Method for constructing photocatalytic fluorescence sensor based on light-responsive nanozymes

Abstract

The present invention discloses a method for constructing a photocatalytic fluorescence sensor based on a light-responsive nanozyme. UCNPs are synthesized by a thermal decomposition method, and Fe3O4@NPC magnetic composite porous material is synthesized by a calcination method. The heterojunction structure of Fe3O4@NPC enhances the photocatalytic activity. 2,7-Dichlorodihydrofluorescein (DCFH) is selected as a highly efficient fluorescence probe for generating ROS during the assembly process. A photocatalytic fluorescence sensor for rapid and highly sensitive detection of aflatoxin B1 is constructed based on UCNPs and Fe3O4@NPC. UCNPs are used as a target-mediated optical regulator to transfer energy to Fe3O4@NPC, inducing the separation of electron-hole pairs on Fe3O4@NPC and initiating a photocatalytic oxidation reaction. The heterostructure of Fe3O4@NPC slows down the recombination rate of electron-hole pairs, promotes the generation of more ROS, oxidizes DCFH into DCF with high fluorescence intensity, thereby amplifying the fluorescence signal difference and improving the detection sensitivity, providing a relatively broad application prospect for the construction of a new type of highly sensitive photocatalytic fluorescence sensor in the future.

Description

Technical Field

[0001] The present invention belongs to the technical field of constructing a photocatalytic fluorescent sensor, and specifically relates to a method for constructing a photocatalytic fluorescent sensor based on a light-responsive nanozyme. Background Art

[0002] With the rapid development of nanotechnology, fluorescence sensors based on FRET technology have been widely used for rapid and sensitive analysis of selected targets. In traditional FRET sensors, the sensitivity of these sensors based on FRET technology is improved by changing the surface structure of nanoparticles, such as by adjusting the size of nanoparticles, enhancing the luminescence of nanoparticles by doping emitting ions, making energy transfer more efficient by adjusting energy acceptors within a shorter spatial distance, or by increasing the spectral overlap range between donor emission and acceptor absorption. However, some of the above methods still have defects, such as: biological molecules and polymers are not stable enough, resulting in insignificant improvement in detection sensitivity; the adjustment of nanoparticle size will inevitably affect the specific surface area of ​​nanoparticles, resulting in severe surface quenching and reduced fluorescence quantum yield; all of these methods are highly dependent on FRET efficiency, and the current FRET efficiency is still unsatisfactory and has little room for improvement, which greatly affects the sensitivity of these biosensors based on FRET technology.

[0003] In the past few years, many researchers have begun to combine UCNPs as light modulators and compound some metal semiconductors or composite nanomaterials to carry out efficient heterogeneous photocatalysis. In this photocatalytic system, UCNPs absorb near-infrared photons and transfer energy to metal semiconductors or composite nanomaterials through an efficient FRET process, ultimately achieving efficient photocatalytic degradation or photodynamic therapy. In addition, the heterojunction can accelerate the effective separation of charge and hole pairs and inhibit their recombination, thereby greatly improving the reaction efficiency. Although this strategy has always been promising in biological applications, the ultrasensitive photocatalytic fluorescence sensor constructed based on the photoresponsive nanozyme Fe3O4@NPC for detecting other trace targets is still imperfect. Therefore, it is of great significance to develop new sensing strategies that are not directly limited by FRET or quenching efficiency, which also provides a good inspiration for constructing a new ultrasensitive fluorescence sensor that fully utilizes the catalytic properties of photoresponsive nanozymes and the advantages of UCNPs optical properties. Summary of the invention

[0004] The object of the present invention is to provide a method for constructing a photocatalytic fluorescence sensor based on light-responsive nanozymes to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The method for constructing a photocatalytic fluorescent sensor based on a light-responsive nanozyme comprises the following steps:

[0007] S1. Synthesis of upconversion nanoparticles: synthesizing oleic acid-modified UCNPs by thermal decomposition method, precipitating the synthesized oleic acid-modified UCNPs by high-speed centrifugation, dispersing the oleic acid-modified UCNPs in chloroform, and dropping them dropwise into a polyacrylic acid ethanol solution, washing them twice with ethanol and then washing them three times with water to obtain UCNPs;

[0008] S2. Preparation of functionalized upconversion nanoparticles: UCNPs were surface-modified with AFB1-specific antibodies by covalent coupling to obtain functionalized UCNPs, i.e., Ab-UCNPs, which were dispersed in phosphate buffer solution and stored at 4°C for future use;

[0009] S3. Synthesis of Fe3O4@NPC: First, the precursor of magnetic nanoporous carbon, NH2-MIL-101 (Fe), was synthesized. NH2-MIL-101 (Fe) was calcined in a vacuum tube furnace at a heating rate of 2°C / min to 350°C, and the temperature was maintained at 350°C for 50 minutes in an air atmosphere. The final product was cooled to room temperature to obtain Fe3O4@NPC, and the N2 gas flow valve was opened during the heating and cooling stages.

[0010] S4. Preparation of functionalized Fe3O4@NPC: functionalizing Fe3O4@NPC to obtain functionalized Fe3O4@NPC, i.e., Ag-Fe3O4@NPC was dispersed in phosphate buffer solution and stored at 4°C for future use;

[0011] S5. Construction of light-responsive nanozyme Fe3O4@NPC photocatalytic fluorescence sensor to detect aflatoxin B1: 2,7-dichlorodihydrofluorescein was used as the fluorescent signal label, where the symbol of 2,7-dichlorodihydrofluorescein is DCFH, which is used to verify the target-induced reactive oxygen free radicals in the sensing system. Reactive oxygen free radicals can catalyze the oxidation of colorless DCFH to generate 2,7-dichlorofluorescein with high fluorescence intensity.

[0012] Preferably, the UCNPs are synthesized by thermal decomposition method as follows: accurately weigh the rare earth chloride complex, stir at room temperature and at a speed of 1500 r / min for 30 min, then evacuate to remove oxygen in the three-necked flask, heat to 150°C in this state, evacuate to remove moisture in the system, then heat to 160°C under a nitrogen atmosphere, maintain stirring at 160°C for 30 min until a clear and transparent solution is formed, cool the reaction solution to 80°C to obtain a three-necked flask reaction system; accurately weigh 0.26 g of sodium hydroxide and 0.26 g of ammonium fluoride and add them to 20 mL of methanol and stir evenly at a stirring temperature of 60°C and a stirring speed of 1500 r / min to obtain liquid A, then drip liquid A dropwise into the three-necked flask reaction system, add liquid A, and stir at a constant temperature of 80°C for 30 min, then heat to 100°C and keep stirring for 30 min to remove excess methanol, evacuate, quickly heat to 300°C and keep at a constant temperature of 60 min, then the heat source was removed and the temperature was cooled to room temperature, and ethanol and cyclohexane solution with a volume ratio of 1:1 were added to synthesize oleic acid-modified UCNPs.

[0013] Preferably, the rare earth chloride complex is configured as yttrium, ytterbium and erbium, and the ratio of yttrium, ytterbium and erbium is 78:20:2, that is, 0.9464g of yttrium chloride hexahydrate, 0.3112g of ytterbium chloride hexahydrate, and 0.015g of erbium chloride hexahydrate are placed in a 100mL three-necked flask, and then 12mL of oleic acid and 30mL of 1-octadecene are added.

[0014] Preferably, when the UCNPs are surface modified, 50 mg of oleic acid-modified UCNPs are dispersed in 10 mL of chloroform solution to obtain a UCNPs chloroform solution, 300 mg of polyacrylic acid is dissolved in 20 mL of ethanol solution to obtain a polyacrylic acid ethanol solution, and then the UCNPs chloroform solution is added dropwise to the polyacrylic acid ethanol solution, stirred for 24 h at room temperature in the dark, and the obtained polyacrylic acid-modified UCNPs are washed alternately with ethanol and ultrapure water for 3 times and stored for later use.

[0015] Preferably, when preparing the functionalized upconversion nanoparticles, 5.5 mg of UCNPs modified with polyacrylic acid are dispersed in 5 mL of phosphate buffer solution, 200 μL of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 120 μL of N-hydroxysuccinimide are added, and the reaction is carried out at room temperature for 30 min. The solution is washed three times with ultrapure water, centrifuged at 2000 g for 8 min to remove unreacted N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and then 2 mL of AFB1-specific antibody is added and incubated on a shaker overnight to produce functionalized UCNPs, wherein the phosphate buffer solution is set to 0.01 mM The pH is 7.4, the symbol of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is EDC, the concentration is 10 mg / mL, and the symbol of N-hydroxysuccinimide is NHS, the concentration is 10 mg / mL.

[0016] Preferably, the synthesis steps of the magnetic nanoporous carbon precursor NH2-MIL-101 (Fe) are as follows: weigh 450.0 mg of 2-aminoterephthalic acid and stir it evenly in 15.0 mL of N,N-dimethylformamide solution, weigh 1350.0 mg of ferric chloride hexahydrate and mix it in 15.0 mL of N,N-dimethylformamide solution and stir it thoroughly to dissolve, then mix the two and stir them thoroughly and put them into a high-pressure reactor, react at 110°C for 20 h, recover the obtained dark brown precipitate by centrifugation, wash it three times with N,N-dimethylformamide solution to remove unreacted ligands, and then dry the sample in a vacuum drying oven at 70°C to obtain a dark brown solid powder, which is stored for later use.

[0017] Preferably, when preparing the functionalized Fe3O4@NPC, 3.6 mg of Fe3O4@NPC is dispersed in 6 mL of phosphate buffer solution to obtain a Fe3O4@NPC solution, 350 μL of EDC and 140 μL of NHS are added to the Fe3O4@NPC solution respectively, and the mixture is shaken at room temperature for 30 min. Then, the mixture is washed with water three times, centrifuged at 3000 g for 5 min, unreacted EDC and NHS are removed, and a mixed solution is obtained. 2 mL of AFB1-specific antigen is added to the mixed solution, and the mixture is shaken overnight. The obtained functional Fe3O4@NPC is dispersed in 5 mL of phosphate buffer solution and stored at 4°C for later use.

[0018] Preferably, when constructing the light-responsive nanozyme Fe3O4@NPC photocatalytic fluorescence sensor for detecting aflatoxin B1, 1 mg of 2, 7-dichlorofluorescein acetate and 1 mL of sodium hydroxide solution are weighed and mixed, and the solution is vigorously stirred for 30 min at room temperature in the dark to obtain DCFH, wherein the symbol of 2, 7-dichlorofluorescein acetate is DCFH-DA. In order to obtain a neutral solution, it is diluted to 40 mL with phosphate buffer solution, divided into several 2 mL centrifuge tubes, wrapped with tin foil to avoid light, and stored at -20°C for later use.

[0019] Preferably, 500 μL Ab-UCNPs, 500 μL Ag-Fe3O4@NPC, 100 μL DCFH and 100 μL AFB1 standard solution of different concentrations are added to a 2 mL centrifuge tube and incubated at room temperature for 60 min to obtain a reaction solution. The entire experimental process needs to be protected from light. The reaction solution is irradiated with a 980 nm near-infrared laser in the dark for 15 min. The final sample is directly analyzed and detected by a fluorescence spectrometer, and the detection limit (LOD) = 3S / M, where S is the standard deviation value of the blank sample, and M is the slope of the standard curve. The excitation wavelength of the fluorescence spectrometer is 480 nm, the emission wavelength is 521 nm, and the gain is set to 9; the concentration of Ab-UCNPs is 1.3 mg / mL, the concentration of Ag-Fe3O4@NPC is 0.6 mg / mL, and the concentration of DCFH is 25 µg / mL.

[0020] The method for constructing a photocatalytic fluorescent sensor based on light-responsive nanozymes proposed in the present invention has the following advantages compared with the prior art:

[0021] 1. The present invention synthesizes UCNPs by thermal decomposition and Fe3O4@NPC magnetic composite porous materials by calcination. The heterojunction structure of Fe3O4@NPC enhances the photocatalytic activity. 2,7-dichlorodihydrofluorescein (DCFH) is selected as an efficient fluorescent probe for generating ROS during the assembly process. A photocatalytic fluorescence sensor for rapid and highly sensitive detection of aflatoxin B1 is constructed based on UCNPs and Fe3O4@NPC. UCNPs are used as target-mediated light regulators to transfer energy to Fe3O4@NPC, inducing the separation of electron-hole pairs on Fe3O4@NPC and starting the photocatalytic oxidation reaction. The heterojunction structure of Fe3O4@NPC slows down the recombination rate of electron-hole pairs, promotes the generation of more ROS, and oxidizes DCFH to a high fluorescence intensity of DCF, thereby amplifying the difference in fluorescence signals and improving the detection sensitivity, providing a relatively broad application prospect for the construction of new high-sensitivity photocatalytic fluorescence sensors in the future.

[0022] 2. The present invention changes the fluorescence intensity of the probe DCF after catalytic oxidation in the detection system by mediating the assembly degree of functionalized Fe3O4@NPC and UCNPs through the target, thereby establishing a new fluorescent sensing system for detecting harmful substances in food, and applying the established sensing system to the detection of actual samples, with good practicality.

[0023] 3. The light-responsive nanozyme Fe3O4@NPC and UCNPs assembly system used in the present invention to detect the target has greatly improved the sensitivity of detecting the target compared with the traditional FRET sensor. The photocatalytic fluorescence sensor based on the light-responsive nanozyme Fe3O4@NPC has excellent sensitivity and low detection limit (LOD) for the determination of the target. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a flowchart of the present invention;

[0025] Figure 2 is a standard curve diagram between the AFB1 concentration of the present invention and the fluorescence intensity of DCF after oxidation;

[0026] Figure 3 is a standard curve diagram between the AFB1 concentration and the up-conversion fluorescence intensity of the present invention;

[0027] Figure 4 This is a schematic diagram of the detection principle of the fluorescent sensor based on light-responsive nanozyme catalysis of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] The present invention provides Figure 1 The method for constructing a photocatalytic fluorescent sensor based on a light-responsive nanozyme comprises the following steps:

[0030] S1. Synthesis of upconversion nanoparticles: Oleic acid-modified UCNPs were synthesized by thermal decomposition method, the synthesized oleic acid-modified UCNPs were precipitated by high-speed centrifugation, the oleic acid-modified UCNPs were dispersed in chloroform, and added dropwise to the polyacrylic acid ethanol solution, washed twice with ethanol and then washed three times with water to obtain UCNPs; Specifically: The rare earth chloride complex was accurately weighed, stirred at room temperature and at a speed of 1500 r / min for 30 min, and then the oxygen in the three-necked flask was removed by vacuum, and the temperature was raised to 150°C in this state, and the water in the system was removed by vacuum, and then the temperature was raised to 160°C under a nitrogen atmosphere, and stirred at 160°C for 30 min to form a clear and transparent solution, and the reaction solution was cooled to 80°C to obtain a three-necked flask reaction system; 0.26 g of sodium hydroxide and 0.26 g of ammonium fluoride were accurately weighed and added to 20 mL of methanol and stirred evenly, and the stirring temperature was 60°C and the stirring speed was 1500 r / min. r / min to obtain liquid A, and then drip liquid A drop by drop into the reaction system in the three-necked flask. After the addition of liquid A is completed, stir at 80°C for 30 min at a constant temperature for solid fluorine, then raise the temperature to 100°C and keep stirring for 30 min, remove excess methanol, evacuate, quickly heat to 300°C and keep the temperature for 60 min, then remove the heat source and cool to room temperature, add ethanol and cyclohexane solution with a volume ratio of 1:1, and synthesize oleic acid-modified UCNPs.

[0031] Among them, the rare earth chloride complex is set to yttrium, ytterbium and erbium, and the ratio of yttrium, ytterbium and erbium is 78:20:2, that is, 0.9464g of yttrium chloride hexahydrate, 0.3112g of ytterbium chloride hexahydrate, and 0.015g of erbium chloride hexahydrate are placed in a 100 mL flask, and then 12 mL of oleic acid and 30 mL of 1-octadecene are added.

[0032] S2. Preparation of functionalized upconversion nanoparticles: UCNPs were surface-modified with AFB1-specific antibodies by covalent coupling to obtain functionalized UCNPs, i.e., Ab-UCNPs were dispersed in phosphate buffer solution and stored at 4°C for future use;

[0033] Specifically, when UCNPs are surface modified, 50 mg of oleic acid-modified UCNPs are dispersed in 10 mL of chloroform solution to obtain a UCNPs chloroform solution, 300 mg of polyacrylic acid is dissolved in 20 mL of ethanol solution to obtain a polyacrylic acid ethanol solution, and then the UCNPs chloroform solution is added dropwise to the polyacrylic acid ethanol solution, and stirred for 24 h at room temperature in the dark. The obtained polyacrylic acid-modified UCNPs are washed alternately with ethanol and ultrapure water for 3 times and stored for later use.

[0034] When preparing the functionalized upconversion nanoparticles, 5.5 mg of UCNPs modified with polyacrylic acid were dispersed in 5 mL of phosphate buffer solution, 200 μL of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (10 mg / mL) and 120 μL of N-hydroxysuccinimide (10 mg / mL) were added, and the mixture was reacted at room temperature for 30 min. The solution was washed three times with ultrapure water, centrifuged at 2000 g for 8 min to remove unreacted N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and then 2 mL of AFB1 specific antibody (8 μg / mL, phosphate buffer solution 0.01 mM, pH = 7.4) was added and incubated on a shaking table overnight to produce functionalized UCNPs (Ab-UCNPs), wherein the phosphate buffer solution was set to 0.01 mM and pH 7.4, the symbol of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is EDC, the concentration is 10 mg / mL, the symbol of N-hydroxysuccinimide is NHS, and the concentration is 10 mg / mL.

[0035] S3. Synthesis of Fe3O4@NPC: First, the precursor of magnetic nanoporous carbon, NH2-MIL-101 (Fe), was synthesized. NH2-MIL-101 (Fe) was calcined in a vacuum tube furnace at a heating rate of 2°C / min to 350°C, and the temperature was maintained at 350°C for 50 minutes in an air atmosphere. The final product was cooled to room temperature to obtain Fe3O4@NPC, and the N2 gas flow valve was opened during the heating and cooling stages.

[0036] The synthesis steps of the magnetic nanoporous carbon precursor NH2-MIL-101 (Fe) are as follows: 450.0 mg of 2-aminoterephthalic acid is weighed and stirred evenly in 15.0 mL of N,N-dimethylformamide solution, 1350.0 mg of ferric chloride hexahydrate is weighed and mixed in 15.0 mL of N,N-dimethylformamide solution and stirred to dissolve, then the two are mixed and stirred sufficiently and put into a high-pressure reactor, reacted at 110° C. for 20 h, the obtained dark brown precipitate is recovered by centrifugation, washed three times with N,N-dimethylformamide solution to remove unreacted ligands, and then the sample is dried in a vacuum drying oven at 70° C. to obtain a dark brown solid powder, which is stored for later use.

[0037] S4. Preparation of functionalized Fe3O4@NPC: functionalizing Fe3O4@NPC to obtain functionalized Fe3O4@NPC, i.e., Ag-Fe3O4@NPC was dispersed in phosphate buffer solution and stored at 4°C for future use;

[0038] When preparing the functionalized Fe3O4@NPC, 3.6 mg of Fe3O4@NPC was dispersed in 6 mL of phosphate buffer solution (0.01 mM, pH=7.4) to obtain a Fe3O4@NPC solution, 350 μL of EDC (10 mg / mL) and 140 μL of NHS (10 mg / mL) were added to the Fe3O4@NPC solution, respectively, and the mixture was shaken at room temperature for 30 min. Then, the mixture was washed with water three times and centrifuged at 3000 g for 5 min to remove unreacted EDC and NHS to obtain a mixed solution. 2 mL of AFB1-specific antigen (8 μg / mL, phosphate buffer solution 0.01 mM, pH=7.4) was added to the mixed solution, and then the mixture was shaken overnight. The obtained functional Fe3O4@NPC (Ag-Fe3O4@NPC) was dispersed in 5 mL of phosphate buffer solution (5 mL, 0.01 mM, pH=7.4) and stored at 4°C for future use.

[0039] S5. Construction of light-responsive nanozyme Fe3O4@NPC photocatalytic fluorescence sensor to detect aflatoxin B1: 2,7-dichlorodihydrofluorescein was used as the fluorescent signal label, where the symbol of 2,7-dichlorodihydrofluorescein is DCFH, which is used to verify the target-induced reactive oxygen free radicals in the sensing system. Reactive oxygen free radicals can catalyze the oxidation of colorless DCFH to generate 2,7-dichlorofluorescein with high fluorescence intensity.

[0040] The light-responsive nanozyme Fe3O4@NPC photocatalytic fluorescence sensor for detecting aflatoxin B1 is constructed by weighing 1 mg of 2, 7-dichlorofluorescein acetate and 1 mL of sodium hydroxide solution (0.1 M) and mixing them. The solution is vigorously stirred for 30 min at room temperature in the dark to obtain DCFH, where the symbol of 2, 7-dichlorofluorescein acetate is DCFH-DA. In order to obtain a neutral solution, it is diluted to 40 mL with phosphate buffer solution (0.01 M, pH=7.4), divided into several 2 mL centrifuge tubes, wrapped with tin foil to avoid light, and stored at -20°C for later use.

[0041] In a 2 mL centrifuge tube, 500 μL Ab-UCNPs (1.3 mg / mL), 500 μL Ag-Fe3O4@NPC (0.6 mg / mL), 100 μL DCFH (120 µg / mL) and 100 μL AFB1 standard solution of different concentrations (final concentration: 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL) were added and incubated at room temperature for 60 min to obtain a reaction solution. The entire experimental process needed to be protected from light. The reaction solution was irradiated with a 980 nm near-infrared laser in the dark for 15 min. The final sample was directly analyzed and detected by a fluorescence spectrometer with a detection limit (LOD) of 3S / M, where S is the standard deviation of the blank sample and M is the slope of the standard curve. The excitation wavelength of the fluorescence spectrometer F96PRO is 480 nm, emission wavelength: 521 nm; the concentration of Ab-UCNPs is 1.3 mg / mL, the concentration of Ag-Fe3O4@NPC is 0.6 mg / mL, and the concentration of DCFH is 120 µg / mL.

[0042] like Figure 2 As shown in the figure, a standard curve was drawn with the concentration of aflatoxin B1 as the horizontal axis and the fluorescence intensity of oxidized DCF as the vertical axis. There was a good linear relationship between the two, and the linear equation was: y = -4283.80 log(x)+4202.85. The detection limit was: LOD=3S / M=3×0.8 / 4283.8=0.56 pg / mL, which was 57 times more sensitive than the traditional FRET sensor.

[0043] Construction of traditional FRET sensor to detect AFB1 (taking UCNPs-AuNPs assembly as an example)

[0044] Functionalization of gold nanoparticles (AuNPs): Take 5 mL of 3 nM AuNPs, add 48 uL of 0.1M potassium carbonate solution, add 2mL of 4 ug / mL antigen, shake for 1 hour, add 400 uL of 1% bovine serum albumin solution to block for 1 hour (divide into 1.5 ml conical bottom centrifuge tubes), centrifuge at 8000 g for 10 min, wash once with ultrapure water, and finally dissolve to 5 mL with ultrapure water.

[0045] Add 500 μL Ab-UCNPs (1.3 mg / mL), 500 μL Ag-AuNPs (3 nM), 100 μL ultrapure water (control) and 100 μL AFB1 standard solution of different concentrations (final concentration: 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL) into a 2 mL centrifuge tube and incubate at room temperature for 60 min. The final sample was directly analyzed and detected by fluorescence spectrometer (excitation wavelength: 980 nm, emission wavelength: 543 nm).

[0046] like Figure 3 As shown in the figure, a standard curve was drawn with the concentration of aflatoxin B1 as the horizontal axis and the upconversion fluorescence intensity as the vertical axis. The linear equation was: y = 51.32 log(x)+1.13, and the detection limit was: LOD=3S / M=3×0.55 / 51.32=0.03 ng / mL.

[0047] The basic principle of constructing a photocatalytic fluorescence sensor for trace detection of AFB1 based on Fe3O4@NPC is as follows: Figure 4 As shown. Specifically, UCNPs are used as energy donors and light source regulators, and Fe3O4@NPC is used as an energy acceptor. UCNPs are modified with antibodies to become functionalized UCNPs, and Fe3O4@NPC is modified with antigens to become functionalized Fe3O4@NPC. When the target AFB1 is not present, functionalized UCNPs and functionalized Fe3O4@NPC form an assembly through specific biorecognition between antibodies and antigens. Under near-infrared light excitation, UCNPs absorb near-infrared photons and excite the electrons of the nanoporous carbon in Fe3O4@NPC to high energy levels through an effective FRET process, generating electrons (e-) and holes (h+) in turn. Due to the energy level difference, the e- of the nanoporous carbon in Fe3O4@NPC will be transferred to the surrounding Fe3O4, and the electric field formed by the heterojunction will also prevent the transfer of h+. Then e- and h+ react with the surrounding O2 / H2O to eventually produce ROS, mainly hydroxyl radicals •OH, which can oxidize the colorless probe DCFH into DCF with high fluorescence, resulting in changes in signal difference amplification. When AFB1 is present, a small amount of target will react competitively with the AFB1-specific antibody, inducing UCNPs-Fe3O4@NPC disassembly, resulting in reduced energy transfer efficiency and low yield of ROS, at which time the fluorescence intensity of DCF is minimal. Therefore, the concentration of AFB1 can be quantitatively analyzed by detecting changes in the DCF fluorescence signal.

[0048] In summary, by modifying specific antibodies and antigens on the surface of upconversion nanoparticles and light-responsive nanozyme materials, the target can be specifically identified; the heterogeneous structure of the light-responsive nanozyme can slow down the recombination rate of electrons and holes on the surface of the material, thereby generating more reactive oxygen free radicals during assembly, amplifying the difference in DCF fluorescence change before and after assembly, and improving the detection sensitivity; and the concentration of the target in the detection system can mediate the fluorescence change of the fluorescent probe DCF before and after the functionalized material assembly. In the absence of the target, the DCF fluorescence value is the highest, and in the presence of the target, the fluorescence value is lower. Using the ROS probe with high fluorescence response as a beacon, a method for detecting the target through a fluorescence spectrometer is constructed, thereby realizing ultra-sensitive and highly specific immune detection of the target.

[0049] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a photocatalytic fluorescent sensor based on a light-responsive nanozyme, characterized in that: The steps include: S1. Synthesis of upconversion nanoparticles: UCNPs modified with oleic acid were synthesized by thermal decomposition method, the synthesized UCNPs modified with oleic acid were precipitated by high-speed centrifugation, washed twice with ethanol and then washed three times with water, and finally dispersed in chloroform to obtain UCNPs; The UCNPs were synthesized by thermal decomposition method as follows: accurately weigh the rare earth chloride complex, stir at room temperature and at a speed of 1500 r / min for 30 min, then evacuate to remove oxygen in the three-necked flask, heat to 150°C in this state, evacuate to remove moisture in the system, then heat to 160°C in a nitrogen atmosphere, maintain stirring at 160°C for 30 min until a clear and transparent solution is formed, cool the reaction solution to 80°C to obtain a three-necked flask reaction system; accurately weigh 0.13 g of sodium hydroxide and 0.13 g of ammonium fluoride and add them to 20 mL of methanol and stir evenly at a stirring temperature of 60°C and a stirring speed of 1500 r / min to obtain liquid A, then drop liquid A into the three-necked flask reaction system, add liquid A drop by drop, stir at 80°C for 30 min, then heat to 100°C and keep stirring for 30 min, remove excess methanol, evacuate, quickly heat to 300°C and keep at 60 min, then the heat source was removed and the temperature was cooled to room temperature, and oleic acid-modified UCNPs were synthesized by adding ethanol and cyclohexane solution with a volume ratio of 1:1; The rare earth chloride complex is set to be yttrium, ytterbium and erbium, and the ratio of yttrium, ytterbium and erbium is 78:20:2, that is, 0.9464g of yttrium chloride hexahydrate, 0.3112g of ytterbium chloride hexahydrate, and 0.015g of erbium chloride hexahydrate are placed in a 100mL three-necked flask, and then 12mL of oleic acid and 30mL of 1-octadecene are added; S2. Preparation of functionalized upconversion nanoparticles: UCNPs were surface-modified with AFB1-specific antibodies by covalent coupling to obtain functionalized UCNPs, i.e., Ab-UCNPs, which were dispersed in phosphate buffer solution and stored at 4°C for future use; When the UCNPs are surface modified, oleic acid-modified UCNPs are dispersed in 10 mL of chloroform solution, the concentration of the chloroform solution is 5 mg / mL, to obtain a UCNPs chloroform solution, polyacrylic acid is dissolved in 20 mL of ethanol solution, the concentration of the ethanol solution is 15 mg / mL, to obtain a polyacrylic acid ethanol solution, and then the UCNPs chloroform solution is added dropwise to the polyacrylic acid ethanol solution, stirred at room temperature in the dark for 24 h, and the obtained polyacrylic acid-modified UCNPs are washed alternately with ethanol and ultrapure water for 3 times, and stored for later use; When the functionalized upconversion nanoparticles are prepared, 5.5 mg of UCNPs modified with polyacrylic acid are dispersed in 5 mL of phosphate buffer solution, 200 μL of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 120 μL of N-hydroxysuccinimide are added, and the mixture is reacted at room temperature for 30 min. The solution is washed three times with ultrapure water, and centrifuged at 2000 g for 8 min to remove unreacted N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. Then, 2 mL of AFB1-specific antibody is added and incubated overnight on a shaking table to produce functionalized UCNPs, wherein the phosphate buffer solution is set to 0.01 mM and pH 7.4, the symbol of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is EDC, the concentration is 10 mg / mL, and the symbol of N-hydroxysuccinimide is NHS, the concentration is 10 mg / mL; S3. Synthesis of Fe3O4@NPC: First, the precursor of magnetic nanoporous carbon, NH2-MIL-101 (Fe), was synthesized. NH2-MIL-101 (Fe) was calcined in a vacuum tube furnace at a heating rate of 2°C / min to 350°C, and the temperature was maintained at 350°C for 50 minutes in an air atmosphere. The final product was cooled to room temperature to obtain Fe3O4@NPC, and the N2 gas flow valve was opened during the heating and cooling stages. The synthesis steps of the magnetic nanoporous carbon precursor NH2-MIL-101 (Fe) are as follows: 450.0 mg of 2-aminoterephthalic acid is weighed and stirred evenly in 15.0 mL of N, N-dimethylformamide solution, 1350.0 mg of ferric chloride hexahydrate is weighed and mixed in 15.0 mL of N, N-dimethylformamide solution and fully stirred to dissolve, then the two are mixed and stirred fully and put into a high-pressure reactor, reacted at 110° C. for 20 h, and the obtained dark brown precipitate is recovered by centrifugation, washed three times with N, N-dimethylformamide solution to remove unreacted ligands, and then the sample is dried in a vacuum drying oven at 70° C. to obtain a dark brown solid powder, which is stored for later use, wherein the mass of 2-aminoterephthalic acid is 2.5 mmol, and the mass of ferric chloride hexahydrate is 5.0 mmol; S4. Preparation of functionalized Fe3O4@NPC: functionalizing Fe3O4@NPC to obtain functionalized Fe3O4@NPC, i.e., Ag-Fe3O4@NPC was dispersed in phosphate buffer solution and stored at 4°C for future use; During the preparation of the functionalized Fe3O4@NPC, 3.6 mg of Fe3O4@NPC was dispersed in 6 mL of phosphate buffer solution to obtain a Fe3O4@NPC solution, 350 μL of EDC and 140 μL of NHS were added to the Fe3O4@NPC solution, and the mixture was shaken at room temperature for 30 min. Then, the mixture was washed with water for 3 times, and centrifuged at 3000 g for 5 min to remove unreacted EDC and NHS to obtain a mixed solution. 2 mL of AFB1-specific antigen was added to the mixed solution, and the mixture was shaken overnight. The obtained functional Fe3O4@NPC was dispersed in 5 mL of phosphate buffer solution and stored at 4°C for future use. S5. Construction of a light-responsive nanozyme Fe3O4@NPC photocatalytic fluorescence sensor for the detection of aflatoxin B1: 2,7-dichlorodihydrofluorescein was used as a fluorescent signal label, wherein the symbol of 2,7-dichlorodihydrofluorescein is DCFH, which is used to verify the reactive oxygen free radicals induced by the target in the sensing system. The reactive oxygen free radicals can catalyze the oxidation of colorless DCFH to generate 2,7-dichlorofluorescein with high fluorescence intensity. The light-responsive nanozyme Fe3O4@NPC photocatalytic fluorescence sensor for detecting aflatoxin B1 is constructed by weighing 1 mg of 2, 7-dichlorofluorescein acetate and 1 mL of sodium hydroxide solution, and stirring the solution vigorously at room temperature for 30 min in the dark to obtain DCFH, wherein the symbol of 2, 7-dichlorofluorescein acetate is DCFH-DA. In order to obtain a neutral solution, it is diluted to 40 mL with phosphate buffer solution, and dispensed into several 2 mL centrifuge tubes, wrapped with tin foil to avoid light, and stored at -20°C for later use; Add 500 μL Ab-UCNPs, 500 μL Ag-Fe3O4@NPC, 100 μL DCFH and 100 μL AFB1 standard solution of different concentrations into a 2 mL centrifuge tube and incubate at room temperature for 60 min to obtain a reaction solution. The entire experimental process needs to be protected from light. The reaction solution is irradiated with a 980 nm near-infrared laser in the dark for 15 min. The final sample is directly analyzed and detected by a fluorescence spectrometer. The detection limit = 3S / M, where S is the standard deviation value of the blank sample, and M is the slope of the standard curve. The excitation wavelength of the fluorescence spectrometer is 480 nm, and the emission wavelength is 521 nm; the concentration of Ab-UCNPs is 1.3 mg / mL, the concentration of Ag-Fe3O4@NPC is 0.6 mg / mL, and the concentration of DCFH is 120 µg / mL.

Images