A dual-mode detection method for food hazards based on a three-dimensional folded paper-based microfluidic analysis device

Abstract

The application belongs to the field of food safety detection, and particularly relates to a food hazard double-mode detection method based on a three-dimensional folded paper-based microfluidic analysis device; the steps are as follows: step one, preparation of fluorescence-enhanced upconversion nanomaterial; step two, surface biological functionalization of the upconversion nanomaterial; step three, preparation of a double-metal organic framework with peroxidase activity; step four, preparation of a double-metal organic framework modified by a target aptamer; step five, preparation of a nano probe formed by coupling of the upconversion nanomaterial and the double-metal organic framework; step six, preparation of a three-dimensional folded paper-based microfluidic analysis device; step seven, rapid detection of food hazards by using the three-dimensional folded paper-based microfluidic analysis device; taking chlorpyrifos as an example, rapid and accurate detection of the content of chlorpyrifos in food is realized; meanwhile, the three-dimensional folded paper-based microfluidic analysis device with double-mode output has the capability of on-site real-time detection of hazards, and has a good practical prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food safety detection, and particularly relates to a food hazard double-mode detection method based on a three-dimensional folded paper-based microfluidic analysis device. BACKGROUND

[0002] Food contains rich nutrients and provides the human body with the required energy, and is an indispensable part of daily life. However, the residues of some hazardous substances in food can cause diseases to humans, and even endanger lives. Generally, foodborne diseases are caused by food or water contaminated by food hazards such as pathogenic bacteria, viruses or agricultural and veterinary drugs entering the human body. Common food hazards include the pesticides chlorpyrifos, imidacloprid and paraquat, and the veterinary drugs chloramphenicol and quinolones.

[0003] However, the long-term and large-scale use of food hazards not only pollutes the ecological environment, but also causes residues in agricultural and fishery products, posing a great threat to the environment and human life safety. Therefore, it is of great significance to develop a rapid and sensitive detection method for food hazards for food safety and human health.

[0004] At present, conventional food hazard detection methods, such as high-performance liquid chromatography and enzyme-linked immunosorbent assay, often face difficulties such as expensive detection instruments and equipment, high cost and complicated steps, and it is difficult to achieve rapid quantitative detection. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a food hazard double-mode detection method based on a three-dimensional folded paper-based microfluidic analysis device to solve the problems of high detection cost, complicated detection steps and unsuitability for on-site detection in the prior art.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0007] A food hazard double-mode detection method based on a three-dimensional folded paper-based microfluidic analysis device, the specific steps are as follows:

[0008] Step 1: Preparation of fluorescence-enhanced upconversion nanomaterials:

[0009] (a) Dissolve yttrium chloride hexahydrate in methanol A, then add oleic acid and 1-octadecene, mix and heat for the first time, stop heating, and wait for the solution to cool to room temperature, then add a mixed solution containing sodium hydroxide, ammonium fluoride and methanol B, heat for the second time, continue to heat for the third time after the second heating, and cool to room temperature after heating, centrifuge to obtain a precipitate, and then wash to obtain upconversion nanoparticle seeds; disperse them in cyclohexane to obtain an upconversion nanoparticle seed solution;

[0010] (b) taking thulium chloride hexahydrate and ytterbium chloride hexahydrate to be dissolved in methanol A, then adding oleic acid and 1-octadecene, mixing and then carrying out the first heating reaction, cooling to room temperature after the reaction is completed, adding the upconversion nanoparticle seed solution prepared in step (a), and dropping a mixed solution of sodium hydroxide, ammonium fluoride and methanol B, carrying out the second heating, continuing to heat for the third heating after the second heating, cooling to room temperature after heating, and then centrifugally separating the precipitate to obtain the core / shell structure upconversion nanoparticles after washing, which are dispersed in cyclohexane to obtain a core / shell structure upconversion nanoparticle solution;

[0011] (c) taking yttrium chloride hexahydrate to be dissolved in methanol A, then adding oleic acid and 1-octadecene, mixing and then carrying out the first heating reaction, cooling after the reaction is completed, adding the core / shell structure upconversion nanoparticle solution prepared in step (b), and dropping a mixed solution of sodium hydroxide, ammonium fluoride and methanol B, carrying out the second heating, then heating for the third heating, cooling to room temperature after heating, and then centrifugally separating the precipitate to obtain the core / shell / shell structure upconversion nanoparticles after the reaction is completed;

[0012] Step two, surface biological functionalization of the upconversion nanomaterial:

[0013] S1, taking the core / shell / shell structure upconversion nanoparticles prepared in step one to be added to a mixed solution of trichloromethane and toluene, then adding a mixed solution of polyacrylic acid and ultrapure water and sealing, and then carrying out the stirring reaction; after the stirring reaction is completed, the mixed solution is centrifugally collected to obtain the precipitate, which is washed with a mixed solution of ethanol and ultrapure water and then centrifugally separated to obtain the water-soluble upconversion nanomaterial modified with carboxyl;

[0014] S2, ultrasonically dissolving the water-soluble upconversion nanomaterial modified with carboxyl in a Tris-HCl buffer solution, then adding a 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide solution and a N-hydroxysuccinimide solution to obtain a mixed solution, and then placing the mixed solution on a shaker to carry out the first incubation; after the incubation, the upconversion nanomaterial with activated carboxyl is obtained by centrifugal separation;

[0015] S3, adding the upconversion nanomaterial with activated carboxyl to a Tris-HCl buffer solution to obtain an upconversion nanomaterial solution with activated carboxyl; then adding the target aptamer complementary strand modified with amino to the upconversion nanomaterial solution with activated carboxyl, and then carrying out the second incubation on a shaker; after the incubation, the precipitate is collected by centrifugal separation, washed with a phosphate buffer solution, and then separated; and then the precipitate is redissolved in a STE buffer solution to obtain the upconversion nanomaterial solution modified with the aptamer complementary strand on the surface;

[0016] Step three, preparation of a bimetallic organic framework (Fe / Zr-MOF) with peroxidase activity:

[0017] Firstly, zirconium (IV) chloride, ferric chloride hexahydrate and 2-amino terephthalic acid were weighed and dissolved in N,N-dimethylformamide to obtain a zirconium chloride solution, a ferric chloride hexahydrate solution and a 2-amino terephthalic acid solution; then the zirconium chloride solution, the ferric chloride hexahydrate solution, the 2-amino terephthalic acid solution, N,N-dimethylformamide and acetic acid were mixed to obtain a mixed solution, which was then transferred to a hydrothermal reactor for heating reaction. After the reaction was completed, the reaction product was collected and washed with a mixed solution of methanol and ethanol and then centrifuged. The precipitate obtained by centrifugation was dried to obtain a yellow-brown powder, which was a bimetallic organic framework with peroxidase activity, denoted as Fe / Zr-MOF;

[0018] Step four, preparation of target aptamer modified Fe / Zr-MOF:

[0019] The Fe / Zr-MOF prepared in step three was weighed and dissolved in Tris-HCl buffer, and then added to STE buffer containing the target aptamer to obtain a mixed solution, which was incubated on a shaker. After incubation, the precipitate was collected by centrifugation and resuspended in STE buffer to obtain a target aptamer modified Fe / Zr-MOF solution;

[0020] Step five, preparation of a nanoprobe formed by coupling of the upconversion nanomaterial and the Fe / Zr-MOF:

[0021] The upconversion nanomaterial solution modified with the complementary strand of the target aptamer prepared in step two was taken, and the target aptamer modified Fe / Zr-MOF solution prepared in step four was added, followed by incubation on a shaker. After the reaction was completed, the precipitate was collected by centrifugation and resuspended in STE buffer to obtain a nanoprobe solution formed by coupling of the upconversion nanomaterial and the Fe / Zr-MOF;

[0022] Step six, preparation of a three-dimensionally folded paper-based microfluidic analysis device:

[0023] Firstly, a paper-based pattern was drawn, which consisted of four parts in sequence, namely a sample injection layer, a transport layer, a detection layer and an anti-pollution layer. The sample injection layer, the transport layer and the detection layer each contained a hydrophilic functional region, and the anti-pollution layer was a hydrophobic barrier region. Then, filter paper was cut and the paper-based pattern was printed on the filter paper using a laser. After printing, the filter paper was subjected to heat curing treatment, and after the treatment, the filter paper was folded to obtain a three-dimensionally folded paper-based microfluidic analysis device composed of a hydrophobic barrier and a hydrophilic functional region;

[0024] The nanoprobe solution prepared in step five was added dropwise to the detection area of the paper-based microfluidic analysis device. Then, a mixed solution of tetramethylbenzidine and hydrogen peroxide was uniformly sprayed onto the detection area, and after it was naturally air-dried, a treated three-dimensionally folded paper-based microfluidic analysis device was obtained.

[0025] Step seven, detection of the content of the food hazards:

[0026] (a) Establishment of standard curve: first, prepare standard solutions of different concentrations of food hazards, and drop them into the sample layer of the three-dimensional folded paper-based microfluidic analysis device treated in step six. The standard solution of food hazards flows from the hydrophilic functional area one to the hydrophilic functional area two of the transport layer, and finally reaches the hydrophilic functional area three of the detection area. Then, incubate at room temperature to allow the food hazards to react with the nano probes, and obtain three-dimensional folded paper-based microfluidic analysis devices of different concentrations. Subsequently, collect the fluorescence signal value and the paper-based image B value of the detection area. Finally, linearly fit the food hazard concentration with the fluorescence signal value and the paper-based image B value, respectively, to establish the standard curve for detecting the content of food hazards;

[0027] (b) Detection of actual samples: pretreat the sample to be tested to obtain a test solution containing food hazards. Then, drop the test solution into the sample layer of the three-dimensional folded paper-based microfluidic analysis device, and incubate at room temperature. Measure the fluorescence signal value and the paper-based color B value of the detection area, and input them into the standard curve for detecting the content of food hazards obtained in step seven (a) to calculate the content of food hazards in the actual sample.

[0028] Preferably, in step one (a), the amount of yttrium chloride hexahydrate, methanol A, oleic acid, 1-octadecene, sodium hydroxide, ammonium fluoride, and methanol B is 303.4 mg: 10 mL: 10 mL: 10 mL: 266.7 mg: 340 mg: 10 mL. The first heating temperature is 160°C, and the time is 30 min. The second heating temperature is 120°C, and the time is 30 min. The third heating temperature is 300°C, and the time is 60 min. The concentration of the upconversion nanoparticle seed solution is 1-2 mg / mL.

[0029] In step one (b), the amount of ytterbium chloride hexahydrate, thulium chloride hexahydrate, methanol A, oleic acid, 1-octadecene, upconversion nanoparticle seed solution, sodium hydroxide, ammonium fluoride, and methanol B is 151.2 mg: 2.9 mg: 10 mL: 6 mL: 16 mL: 2 mL: 39.9 mg: 55.5 mg: 10 mL. The first heating temperature is 160°C, and the time is 30 min. The second heating temperature is 120°C, and the time is 30 min. The third heating temperature is 300°C, and the time is 60 min. The concentration of the core / shell structure upconversion nanoparticle solution is 1-2 mg / mL.

[0030] In step one, the amount of yttrium chloride hexahydrate, methanol A, oleic acid, 1-octadecene, core / shell structure upconversion nanoparticle solution, sodium hydroxide, ammonium fluoride and methanol B is 121.3 mg: 10 mL: 3 mL: 8 mL: 4 mL: 39.9 mg: 5 mg: 5 mL; the first heating temperature is 160°C, and the time is 30 min; the second heating temperature is 120°C, and the time is 30 min; the third heating temperature is 300°C, and the time is 40 min.

[0031] In steps (a)-(c), the centrifugal separation conditions are as follows: the rotation speed is 8000-9000 rpm, and the time is 10-15 min; the cleaning is performed by using a cyclohexane and ethanol mixed solution with a volume ratio of 1:1.

[0032] Preferably, in S1 of step two, the amount of core / shell / shell structure upconversion nanomaterial, chloroform, toluene, polyacrylic acid and ultrapure water is 50 mg: (2-6) mL: (4-10) mL: (300-400) mg: (15-20) mL; the stirring reaction time is 24-48 h, the volume ratio of ethanol and ultrapure water is 1:1, and the centrifugal separation conditions are as follows: the rotation speed is 8000-10000 rpm, and the time is 8-10 min.

[0033] In S2 of step two, the amount of water-soluble upconversion nanomaterial modified with carboxyl, Tris-HCl buffer solution, 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide solution and N-hydroxysuccinimide solution is 10 mg: 10 mL: 0.5 mL: 0.5 mL, wherein the concentration of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide solution is 2 mg / mL, and the concentration of N-hydroxysuccinimide solution is 1 mg / mL; the pH of Tris-HCl is 6; the first incubation temperature is 25°C, and the time is 1-2 h, and the shaking speed is 300-400 rpm per minute;

[0034] In S3 of step two, the amount of activated carboxyl upconversion nanomaterial solution and amino-modified target aptamer complementary strand is 10 mg: 20 μL, wherein the concentration of activated carboxyl upconversion nanomaterial solution is 1-2 mg / mL, and the concentration of amino-modified target aptamer complementary strand is 100 μM; the pH of Tris-HCl is 7.4; the second incubation temperature is 25°C, and the time is 2-4 h, and the shaking speed is 200-300 rpm; and the concentration of surface-modified aptamer complementary strand upconversion nanomaterial solution is 1 mg / mL.

[0035] Preferably, in step three, the amounts of the zirconium chloride solution, the ferric chloride hexahydrate solution, the 2-amino terephthalic acid solution, the N,N-dimethylformamide and the acetic acid are in the ratio of 5.0 mL:5.0 mL:10.0 mL:5.0 mL:5.0 mL; wherein the concentrations of the zirconium chloride solution, the ferric chloride hexahydrate solution and the 2-amino terephthalic acid solution are all 0.5 mmol;

[0036] The heating reaction condition is 120℃ for 24h; the volume ratio of the methanol and the ethanol is 1:1; the centrifugal separation condition is a rotation speed of 6000-8000rpm for 10-15min.

[0037] Preferably, in step four, the amounts of the Fe / Zr-MOF, the Tris-HCl buffer solution, the target aptamer and the STE buffer solution are in the ratio of 2.0 mg:1.0 mL:10.0 μL:2.0 mL; the pH of the STE buffer solution is 7.4 and the molar concentration is 10 mmol / L; the concentration of the target aptamer is 100 μM; the incubation reaction condition is 25℃ for 12h; the centrifugal separation condition is a rotation speed of 6000-8000rpm for 8-10min; and the concentration of the target aptamer modified Fe / Zr-MOF solution is 2 mg / mL.

[0038] Preferably, in step five, the amounts of the upconversion nanomaterial solution with the surface modified with the complementary strand of the target aptamer, the target aptamer modified Fe / Zr-MOF solution and the STE buffer solution are in the ratio of 2.0 mL:1.0 mL:3.0 mL; the pH of the STE buffer solution is 7.4 and the molar concentration is 10 mmol / L; the incubation reaction condition is 25℃ for 2h; the centrifugal separation condition is a rotation speed of 6000-8000rpm for 8-10min; and the concentration of the nanoprobes solution is 1.5 mg / mL.

[0039] Preferably, in step six, the filter paper is Whatman No. 1 filter paper with a size of 20x20mm; the paper-based pattern is composed of four parts, the first part is a sample loading layer containing a circular hydrophilic functional area one with a diameter of 12mm; the second part is a transport layer containing a circular hydrophilic functional area two with a diameter of 8mm; the third part is a detection layer containing a circular hydrophilic functional area three with a diameter of 6mm; and the fourth part is an anti-pollution layer, all of which are composed of hydrophobic barriers; wherein the hydrophilic functional area one, the hydrophilic functional area two and the hydrophilic functional area three are independent of each other; the temperature of the heat curing treatment is 180-200℃ and the curing time is 4-5h; the concentration of the tetramethylbenzidine solution is 30 mmol / L; and the amounts of the nanoprobes solution, the tetramethylbenzidine solution and the hydrogen peroxide are in the ratio of 5uL:1uL:1uL, and the mass fraction of the hydrogen peroxide is 30%.

[0040] The folding is respectively at the connection of the sample layer, the conveying layer, the detection layer and the anti-pollution layer, and is alternately folded in clockwise and counterclockwise directions.

[0041] Preferably, in the step (a) of the seventh step, the food hazards include but are not limited to chlorpyrifos; the dropping amount of the food hazard standard solution is 20uL, and the concentration range of the food hazard standard solution is 0.05-500ng / mL; the incubation time at room temperature is 15min; in the step (b), the dropping amount of the to-be-tested solution is 20uL; and the incubation time is 15min.

[0042] The present application discloses the following technical effects:

[0043] 1. The present application discloses a synthesis method of a core-shell structure upconversion nanomaterial, and based on the enhancement of the upconversion fluorescence signal, the sensitivity of the detection system is improved; in addition, in the detection system based on fluorescence energy resonance transfer, the fluorescence donor is used, and a basis for realizing double-mode detection of food hazards is provided.

[0044] 2. The present application introduces a new bimetallic organic framework composed of various inorganic metal ions and organic ligands; it has significant peroxidase activity, can be used as a catalyst in a colorimetric detection system, and can be used as a quencher in a fluorescence detection system, successfully connects the fluorescence-colorimetric double-mode detection system, and shows its unique transmission ability.

[0045] 3. The present application discloses a detection method of food hazards, specifically a detection area is prepared on a paper base by laser printing combined with heat curing technology, the prepared nanoprobes are deposited in the microfluidic paper base detection area, based on the specific recognition of aptamers to targets leading to the change of characteristic fluorescence signals, the quantitative detection of targets is realized, only a very small amount of sample liquid (20uL) is needed, and high sensitivity real-time detection of food hazards can be realized.

[0046] 4. The specific detection system constructed by the present application has strong fluorescence response to different food hazards, can effectively eliminate background fluorescence and the interference of other molecules, has high specificity for hazard detection, can realize high sensitivity detection of hazard content, and can detect different hazards by replacing different aptamer sequences, overcoming the shortcomings of traditional methods, and is crucial for ensuring food and environmental safety. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1A schematic diagram of a three-dimensional folded paper-based microfluidic analytical device for food hazard detection;

[0048] Figure 2 A characterization diagram of the upconversion nanomaterial prepared in Example 1; wherein A is a transmission electron microscope diagram of the core / shell / shell structure upconversion nanomaterial, and B is a transmission electron microscope diagram of the Fe / Zr-MOF material;

[0049] Figure 3 A is a structural schematic diagram of a three-dimensional folded paper-based microfluidic analytical device, wherein ① is a hydrophilic functional area one, ② is a hydrophilic functional area two, and ③ is a hydrophilic functional area three; B is a scanning electron microscope diagram of different functional areas of the three-dimensional folded paper-based microfluidic analytical device;

[0050] Figure 4 A standard curve established for detecting different concentrations of chlorpyrifos in Example 1; wherein A is an upconversion fluorescence spectrum of the detection area under different concentrations of chlorpyrifos; B is a standard curve diagram based on the fluorescence signal value; C is the ultraviolet absorbance value of the detection area under different concentrations of chlorpyrifos; D is a standard curve diagram based on the color B value;

[0051] Figure 5 Specificity and anti-interference analysis of the three-dimensional folded paper-based microfluidic analytical device; wherein A is a molecular ball stick model of chlorpyrifos and commonly used veterinary drugs, and B is a result diagram of the specificity analysis and the anti-interference analysis. DETAILED DESCRIPTION

[0052] The terms described in the present application are only for describing the specific embodiments, and are not intended to limit the present application. In addition, for the numerical range in the present application, it is understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range of values and any other stated value or stated range of values is also included within the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between any document incorporated by reference and the present specification, the present specification controls.

[0054] Many modifications and variations to the illustrative embodiments described herein will be apparent to those skilled in the art from consideration of the specification and practice of the subject technology. Additional embodiments of the technology will be apparent to those skilled in the art from consideration of the specification and practice of the subject technology. It is intended that the specification and examples be considered as exemplary only, with a true scope of the subject technology being indicated by the following claims.

[0055] The method of the present application is a general detection method for food hazards, including any one of veterinary drugs, pesticides, pathogenic bacteria and toxins; wherein the veterinary drugs include malachite green, quinolones, tetracycline antibiotics; the pesticides include carbendazim, paraquat, organophosphorus pesticides such as chlorpyrifos, the pathogenic bacteria include escherichia coli, staphylococcus aureus; the toxins include aflatoxin, zearalenone, fumonisin. The target aptamer and the target aptamer complementary chain corresponding thereto can be directly purchased, and the aptamer and the aptamer complementary chain corresponding to the replacement of different hazards in the method of the present application can realize the detection of the corresponding hazards.

[0056] The food hazards of the present application take chlorpyrifos as an example for specific embodiment presentation; chlorpyrifos is purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., and the target aptamer and the target aptamer complementary chain thereof are purchased from Shengong Bioengineering (Shanghai) Co., Ltd., and the specific sequences are as follows: chlorpyrifos aptamer, 5'-CCT GCC ACG CTC CGC AAG CTT AGG GTT ACG CCT GCA GCG ATT CTT GAT CGC-3'; chlorpyrifos aptamer complementary chain, 5'-GCG ATC AAG CGC TGC AGG CGT AAC CCT AAG CTT GCG GAG CGT GGC AGG-NH2-3'. The present application does not involve a sequence listing, and the provided sequence is used as a conventional primer.

[0057] Figure 1 A schematic diagram of the three-dimensional folded paper-based microfluidic analysis device for food hazard detection is constructed; the specific steps are described in detail in the embodiment.

[0058] Embodiment 1:

[0059] Step one, preparation of fluorescence-enhanced upconversion nanomaterials:

[0060] (a) Accurately weigh 1 mmol yttrium chloride hexahydrate and dissolve in 10 mL methanol solution, then add 10 mL oleic acid and 10 mL 1-octadecene, mix and heat at 160 °C for 30 min, after stopping heating, cool the solution to room temperature, then add a methanol solution containing 0.2667 g sodium hydroxide and 0.34 g ammonium fluoride, and heat at 120 °C for 30 min, then further heat the mixed solution at 300 °C for 30 min, after heating, cool to room temperature, add 25 mL ethanol, centrifuge at 10000 rpm for 10 min to obtain the precipitate, then wash with a 1:1 mixture of cyclohexane and ethanol to obtain the upconversion nanoparticle seed;

[0061] (b) Accurately weigh 0.39 mmol thulium chloride hexahydrate and 0.01 mmol ytterbium chloride hexahydrate and dissolve in 10 mL methanol solution, then add 6 mL oleic acid and 16 mL 1-octadecene, mix and heat at 160 °C for 30 min, after the reaction is completed, cool to room temperature, add 2 mL of the upconversion nanoparticle seed solution prepared in step (a), and dropwise add a 10 mL methanol solution containing 0.0399 g sodium hydroxide and 0.0555 g ammonium fluoride, heat at 120 °C for 30 min, then further heat the mixed solution at 300 °C for 60 min, after heating, cool to room temperature, add 25 mL ethanol, centrifuge at 10000 rpm for 10 min to obtain the precipitate, then wash with a 1:1 mixture of cyclohexane and ethanol to obtain the core / shell upconversion nanoparticle; disperse it in 8 mL cyclohexane to obtain the core / shell upconversion nanoparticle solution;

[0062] (c) Accurately weigh 0.4 mmol yttrium chloride hexahydrate and dissolve in 10 mL methanol solution, then add 3 mL oleic acid and 8 mL 1-octadecene, mix and heat at 160 °C for 30 min, after the reaction is completed, cool, add 4 mL of the core / shell upconversion nanoparticle solution prepared in step (b), and dropwise add a 5 mL methanol solution containing 0.0399 g sodium hydroxide and 0.0555 g ammonium fluoride, and heat at 120 °C for 30 min, then further heat the mixed solution at 300 °C for 60 min, after heating, cool to room temperature, after the reaction is completed, add 25 mL ethanol, centrifuge at 10000 rpm for 10 min to obtain the precipitate, then wash with a 1:1 mixture of cyclohexane and ethanol to obtain the core / shell / shell upconversion nanoparticle, and dry at 60 °C for standby use;

[0063] Step two, surface biological functionalization of upconversion nanomaterials:

[0064] Accurately weigh 50.0 mg of the core / shell / shell structured upconversion nanoparticles prepared in step one into a mixed solution containing 6.0 mL of chloroform and 4.0 mL of toluene and ultrasonically treat for 10 min, then add a 20.0 mL aqueous solution containing 300.0 mg of polyacrylic acid and seal, and stir vigorously for 48 h; after the end, use a mixed solution of ethanol and ultrapure water in a volume ratio of 1:1 to clean and centrifuge, to obtain water-soluble upconversion nanomaterials modified with carboxyl; weigh 10 mg of the above upconversion nanomaterials, ultrasonically dissolve in 10 mL of Tris-HCl buffer solution with pH = 6, and then add 0.5 mL of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide solution with a concentration of 2 mg / mL and 0.5 mL of N-hydroxysuccinimide solution with a concentration of 1 mg / mL, and then place the mixed solution in a shaking bed at 25°C for 2 h; centrifugal separation to obtain upconversion nanomaterials with activated carboxyl groups;

[0065] Add 20 μL of amino-modified target aptamer complementary strands with a concentration of 100 μM to the activated carboxyl upconversion nanomaterial solution, incubate at 25°C on a shaking bed for 2 h, and then separate by washing twice with phosphate buffer solution, and re-dissolve the precipitate in 10 mL of STE buffer solution to obtain an upconversion nanomaterial solution modified with aptamer complementary strands on the surface;

[0066] Step three, preparation of a double-metal organic framework (Fe / Zr-MOF) with peroxidase activity:

[0067] Weigh zirconium (IV) chloride, iron chloride hexahydrate, and 2-amino terephthalic acid, and dissolve them in N,N-dimethylformamide to prepare solutions with a concentration of 0.5 mmol; then mix 5 mL of the zirconium chloride solution, 5 mL of the iron chloride hexahydrate solution, 10 mL of the 2-amino terephthalic acid solution, 5 mL of N,N-dimethylformamide, and 5 mL of acetic acid, and then transfer the mixed solution to a hydrothermal reaction kettle, and react at 120°C for 12 h; after the reaction is completed, cool to room temperature, collect the reaction product, and clean and centrifuge with a mixed solution of methanol and ethanol; collect the precipitate obtained by centrifugation and dry in an oven at 60°C overnight to obtain a yellow-brown powder, which is a double-metal organic framework with peroxidase activity, denoted as Fe / Zr-MOF

[0068] Figure 2 Characterization chart of the upconversion nanomaterials prepared in Example 1; wherein A is a transmission electron microscope chart of the core / shell / shell structured upconversion nanomaterials, and B is a transmission electron microscope chart of the Fe / Zr-MOF material;

[0069] It can be seen that the upconversion nanomaterials synthesized by high-temperature thermal decomposition method have uniform particle size, and the average diameter is about 45 nm, indicating that the shell successfully wraps the core; B is the transmission electron micrograph of Fe / Zr-MOF material, it can be seen that Fe / Zr-MOF material is well dispersed in the solution, uniform in size and shape, and presents an octahedral shape.

[0070] Step four, preparation of target aptamer modified Fe / Zr-MOF:

[0071] Accurately weigh 2 mg of Fe / Zr-MOF powder and dissolve it in 1 mL of Tris-HCl buffer with pH=7.4, then add 2.0 mL of STE buffer dissolved with 10 μL of target aptamer, and then incubate the mixed solution in a shaking bed at room temperature for 12 h. Remove the unbound chemicals and target aptamer by centrifugation, collect the precipitate and resuspend it in 1 mL of STE buffer to obtain a target aptamer modified Fe / Zr-MOF solution with a concentration of 2 mg / mL;

[0072] Step five, preparation of nanoprobes formed by coupling upconversion nanomaterials and Fe / Zr-MOF:

[0073] Take 2 mL of upconversion nanomaterial solution surface modified with target aptamer complementary strands, add 1 mL of target aptamer modified Fe / Zr-MOF solution prepared in step four, and incubate in a shaking bed at 25°C for 2 h; after the reaction is completed, centrifuge and suspend the remaining material in 3 mL of STE buffer to obtain a nanoprobe solution formed by coupling upconversion nanomaterials and Fe / Zr-MOF, with a concentration of 1.5 mg / mL;

[0074] Step six, preparation of a three-dimensionally folded paper-based microfluidic analysis device:

[0075] First, draw a paper-based pattern, which is composed of four parts in order: a sample layer, a transport layer, a detection layer, and an anti-pollution layer, wherein the sample layer, the transport layer, and the detection layer all contain a hydrophilic functional area, and the anti-pollution layer is a hydrophobic barrier area; the sample layer contains a circular hydrophilic functional area one with a diameter of 12 mm; the transport layer contains a circular hydrophilic functional area two with a diameter of 8 mm; the detection layer contains a circular hydrophilic functional area three with a diameter of 6 mm, wherein the hydrophilic functional area one, the hydrophilic functional area two, and the hydrophilic functional area three are inkjet printing-free areas and are independent of each other;

[0076] Cutting Whatman No. 1 filter paper with the size of 20 x 20 mm; printing the paper-based pattern drawn by Microsoft PowerPoint 2022 software on the filter paper using a laser printer; then placing the filter paper in a blast drying oven for heat curing treatment, the heat curing temperature is 200℃, and the curing time is 4h; after treatment, folding the filter paper in the clockwise and counterclockwise directions alternately according to the connection of the sample layer, the transport layer, the detection layer and the anti-pollution layer, and the three-dimensional folded paper-based microfluidic analysis device composed of the hydrophobic barrier and the hydrophilic functional area is obtained after folding;

[0077] Dropping 20uL of the nanoprobes solution prepared in step five into the detection area of the paper-based microfluidic analysis device; then, uniformly spraying the mixed solution of tetramethyl benzidine solution and 30% hydrogen peroxide into the detection area, and after natural air drying, the ready-to-use three-dimensional folded paper-based microfluidic analysis device is prepared;

[0078] Figure 3 Fig. A is a structural schematic diagram of the three-dimensional folded paper-based microfluidic analysis device, and Fig. B is a scanning electron microscope image of different functional areas of the three-dimensional folded paper-based microfluidic analysis device;

[0079] It can be clearly seen that the three-dimensional folded paper-based microfluidic analysis device is composed of four layers, the first layer is the sample layer, which contains a circular hydrophilic functional area one with a diameter of 12mm, and the scanning electron microscope image of the hydrophilic functional area one shows the morphology of the original paper base, the sample solution can be added dropwise from here; the second layer is the transport layer, which contains a circular hydrophilic functional area two with a diameter of 8mm, the sample solution can filter out macromolecular interfering substances and balance the sample solution when passing through this area; the third layer is the detection layer, which contains a circular hydrophilic functional area three with a diameter of 6mm, this area is loaded with nanoprobes solution to detect food hazards in the sample solution, and the scanning electron microscope image can clearly show that there are nanomaterials on the surface of the paper base; the fourth layer is the anti-pollution layer, which is composed of filter paper treated as a hydrophobic barrier, and from the scanning electron microscope image of the fourth layer, it can be seen that after the inkjet printed paper base is treated at high temperature, the graphite gradually melts and penetrates into the lower layer, and the fiber pores are completely filled.

[0080] Step seven, detection of the content of food hazards, taking chlorpyrifos as an example:

[0081] (a) Establishment of standard curves: First, standard solutions of chlorpyrifos at different concentrations (0.05-500 ng / mL) were prepared and added dropwise to the hydrophilic functional zone 1 of the sample introduction layer in the three-dimensional folded paper-based microfluidic analyzer obtained in step six. The samples flowed downward from the hydrophilic functional zone 1 through the circular hydrophilic functional zone 2 of the transport layer and finally reached the hydrophilic functional zone 3 of the detection zone. The samples were incubated at room temperature to allow the chlorpyrifos to react with the nanoprobes, resulting in three-dimensional folded paper-based microfluidic analyzers with different concentrations. Subsequently, a 980 nm laser was used as the excitation source to collect the fluorescence signal value and the B value of the paper-based image in the detection zone. Three points were selected in each detection zone as the average value of the fluorescence and image detection results. The concentration of chlorpyrifos was linearly fitted with the fluorescence signal value and the B value of the paper-based image to establish a standard curve for the detection of chlorpyrifos content.

[0082] (b) Detection of actual samples: The actual sample is pretreated to extract a test solution containing chlorpyrifos; the test solution is added dropwise to the sample layer of the three-dimensional folded paper-based microfluidic analyzer and incubated at room temperature; the fluorescence signal value and the paper-based color B value of the detection area are measured and respectively substituted into the chlorpyrifos content detection standard curve obtained in step seven (a) to calculate the chlorpyrifos content in the actual sample;

[0083] Figure 4 The standard curves for detecting different concentrations of chlorpyrifos in Example 1 are shown below; where A is the upconversion fluorescence spectrum of the detection area with different concentrations of chlorpyrifos; B is the standard curve based on the fluorescence signal value; C is the ultraviolet absorbance value of the detection area with different concentrations of chlorpyrifos; and D is the standard curve based on the color B value.

[0084] from Figure 4 As shown in Figure A, the fluorescence signal at 450 nm continuously increases with the increase of chlorpyrifos concentration; the standard curve for chlorpyrifos content detection was obtained by linear fitting: y = 6383.1x + 32236( Figure 4 B), Correlation coefficient R 2 =0.9966, the limit of detection (LOD) is 0.028 ng / mL, and the linear range is 0.05–500 ng / mL; furthermore, the absorbance value in the detection region changes accordingly with increasing chlorpyrifos concentration. Figure 4 C); Taking the B value in the color characteristic values ​​as an example, the standard curve for detection was obtained by linear fitting with the chlorpyrifos content: y = 12.1x + 104.9 ( Figure 4 D), correlation coefficient R 2 =0.9833, the limit of detection (LOD) was 0.043 ng / mL, and the linear range was 0.05–500 ng / mL.

[0085] (1) Detection of chlorpyrifos content in matcha;

[0086] Take 5 g of matcha powder in a 50 mL centrifuge tube, add 10 mL of ultrapure water and soak for 10 min, then add 30 mL of ethyl acetate, homogenate for 2 min, centrifuge at 5000 r / min for 5 min, collect the supernatant with anhydrous sodium sulfate column in a 250 mL concentration bottle, add 30 mL of ethyl acetate to the residue after centrifugation for re-extraction, then dehydrate with anhydrous sodium sulfate column and mix with the extract in a 250 mL concentration bottle; add 30 mL of ethyl acetate, dehydrate with anhydrous sodium sulfate column, re-extract the residue, and put the combined extract into a 250 mL flask, concentrate to dryness, then dilute to 2 mL with ethyl acetate-hexane solution, and finally filter with a 0.22 μm organic filter membrane to obtain the test solution;

[0087] Take 20 μL of the test solution for detection, drop it into the sample layer of the three-dimensional folded paper-based microfluidic analysis device, incubate at room temperature for 15 min, and then measure the fluorescence signal and color data (B value in RGB) of the detection zone. The above-constructed standard curve is brought in to calculate the content of chlorpyrifos in matcha as 9.58 and 10.14 ng / mL, respectively;

[0088] Table 1 Comparison of detection results of chlorpyrifos in matcha samples by the present method and HPLC method

[0089]

[0090] RSD a : relative standard deviation;

[0091] P b1 : two-tailed t-test results between fluorescence and HPLC methods;

[0092] P b2 : two-tailed t-test results between image and HPLC methods;

[0093] The detection amount of chlorpyrifos in matcha: X = (c x V x 1000) / (m x 1000), wherein:

[0094] X: the detection amount of chlorpyrifos in matcha, in units of micrograms per kilogram (μg / kg);

[0095] c: the detection concentration of chlorpyrifos in matcha, in units of nanograms per milliliter (ng / mL);

[0096] V: sample volume, in units of milliliters (mL);

[0097] m: the mass of matcha, in units of grams (g).

[0098] From Table 1, there is no significant difference between the HPLC method and the two detection modes of the three-dimensional folded paper-based microfluidic analytical device constructed (p>0.05), the results are accurate and reliable, which supports the practicability of the double-mode method for detecting chlorpyrifos in matcha samples.

[0099] (2) Detection of chlorpyrifos content in apples;

[0100] First, the apple sample was homogenized using a homogenizer, then 5g was weighed into a 50mL centrifuge tube; after adding 50mL acetonitrile and 6g sodium chloride, the mixture was vortexed for 2min and centrifuged at 4000r / min for 5min to take the supernatant; and the supernatant was dried under nitrogen at 80℃, then resuspended with 2mL methanol, and after resuspension, filtered with a 0.22μm organic filter membrane to remove suspended solids and impurities to obtain the test solution;

[0101] 20μL of the test solution was taken for detection, which was added dropwise to the sample layer of the three-dimensional folded paper-based microfluidic analytical device, and incubated at room temperature for 15min; after incubation, the fluorescence signal and color data (B value in RGB) of the detection zone were measured, and the standard curve constructed above was introduced to calculate the chlorpyrifos content in apples as 9.68 and 10.27ng / mL, respectively;

[0102] Table 2 Comparison of the detection results of chlorpyrifos in apple samples by the present method and HPLC method

[0103]

[0104]

[0105] a RSD: relative standard deviation;

[0106] b1 P: two-tailed t-test results between fluorescence and HPLC methods;

[0107] b2 P: two-tailed t-test results between image and HPLC methods;

[0108] Detection amount of chlorpyrifos in apples: X=(c×V×1000) / (m×1000) In the formula:

[0109] X: detection amount of chlorpyrifos in apples, unit: micrograms per kilogram (μg / kg);

[0110] c: detection concentration of chlorpyrifos in apples, unit: nanograms per milliliter (ng / mL);

[0111] V: sample constant volume, unit: milliliter (mL);

[0112] m: the mass of the apple, in grams (g).

[0113] As can be seen from Table 2, there was no significant difference between the HPLC method and the two detection modes of the three-dimensional folded paper-based microfluidic analytical device constructed (p>0.05), the results were accurate and reliable, and the practicability of the double-mode method for detecting chlorpyrifos in apple samples was supported.

[0114] Comparative Example 1:

[0115] Unlike Example 1, the detection object was 50 ng / mL of methy lchlorpyrifos.

[0116] Comparative Example 2:

[0117] Unlike Example 1, the detection object was 50 ng / mL of profenofos.

[0118] Comparative Example 3:

[0119] Unlike Example 1, the detection object was 50 ng / mL of diazinon.

[0120] Comparative Example 4:

[0121] Unlike Example 1, the detection object was 50 ng / mL of sulfotep.

[0122] Comparative Example 5:

[0123] Unlike Example 1, the detection object was 50 ng / mL of glyphosate.

[0124] Comparative Example 6:

[0125] Unlike Example 1, the detection object was 50 ng / mL of acetamiprid.

[0126] Comparative Example 7:

[0127] Unlike Example 1, the detection object was 50 ng / mL of bifenthrin.

[0128] Comparative Example 8:

[0129] Unlike Example 1, the detection object was 50 ng / mL of imidacloprid.

[0130] Comparative Example 9:

[0131] Unlike Example 1, the detection object was a mixed solution of 50 ng / mL of methy lchlorpyrifos and 5 ng / mL of chlorpyrifos.

[0132] Comparative Example 10:

[0133] The difference from Example 1 is that the test object is a mixed solution of 50 ng / mL profenofos and 5 ng / mL chlorpyrifos.

[0134] Comparative Example 11:

[0135] The difference from Example 1 is that the test object is a mixed solution of 50 ng / mL diazinon and 5 ng / mL chlorpyrifos.

[0136] Comparative Example 12:

[0137] The difference from Example 1 is that the test object is a mixed solution of 50 ng / mL fenitrothion and 5 ng / mL chlorpyrifos.

[0138] Comparative Example 13:

[0139] The difference from Example 1 is that the test object is a mixed solution of 50 ng / mL glyphosate and 5 ng / mL chlorpyrifos.

[0140] Comparative Example 14:

[0141] The difference from Example 1 is that the test object is a mixed solution of 50 ng / mL acetamiprid and 5 ng / mL chlorpyrifos.

[0142] Comparative Example 15:

[0143] The difference from Example 1 is that the test object is a mixed solution of 50 ng / mL bifenthrin and 5 ng / mL chlorpyrifos.

[0144] Comparative Example 16:

[0145] The difference from Example 1 is that the test object is a mixed solution of 50 ng / mL imidacloprid and 5 ng / mL chlorpyrifos.

[0146] Figure 5 In section A, a molecular ball-and-stick model of chlorpyrifos and eight similar or commonly used pesticides is used; the fluorescence signal characteristic values ​​of comparative examples 1-16 are shown in [reference needed]. Figure 5 B. By Figure 5 As can be seen from B, when the constructed detection method is applied to the detection of other pesticides—methyl chlorpyrifos, profenofos, diazinon, fenitrothion, glyphosate, acetamiprid, bifenthrin, and imidacloprid—even at a concentration 10 times that of chlorpyrifos (CPF), no significant change in the fluorescence signal of the system is observed. Only when a chlorpyrifos solution is added does the fluorescence intensity of the system show a significant change. Therefore, the detection method constructed in this invention exhibits high specificity and high sensitivity for chlorpyrifos.

[0147] Description: The above examples are only used to illustrate the technical solutions described in the present application and do not limit the present application; therefore, although the present application has been described in detail with reference to the above various embodiments, those of ordinary skill in the art should understand that the present application can still be modified or equivalently replaced; and all technical solutions and improvements that do not deviate from the spirit and scope of the present application should be covered within the scope of the claims of the present application.

Claims

1. A dual-mode detection method for food hazards based on a three-dimensional folded paper-based microfluidic analytical device, characterized in that, The method comprises the following steps: Step one, preparation of fluorescence enhanced upconversion nanomaterials; (a) Dissolve yttrium chloride hexahydrate in methanol A, then add oleic acid and 1-octadecene, mix and heat for the first time. After stopping heating, cool the solution to room temperature, then add a mixed solution containing sodium hydroxide, ammonium fluoride and methanol B, heat for the second time, continue to heat for the third time after the second heating, cool to room temperature after heating, centrifugal separation to obtain precipitate, and then clean and obtain upconversion nanoparticle seeds after washing; (b) Dissolve thulium chloride hexahydrate and ytterbium chloride hexahydrate in methanol A, then add oleic acid and 1-octadecene, mix and heat for the first time. After the reaction is completed and the solution is cooled to room temperature, add the upconversion nanoparticle seed solution prepared in step (a), and drop the mixed solution of sodium hydroxide, ammonium fluoride and methanol B, heat for the second time, continue to heat for the third time after the second heating, cool to room temperature after heating, centrifugal separation to obtain precipitate, and then clean and obtain core / shell structure upconversion nanoparticles after washing; (c) Dissolve yttrium chloride hexahydrate in methanol A, then add oleic acid and 1-octadecene, mix and heat for the first time. After the reaction is completed and the solution is cooled, add the core / shell structure upconversion nanoparticle solution prepared in step (b), and drop the mixed solution of sodium hydroxide, ammonium fluoride and methanol B, heat for the second time, then heat for the third time, cool to room temperature after heating, centrifugal separation to obtain precipitate after the reaction is completed, and then clean and obtain core / shell / shell structure upconversion nanoparticles after washing; Step two, surface biological functionalization of upconversion nanomaterials; S1, weigh the core / shell / shell structure upconversion nanoparticles prepared in step one into a mixed solution of trichloromethane and toluene, then add a mixed solution of polyacrylic acid and ultrapure water and seal, and stir for reaction; After the stirring reaction is completed, the mixed solution is centrifuged to collect the precipitate, and the precipitate is washed with a mixed solution of ethanol and ultrapure water and centrifuged to obtain water-soluble upconversion nanomaterials modified with carboxyl groups; S2, ultrasonically dissolve the water-soluble upconversion nanomaterials modified with carboxyl groups in Tris-HCl buffer solution, then add 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide solution and N-hydroxysuccinimide solution to obtain a mixed solution, and place the mixed solution in a shaker for first incubation; After incubation, centrifugal separation is performed to obtain upconversion nanomaterials with activated carboxyl groups; S3, add the upconversion nanomaterials with activated carboxyl groups to Tris-HCl buffer solution to obtain an upconversion nanomaterial solution with activated carboxyl groups; Then add the target aptamer complementary strand modified with amino groups to the upconversion nanomaterial solution with activated carboxyl groups, and incubate on a shaker for the second time. After incubation, the precipitate is collected by centrifugal separation, washed with phosphate buffer solution, and then separated. The precipitate is redissolved in STE buffer solution to obtain an upconversion nanomaterial solution modified with aptamer complementary strands on the surface; Step three, preparation of a bimetallic organic framework with peroxidase activity; First, weigh the zirconium chloride, ferric chloride hexahydrate and 2-amino terephthalic acid, respectively, and dissolve them in N,N-dimethylformamide to obtain a zirconium chloride solution, a ferric chloride hexahydrate solution and a 2-amino terephthalic acid solution; then mix the zirconium chloride solution, the ferric chloride hexahydrate solution, the 2-amino terephthalic acid solution, N,N-dimethylformamide and acetic acid to obtain a mixed solution, and then transfer it to a hydrothermal reactor for heating reaction; after the reaction is completed, cool it to room temperature, collect the reaction product, and wash it with a mixture of methanol and ethanol and centrifuge it; collect the precipitate obtained by centrifugation, dry it to obtain a yellow-brown powder, which is a bimetallic organic framework with peroxidase activity, denoted as Fe / Zr-MOF; Step four, preparation of target aptamer modified Fe / Zr-MOF: Weigh the Fe / Zr-MOF prepared in step three and dissolve it in Tris-HCl buffer, then add it to the STE buffer containing the target aptamer to obtain a mixed solution, and incubate it in a shaker; After incubation, collect the precipitate by centrifugation and resuspend it in STE buffer to obtain a target aptamer modified Fe / Zr-MOF solution; Step five, preparation of a nanoprobe formed by coupling upconversion nanomaterials with Fe / Zr-MOF: Take the upconversion nanomaterial solution modified with the complementary strand of the target aptamer prepared in step two, add the target aptamer modified Fe / Zr-MOF solution prepared in step four, and then incubate it in a shaker; after the reaction is completed, centrifuge it, collect the precipitate, and suspend it in STE buffer to obtain a nanoprobe solution formed by coupling upconversion nanomaterials with Fe / Zr-MOF; Step six, preparation of a three-dimensionally folded paper-based microfluidic analysis device: First, draw a paper-based pattern, which consists of four parts in order: a sample injection layer, a transport layer, a detection layer and a pollution prevention layer, wherein the sample injection layer, the transport layer and the detection layer all contain a hydrophilic functional area, and the pollution prevention layer is a hydrophobic barrier area; then cut the filter paper and use a laser to print the paper-based pattern on the filter paper; after printing, the filter paper is subjected to heat curing treatment, and then it is folded to obtain a three-dimensionally folded paper-based microfluidic analysis device composed of a hydrophobic barrier and a hydrophilic functional area; Add the nanoprobe solution prepared in step five to the detection area of the paper-based microfluidic analysis device; then, uniformly spray a mixed solution of tetramethylbenzidine and hydrogen peroxide onto the detection area, and after it is naturally dried, a treated three-dimensionally folded paper-based microfluidic analysis device is obtained; Step seven, detection of the content of hazardous substances in food: (a) Standard curve establishment: first, prepare food hazard standard solution with different concentrations, and drop into the sample layer of the three-dimensional folded paper-based microfluidic analysis device after treatment in step six, respectively. The food hazard standard solution flows from the hydrophilic functional area one to the hydrophilic functional area two of the transport layer, and finally reaches the hydrophilic functional area three of the detection area. Then, incubate at room temperature to make the food hazard react with the nano probe, and obtain the three-dimensional folded paper-based microfluidic analysis device with different concentrations. Then, collect the fluorescence signal value and the paper-based image B value of the detection area. Finally, linearly fit the food hazard concentration with the fluorescence signal value and the paper-based image B value, respectively, to establish the standard curve for detecting the content of food hazards; (b) Actual sample detection: pretreat the sample to be tested to obtain a solution containing food hazards. Then, drop the solution into the sample layer of the three-dimensional folded paper-based microfluidic analysis device, and incubate at room temperature. Then, measure the fluorescence signal value and the paper-based color B value of the detection area, and input them into the standard curve for detecting the content of food hazards obtained in step (a) to calculate the content of food hazards in the actual sample.

2. The method according to claim 1, wherein, In (a) of step one, the amount of yttrium chloride hexahydrate, methanol A, oleic acid, 1-octadecene, sodium hydroxide, ammonium fluoride and methanol B is 303.4 mg: 10 mL: 10 mL: 10 mL: 266.7 mg: 340 mg: 10 mL. The first heating temperature is 160 ℃, and the time is 30 min. The second heating temperature is 120 ℃, and the time is 30 min. The third heating temperature is 300 ℃, and the time is 60 min. The concentration of the upconversion nanoparticle seed solution is 1-2 mg / mL. In step (b), the amount of ytterbium chloride hexahydrate, thulium chloride hexahydrate, methanol A, oleic acid, 1-octadecene, upconversion nanoparticle seed solution, sodium hydroxide, ammonium fluoride and methanol B is 151.2 mg: 2.9 mg: 10 mL: 6 mL: 16 mL: 2 mL: 39.9 mg: 55.5 mg: 10 mL. The first heating temperature is 160 ℃, and the time is 30 min. The second heating temperature is 120 ℃, and the time is 30 min. The third heating temperature is 300 ℃, and the time is 60 min. The concentration of the core / shell structure upconversion nanoparticle solution is 1-2 mg / mL. In step (c), the amount of yttrium chloride hexahydrate, methanol A, oleic acid, 1-octadecene, core / shell structure upconversion nanoparticle solution, sodium hydroxide, ammonium fluoride and methanol B is 121.3 mg: 10 mL: 3 mL: 8 mL: 4 mL: 39.9 mg: 5 mg: 5 mL. The first heating temperature is 160 ℃, and the time is 30 min. The second heating temperature is 120 ℃, and the time is 30 min. The third heating temperature is 300 ℃, and the time is 40 min. The centrifugal separation conditions in (a)-(c) of step one are all 8000-9000 rpm for 10-15 min; the washing is all by using a cyclohexane and ethanol mixed solution with a volume ratio of 1:1 for centrifugal washing.

3. The method according to claim 1, wherein the method is a dual-mode detection method for food hazards based on a three-dimensional folded paper-based microfluidic analytical device. In S1 of step two, the amount ratio of the core / shell / shell structured upconversion nanomaterial, chloroform, toluene, polyacrylic acid and ultrapure water is 50 mg:(2-6 mL):(4-10 mL):(300-400 mg):(15-20 mL); the stirring reaction time is 24-48 h, the volume ratio of ethanol and ultrapure water is 1:1; the centrifugal separation condition is 8000-10000 rpm for 8-10 min.

4. The method according to claim 1, wherein, In S2 of step two, the amount ratio of the water-soluble upconversion nanomaterial modified with carboxyl, Tris-HCl buffer solution, 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide solution and N-hydroxysuccinimide solution is 10 mg:10 mL:0.5 mL:0.5 mL, wherein the concentration of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide solution is 2 mg / mL, the concentration of N-hydroxysuccinimide solution is 1 mg / mL; the pH of Tris-HCl is 6; the first incubation temperature is 25 ℃, the time is 1-2 h, and the shaking bed speed is 300-400 rpm.

5. The method according to claim 1, wherein, In S3 of step two, the amount ratio of the upconversion nanomaterial solution with activated carboxyl and the target aptamer complementary strand modified with amino is 10 mg:20 μL, wherein the concentration of the upconversion nanomaterial solution with activated carboxyl is 1-2 mg / mL; the concentration of the target aptamer complementary strand modified with amino is 100 μM; the pH of Tris-HCl is 7.4; the second incubation temperature is 25 ℃, the time is 2-4 h, and the shaking bed speed is 200-300 rpm; the concentration of the upconversion nanomaterial solution of the surface modified aptamer complementary strand is 1 mg / mL.

6. The method according to claim 1, wherein, In step three, the amount ratio of the zirconium chloride solution, the ferric chloride hexahydrate solution, the 2-amino terephthalic acid solution, N,N-dimethylformamide and acetic acid is 5.0 mL:5.0 mL:10.0 mL:5.0 mL:5.0 mL; wherein the concentration of the zirconium chloride solution, the ferric chloride hexahydrate solution and the 2-amino terephthalic acid solution is all 0.5 mmol; The heating reaction condition is 120 ℃ for 24 h; the volume ratio of methanol and ethanol is 1:1; the centrifugal separation condition is 6000-8000 rpm for 10-15 min.

7. The method according to claim 1, wherein, In step four, the amount of Fe / Zr-MOF, Tris-HCl buffer, target aptamer and STE buffer is 2.0 mg: 1.0 mL: 10.0 μL: 2.0 mL; the pH of the STE buffer is 7.4, and the molar concentration is 10 mmol / L; the concentration of the target aptamer is 100 μM; the incubation reaction condition is 25 ℃ for 12 h; the centrifugal separation condition is 6000-8000 rpm for 8-10 min; and the concentration of the target aptamer modified Fe / Zr-MOF solution is 2 mg / mL.

8. The method according to claim 1, wherein, In step five, the amount of the upconversion nanomaterial solution modified with the target aptamer complementary strand, the target aptamer modified Fe / Zr-MOF solution and the STE buffer is 2.0 mL: 1.0 mL: 3.0 mL; the pH of the STE buffer is 7.4, and the molar concentration is 10 mmol / L; the incubation reaction condition is 25 ℃ for 2 h; the centrifugal separation condition is 6000-8000 rpm for 8-10 min; and the concentration of the nanoprobes solution is 1.5 mg / mL.

9. The method according to claim 1, wherein, In step six, the filter paper is Whatman No. 1 filter paper, and the size is 20×20 mm; the paper-based pattern is composed of four parts, the first part is a sample loading layer containing a circular hydrophilic functional area one with a diameter of 12 mm; the second part is a transport layer containing a circular hydrophilic functional area two with a diameter of 8 mm; the third part is a detection layer containing a circular hydrophilic functional area three with a diameter of 6 mm; and the fourth part is a pollution prevention layer, which is composed of a hydrophobic barrier; wherein the hydrophilic functional area one, the hydrophilic functional area two and the hydrophilic functional area three are independent of each other; the heat curing temperature is 180-200 ℃, and the curing time is 4-5 h; the concentration of the tetramethylbenzidine solution is 30 mmol / L; the amount of the nanoprobes solution, the tetramethylbenzidine solution and 30% hydrogen peroxide is 5 uL: 1 uL: 1 uL, and the mass fraction of hydrogen peroxide is 30%. The folding is clockwise and counterclockwise in the connection of the sample loading layer, the transport layer, the detection layer and the pollution prevention layer.

10. The method according to claim 1, wherein, In step seven (a), the food hazards include but are not limited to chlorpyrifos; the dropwise addition amount of the food hazard standard solution is 20 uL, and the concentration of the food hazard standard solution ranges from 0.05 ng / mL to 500 ng / mL; the incubation time at room temperature is 15 min; in step seven (b), the dropwise addition amount of the test solution is 20 uL; and the incubation time is 15 min.

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