 0.1 ng / mL.

[0024] (3) High specificity: Aptamers, as recognition elements, have excellent specific recognition capabilities. Combined with a competitive immune response system, they can effectively distinguish between target toxins and non-target substances.

[0025] (4) Low cost and easy operation: aptamers can be obtained through chemical synthesis, which is cheaper than traditional antibodies; the sensor preparation process is simple and the detection process does not require complicated instruments, making it suitable for rapid on-site screening.

[0026] (5) High versatility: The interface regulation strategy of the present invention can be extended to the detection of other fungal toxins (such as AFB2, AFG1, AFG2, etc.) or other small molecule targets, which can be achieved by changing the corresponding aptamer.

[0027] (6) Convenient signal acquisition: The chemiluminescence signal is acquired by direct coupling of optical fiber to photomultiplier tube or optical fiber spectrometer, which has the characteristics of real-time, in-situ and high signal-to-noise ratio. Attached Figure Description

[0028] Figure 1 The fabrication process and method of a quartz fiber MOF sensor; Figure 2 The results of AFB1 detection are for ZIF-8-COOH as the solid support; Figure 3 Curve showing the effect of pH value on luminescence intensity; Figure 4 Effect curve of aptamer concentration on chemiluminescence intensity; Figure 5 Curve showing the effect of competitive binding time on chemiluminescence intensity; Figure 6 Effect curve of rabbit anti-OVA antibody concentration on chemiluminescence intensity; Figure 7 Curve showing the effect of HRP-labeled antibody concentration on chemiluminescence intensity; Figure 8 Luminescence intensity curve of AFB1 sample solution under ZIF-8-COOH support; Figure 9 AFB1 sample solution calibration under ZIF-8-COOH support. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the reagents and instruments used in the following embodiments are all commercially available conventional products.

[0030] Example 1: Fabrication of a quartz fiber-ZIF-8 aptamer sensor. Please refer to [link to documentation]. Figure 1 .

[0031] 1.1 Surface Pretreatment and Amination of Quartz Optical Fibers Select a quartz multimode optical fiber with a core diameter of 400-600 μm. Carefully remove the plastic cladding layer from approximately 2-3 cm of one end of the fiber using wire strippers, avoiding damage to the fiber core. Immerse the fiber with the cladding removed in deionized water, ultrasonically clean for 2 minutes, and then dry the surface with high-purity nitrogen gas.

[0032] Secure the cleaned and dried optical fiber to the sample holder and place it into the plasma cleaning chamber. Set the processing parameters as follows: oxygen flow rate 30 sccm, chamber pressure 0.3 mbar, RF power 100 W, and processing time 5 min. Remove the optical fiber immediately after processing.

[0033] Preparation of APTES silanization solution: 2 mL of APTES was added to 98 mL of anhydrous ethanol, mixed well, and then 200 μL of glacial acetic acid was added as a catalyst and vortexed to mix. The oxygen plasma-treated silica optical fiber was completely immersed in the APTES solution and incubated at 25°C in the dark for 2 h. After the reaction was completed, the optical fiber was removed and washed three times with anhydrous ethanol and twice with ultrapure water, and then air-dried at 25°C overnight for 12-16 h to obtain surface-aminated silica optical fiber.

[0034] 1.2 Pretreatment and activation of carboxylated ZIF-8 Take 200 μL of a 2 mg / mL ZIF-8-COOH dispersion and place it in a centrifuge tube. Vortex for 15 s to ensure thorough dispersion. Add 1 mL of MES buffer (pH 5.0), vortex for 15 s, and centrifuge at 4°C and 10,000 rpm for 10 min. Discard the supernatant. Repeat the washing procedure three times to obtain a pure ZIF-8-COOH precipitate.

[0035] Accurately weigh 10 mg each of EDC and NHS powder, and dissolve them separately in 1 mL of MES buffer (pH 5.0) to prepare an activation solution of 10 mg / mL. Resuspend the washed ZIF-8-COOH precipitate in 100 μL of MES buffer (pH 5.0), then add 100 μL of EDC solution and 100 μL of NHS solution sequentially. Vortex to mix well, and place on a ZW-A type micro-shaker for activation at 25℃ in the dark for 30 min.

[0036] 1.3 Covalent fixation of carboxylated ZIF-8 on optical fiber surface The activated ZIF-8 mixture was vortexed for 10 seconds, and the aminated silica optical fiber was completely immersed in the mixture and allowed to stand for 3 hours at 25°C in the dark. After the reaction, the optical fiber was removed and rinsed three times with 200 μL of PBST washing solution (PBS buffer containing 0.05% Tween-20). The optical fiber was then immersed in 1% BSA blocking solution and blocked at 25°C for 1 hour. After blocking, it was washed three times with PBST and dried with nitrogen to obtain the fiber-ZIF-8 sensing probe.

[0037] 1.4 Reactivation of carboxyl groups and aptamer fixation on the surface of optical fiber-ZIF-8 Mix 100 μL of MES buffer (pH 5.0), 100 μL of EDC solution (10 mg / mL), and 100 μL of NHS solution (10 mg / mL), immerse the fiber-ZIF-8 probe in the mixture, and incubate at 25°C in the dark for 30 min to reactivate the carboxyl groups on the ZIF-8 surface. After removal, wash three times with 200 μL of PBST.

[0038] The activated optical fiber was immersed in 200 μL of AFB1 amino aptamer solution (50 nM, dissolved in PBS buffer, pH 7.4) and incubated overnight at 4°C for 12–16 h. After incubation, the optical fiber was removed and washed three times with 200 μL of PBST. The optical fiber was then immersed in 200 μL of 1% BSA blocking solution and incubated at 37°C for 30 min to block the unreacted activated carboxyl groups. After blocking, the fiber was washed three times with PBST and dried under nitrogen to obtain the optical fiber-ZIF-8-aptamer sensor.

[0039] To verify the feasibility of the aptamer-antibody sandwich method based on the ZIF-8-COOH carrier for AFB1 detection, ZIF-8-COOH was used as the solid-phase carrier. Different concentrations of AFB1 reagent were detected according to the experimental method described above. AFB1 reagent at concentrations of 0, 0.25, 0.5, 1, 5, 10, and 100 ng / mL were taken and sequentially processed through aptamer coating, competitive reaction, antibody binding, and chemiluminescence detection. Chemiluminescence signals at each concentration were continuously collected for 10 minutes, and the change in luminescence intensity over time was recorded. The feasibility of the detection was verified by analyzing the differences and fluctuations in the luminescence curves corresponding to different concentrations of AFB1. For the AFB1 detection results using ZIF-8-COOH as the solid-phase carrier, please refer to [link to relevant documentation]. Figure 2 .

[0040] Example 2: Optimization of Sensor Detection Conditions To optimize the sensor's detection performance, key experimental parameters affecting chemiluminescence intensity were systematically analyzed. Optimization experiments were conducted using ZIF-8-COOH as the solid-phase support and 10 ng / mL AFB1 as the detection target.

[0041] 2.1 Optimization of pH value for carboxyl group activation on ZIF-8 surface.

[0042] The carboxyl groups on the ZIF-8-COOH surface were activated under different pH conditions (4, 5, 6, 7, 8, 9), while other conditions were the same as in Example 1. Please refer to Example 1. Figure 3The chemiluminescence intensity initially increases and then decreases with pH. At pH 4 and 5, the ZIF-8 material rapidly decomposes in acidic environments (pH < 5.5), leading to structural failure and low luminescence intensity. The luminescence intensity reaches its peak at pH 6. At pH > 6, EDC rapidly hydrolyzes and deactivates in an alkaline environment, reducing carboxyl activation efficiency and causing a gradual decrease in luminescence intensity. Considering both the pH sensitivity and carboxyl activation efficiency of the ZIF-8 material, pH 6 was determined to be the optimal activation condition.

[0043] 2.2 Optimization of aptamer concentration.

[0044] Different aptamer concentrations were set (1, 10, 50, 100, 200, 300 nM), with other conditions the same as in Example 1. Please refer to [link to Example 1]. Figure 4 The chemiluminescence intensity initially increased and then decreased with increasing aptamer concentration. The luminescence intensity reached its highest value at an aptamer concentration of 50 nM. Below 50 nM, the binding sites were unsaturated, resulting in weaker luminescence intensity. Above 50 nM, aptamers accumulated on the ZIF-8 surface due to steric hindrance, affecting the competitive binding of AFB1 and AFB1-OVA, thus decreasing the luminescence intensity. The optimal aptamer concentration was determined to be 50 nM.

[0045] 2.3 Optimization of competitive response time.

[0046] Different competition binding times were set (20, 30, 40, 50, 60, 70 min), with other conditions the same as in Example 1. Please refer to [link to example]. Figure 5 The chemiluminescence intensity initially increased and then decreased with increasing competitive binding time. The luminescence intensity reached its maximum at a competitive binding time of 40 min; when the time was less than 40 min, competitive binding was insufficient; when the time was greater than 40 min, non-specific binding increased, and the luminescence intensity decreased. The optimal competitive binding time was determined to be 40 min.

[0047] 2.4 Optimization of rabbit anti-OVA antibody concentration.

[0048] Different concentrations of rabbit anti-OVA antibody were set (0.1, 0.5, 1, 1.5, 2, 2.5 μg / mL), with other conditions the same as in Example 1. Please refer to [link to Example 1]. Figure 6 The luminescence intensity reached its maximum at an antibody concentration of 0.5 μg / mL; when the concentration was too low, the AFB1-OVA antigen site was not fully occupied; when the concentration was too high, steric hindrance occurred, hindering the effective binding of the HRP-labeled antibody. The optimal rabbit anti-OVA antibody concentration was determined to be 0.5 μg / mL.

[0049] 2.5 Optimization of HRP-labeled antibody concentration.

[0050] Different concentrations of HRP-labeled goat anti-rabbit IgG were set (100, 200, 300, 400, 500, 600 ng / mL), with other conditions the same as in Example 1. Please refer to [link to Example 1]. Figure 7 The chemiluminescence intensity increased with increasing HRP concentration, but plateaued after 200 ng / mL. Signal amplification was insufficient below 200 ng / mL; non-specific adsorption increased and background signal rose above 200 ng / mL. The optimal HRP-labeled antibody concentration was determined to be 200 ng / mL.

[0051] Example 3: Establishment of AFB1 standard curve and calculation of detection limit.

[0052] Under the optimized conditions described above (pH 6, aptamer concentration 50 nM, competitive reaction time 40 min, rabbit anti-OVA antibody concentration 0.5 μg / mL, HRP-labeled antibody concentration 200 ng / mL), different concentrations of AFB1 standard solutions (0, 0.25, 0.5, 1, 5, 10, 100 ng / mL) were detected using ZIF-8-COOH as the solid-phase support. Three replicates were set up for each experiment. Chemiluminescence signals were continuously collected for 10 min, and the change curve of luminescence intensity over time was recorded. Please refer to [link to relevant documentation]. Figure 8 .

[0053] A chemiluminescence intensity curve was constructed with AFB1 concentration on the x-axis and chemiluminescence count on the y-axis. Within the range of 0.25–100 ng / mL, the chemiluminescence intensity decreased significantly with increasing AFB1 concentration. The equation for the chemiluminescence curve was obtained by fitting the data using a four-parameter logistic regression: (1) Using the chemiluminescence value of zero-concentration AFB1 as a benchmark, the relative luminescence intensity (B / B0) of different concentrations of AFB1 was calculated, where B is the chemiluminescence count value of standard samples with different concentrations of AFB1, and B0 is the chemiluminescence count value of blank standard samples. (2) Plot a calibration curve with the logarithm of concentration on the x-axis and relative luminescence intensity on the y-axis. Please refer to [link to relevant documentation]. Figure 9 Through linear fitting, the equation of the calibration curve is obtained as follows: (3) Based on the detection limit formula LOD = 3.3δ / S (where δ is the standard deviation of the intercept and S is the slope of the standard curve), the detection limit of this sensor was calculated to be 0.058 ng / mL. The results indicate that the sensor constructed using ZIF-8-COOH as a solid-phase support to coat the aptamer exhibits excellent accuracy, linearity (R² = 0.998), and sensitivity.

[0054] Example 4: Evaluation of Sensor Specificity To evaluate the specificity of the sensor, solutions of AFB1, AFB2, AFG1, AFG2, and ochratoxin A (OTA) at concentrations of 10 ng / mL were prepared and subjected to chemiluminescence detection according to the detection method described in Example 3. The relative luminescence intensity (B / B0) of each toxin was calculated. The results showed that the relative luminescence intensity of AFB1 was significantly lower than that of other fungal toxins, indicating that the sensor has good specific recognition ability for AFB1 and no obvious cross-reactivity with AFB1 structural analogs and other common fungal toxins.

[0055] Example 5: Evaluation of Sensor Repeatability and Stability Repeatability test: Five fiber-ZIF-8 aptamer sensors prepared in the same batch were used to detect AFB1 standard solutions at concentrations of 1 ng / mL and 10 ng / mL, respectively. Each concentration was measured three times, and the relative standard deviation (RSD) was calculated. The results showed that the intra-batch RSD was ≤ 6.5% and the inter-batch RSD was ≤ 8.2%, indicating that the sensor has good repeatability.

[0056] Stability test: The prepared sensor was stored at 4℃ and removed on days 1, 3, 5, 7, 14, 21, and 28, respectively, and tested with a 10 ng / mL AFB1 standard solution. The results showed that after 28 days of storage at 4℃, the luminescence intensity of the sensor remained above 87% of its initial value, indicating that the sensor has good storage stability.

[0057] Example 6: Spike Recovery Experiment of Actual Stored Grain Samples Blank corn samples were extracted and purified, and then AFB1 standard solution was added to achieve final AFB1 concentrations of 0.5 ng / mL, 2 ng / mL, and 8 ng / mL, respectively. The detection method described in Example 3 was used for determination, with three parallel samples for each concentration. The spiked recovery rate and RSD were calculated. The results are shown in Table 1.

[0058] Table 1

[0059] The results showed that the spiked recovery rate was 92.0%–104.0% and the RSD was ≤ 6.5%. This sensor can be used for the effective detection of AFB1 in actual stored grain samples.

[0060] Example 7: Comparative Experiment of Different MOF Materials Fiber-MOFs-aptamer sensors were prepared according to the method of Example 1, using MIL-101-COOH and UiO-66-COOH respectively instead of ZIF-8-COOH, and AFB1 (10 ng / mL) was detected according to the method of Example 3. The results showed that the ZIF-8-COOH sensor produced the highest chemiluminescence intensity, the widest linear range (0.25~100 ng / mL), and the lowest detection limit (0.058 ng / mL), indicating that ZIF-8-COOH is the preferred MOF material of this invention.

[0061] Example 8: Comparative Experiment of Different Fiber Optic Types Sensors were fabricated using plastic optical fibers and hollow optical fibers instead of quartz multimode optical fibers, respectively, following the method in Example 1. Results showed that quartz multimode optical fiber exhibited the lowest optical transmission loss and the best surface modification stability, resulting in the optimal chemiluminescence signal acquisition performance, making it the preferred optical fiber substrate material for this invention.

[0062] Example 9: Comparative Experiment of Different Aptamer Sequences AFB1 amino aptamers with different sequences (Sequence A, Sequence B) were immobilized at the ZIF-8 fiber interface according to the method in Example 1. The results showed that the aptamer sequence selected in this invention (50 nM, coating at 4℃ for 12-16 h) had the highest binding efficiency and the lowest detection limit.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A competitive chemiluminescence sensor based on a quartz fiber-ZIF-8-aptamer interface, characterized in that, The sensor includes: a quartz optical fiber substrate; a carboxylated ZIF-8 (ZIF-8-COOH) covalently fixed to the surface of the quartz optical fiber; and an AFB1 amino aptamer covalently coupled to the surface of ZIF-8 via amide bonds.

2. The sensor according to claim 1, characterized in that, The surface of the quartz optical fiber substrate is activated by oxygen plasma and treated with APTES silanization to introduce amino functional groups, which are used to form amide bonds with the activated ZIF-8-COOH.

3. The sensor according to claim 1, characterized in that, After being activated by EDC / NHS, the ZIF-8-COOH undergoes an amide reaction with the amino groups on the surface of the quartz optical fiber and is covalently fixed.

4. The sensor according to claim 1, characterized in that, The AFB1 amino aptamer is covalently coupled to the ZIF-8-fiber interface via amide bonds after reactivating the remaining carboxyl groups on the ZIF-8 surface.

5. A method for manufacturing a sensor according to any one of claims 1-4, characterized in that, Includes the following steps: Pretreatment and amination modification of silica optical fiber surface; pretreatment of carboxylated ZIF-8, activation of surface carboxyl groups and covalent fixation on optical fiber surface; reactivation of residual carboxyl groups on optical fiber surface after fixation of ZIF-8; covalent coupling of AFB1 amino aptamer and closure of unreacted sites.

6. The preparation method according to claim 5, characterized in that, Step (a) specifically includes: ultrasonically cleaning the quartz optical fiber with the cladding removed in deionized water for 2 min and then drying it; after oxygen plasma treatment (30 sccm, 0.3 mbar, 100 W, 5 min), immediately immersing it in APTES silanization solution (2 mL APTES + 98 mL anhydrous ethanol + 200 μL glacial acetic acid) and incubating it at 25°C in the dark for 2 h; washing it three times with anhydrous ethanol and twice with ultrapure water, and then air-drying it at 25°C overnight for 12-16 h.

7. The preparation method according to claim 5, characterized in that, The activation and immobilization of carboxylated ZIF-8 in step (b) specifically includes: taking 200 μL of ZIF-8-COOH dispersion with a concentration of 2 mg / mL, washing it 3 times with MES buffer (pH 5.0) and resuspending it in 100 μL of MES; adding 100 μL of EDC solution (10 mg / mL) and 100 μL of NHS solution (10 mg / mL), and activating it by shaking at 25°C in the dark for 30 min; immersing the aminated silica optical fiber in the activated ZIF-8 mixture and soaking it at 25°C in the dark for 3 h; taking it out and rinsing it 3 times with PBST washing solution, then immersing it in 1% BSA blocking solution and blocking it at 25°C for 1 h, washing it and drying it with nitrogen.

8. A method for detecting aflatoxin B1 using the sensor according to any one of claims 1-4, characterized in that, Includes the following steps: Optical fibers immobilized with aptamers were immersed in mixtures containing different concentrations of AFB1 standard solutions and a fixed concentration of AFB1-OVA complete antigen to conduct indirect competitive reactions. The optical fibers after the competitive reactions were then incubated sequentially with rabbit anti-OVA polyclonal antibody and HRP-labeled goat anti-rabbit IgG. The incubated optical fibers were then immersed in a chemiluminescent substrate mixture, and chemiluminescent signals were collected under light-protected conditions. Based on the negative correlation between luminescence intensity and AFB1 concentration, a standard curve was plotted, and the AFB1 content in the test sample was calculated.

9. The detection method according to claim 8, characterized in that, The chemiluminescent substrate mixture solution is a Tris-HCl buffer solution containing 2 mM luminol, 0.5 mM p-iodophenol and 2 mM H2O2, with a pH of 8.5; the luminescence signal is acquired using a photomultiplier tube or a fiber optic spectrometer.

10. The application of the sensor according to any one of claims 1-4 or the detection method according to any one of claims 8-9 in the detection of mycotoxins, characterized in that, The fungal toxins include aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, and other small molecule fungal toxins that can specifically bind to aptamers.

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