A dual-mode detection method for food hazards based on a multi-channel microfluidic paper-based chip

Abstract

The application belongs to the field of food safety detection, and particularly relates to a food hazard detection method based on a multi-channel microfluidic paper-based chip. First, oil-soluble upconversion nanomaterial, water-soluble upconversion nanomaterial and upconversion fluorescent probes are prepared; and magnetic nanomaterial is prepared. Then, nanocomposite material with high quenching activity is prepared based on the magnetic nanomaterial. Further, target aptamer modified nanocomposite material is prepared. The nanoprobes are formed by coupling the upconversion nanomaterial and the nanocomposite material. Finally, the multi-channel paper-based microfluidic chip is prepared by using the paper-based microfluidic chip and the nanoprobes, so as to realize detection of the content of hazards in food. The application takes glyphosate, acetamiprid and malathion as examples, realizes detection of the content of corresponding hazards in food by constructing a standard curve, is not only fast but also accurate, and has good practical prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of food safety detection, and particularly relates to a food hazard detection method based on a multi-channel microfluidic paper-based chip. BACKGROUND

[0002] Food safety is related to human health and social stability. With the change of the times and the development of global economic trade, food safety is increasingly concerned, and has become one of the most prominent public health problems in the world. Chemical hazards are the main food safety hazard factors leading to food poisoning and foodborne diseases, mainly including pesticides, veterinary drugs, heavy metals, allergens, food additives, etc. Foods may be contaminated in the production, harvesting, processing and transportation and storage of raw materials, among which common food hazards include glyphosate, acetamiprid, malathion, paraquat and other pesticides, and chloramphenicol, malachite green and other veterinary drugs.

[0003] Long-term accumulation of food hazards in the human body may cause a series of health hazards, including organ damage, impaired intelligence, kidney failure, and even endanger life. Therefore, it is of great significance to develop a rapid, specific and sensitive detection method for food hazards for food safety and human health.

[0004] At present, conventional food hazard detection methods, such as gas chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay, etc., have the disadvantages of high pretreatment requirement, expensive instrument price, complicated operation steps and long sample detection time, etc., and cannot meet the requirements of on-site rapid detection. SUMMARY

[0005] In view of the deficiencies of the prior art, the application provides a food hazard dual-mode detection method based on a multi-channel microfluidic paper base, so as to solve the problems of high detection cost and complicated detection steps in the prior art, and realize rapid and on-site detection of food hazards.

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

[0007] A food hazard dual-mode detection method based on a multi-channel microfluidic paper base, and the specific steps are as follows:

[0008] Step 1: Preparation of oil-soluble upconversion nanomaterials:

[0009] Yttrium chloride hexahydrate, ytterbium chloride hexahydrate and thulium chloride hexahydrate are added to a certain volume of methanol A and ultrasonically dissolved, then oleic acid and 1-octadecene are added and mixed thoroughly, and then the first heating and stirring is carried out under a closed condition. After stirring, it is cooled to room temperature. The closed condition is removed, and then methanol B containing sodium hydroxide and ammonium fluoride is added dropwise. The second heating and stirring is carried out under a closed condition. After stirring, it is cooled to room temperature. The precipitate is obtained by centrifugal separation. The first washing is carried out using a mixture of ultrapure water and ethanol. Then, the second washing is carried out using a cyclohexane solution. After drying, the oleic acid-coated upconversion nanomaterial is obtained and is ready for use.

[0010] Step two, preparation of water-soluble upconversion nanomaterial:

[0011] The oleic acid-coated upconversion nanoparticles prepared in step one are weighed and dispersed in a hydrochloric acid solution and ultrasonically treated. After treatment, ethanol is added and ultrasonically dispersed uniformly. Then, ultrapure water and ammonia water are added and stirred uniformly under a certain temperature condition. After uniform stirring, tetraethyl orthosilicate is added for the first reaction, and 3-aminopropyl triethoxysilane is added for the second reaction. After reaction, centrifugal separation is carried out. The obtained product is washed with ultrapure water. After washing, it is dispersed in ultrapure water to obtain a water-soluble upconversion nanomaterial solution.

[0012] Step three, preparation of upconversion nanomaterial fluorescent probe:

[0013] S1, the upconversion nanomaterial solution prepared in step two is added to a glutaraldehyde aqueous solution and an activation reaction is carried out on a shaking bed. After activation reaction, centrifugal separation is carried out to obtain the upconversion nanomaterial. Then, it is dispersed in a phosphate buffer A. Then, the target aptamer complementary chain solution A is added, and incubation is carried out on a shaking bed. After incubation, centrifugal separation is carried out. The obtained precipitate is washed with a phosphate buffer and then centrifugally collected. Then, the precipitate is redissolved in a phosphate buffer to obtain a dispersion liquid, which is recorded as upconversion fluorescent probe solution A.

[0014] S2, the same as S1, except that the target aptamer complementary chain solution A is replaced by the target aptamer complementary chain solution B and the target aptamer complementary chain solution C. The rest of the operations are the same. Finally, the upconversion fluorescent probe solution B and the upconversion fluorescent probe solution C are obtained.

[0015] Step four, preparation of magnetic nanomaterial (Fe3O4):

[0016] Firstly, the trichloride iron hexahydrate, poly(4-styrene sulfonic acid-co-maleic acid) sodium salt, anhydrous sodium acetate and ethylene glycol were mixed and then subjected to the first heating and stirring reaction. After the reaction, sodium hydroxide was added and subjected to the second stirring until dissolved. Then, the obtained solution was transferred into a stainless steel high-pressure reaction kettle lined with polytetrafluoroethylene for hydrothermal reaction. After the reaction, it was cooled to room temperature, and the reaction product was collected and washed with a mixed solution of ethanol and pure water and then centrifuged. The collected precipitate was dried in a vacuum oven overnight and marked as Fe3O4 for standby use.

[0017] Step five, preparation of nanocomposite (Fe3O4@Cu) with high quenching activity:

[0018] The magnetic nanomaterial prepared in step four was weighed and dispersed in a copper sulfate solution for the first heating and stirring reaction, and then sodium borohydride was added for the second stirring reaction. The precipitate was obtained by magnetic separation and washed with a mixed solution of ethanol and pure water and then centrifuged. The precipitate obtained by centrifugation was dried in vacuum to obtain nanocomposite with high quenching activity, marked as Fe3O4@Cu.

[0019] Step six, preparation of target aptamer modified Fe3O4@Cu:

[0020] S1, the Fe3O4@Cu material prepared in step five was dissolved in phosphate buffer A, and 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide solution and N-hydroxy succinimide solution were added for activation reaction on a shaker. After activation, the target A aptamer solution was added and incubated on a shaker. After incubation, it was washed with phosphate buffer and centrifuged. The precipitate was re-dissolved in phosphate buffer B to obtain the dispersion, marked as Fe3O4@Cu solution A.

[0021] S2, the same as S1, except that the target A aptamer solution was replaced by target B aptamer solution and target C aptamer solution. The rest of the operation was the same, and finally Fe3O4@Cu solution B and Fe3O4@Cu solution C were obtained.

[0022] Step seven, preparation of nanoprobes formed by coupling of upconversion fluorescent nanomaterials and Fe3O4@Cu:

[0023] S1, the upconversion fluorescent probe solution A with target aptamer complementary chain modified on the surface prepared in step three was taken, and the target aptamer modified Fe3O4@Cu solution A prepared in step six was added, and then placed on a shaker for incubation. After the reaction, it was centrifuged, and the precipitate was suspended in phosphate buffer A to obtain the nanoprobes formed by coupling of upconversion fluorescent nanomaterials and Fe3O4@Cu.

[0024] S2, the same as S1, the difference is that the up-conversion fluorescent probe solution A is replaced by up-conversion fluorescent probe solution B and up-conversion fluorescent probe solution C; the target aptamer modified Fe3O4@Cu solution A is replaced by target aptamer modified Fe3O4@Cu solution B and target aptamer modified Fe3O4@Cu solution C; the rest of the operation is the same, finally get nano probe solution B and nano probe solution C;

[0025] Step eight, preparation of multi-channel microfluidic paper-based chip:

[0026] Cut the filter paper, and print the prepared paper pattern on the filter paper using a laser toner printer, then place the filter paper in a forced air drying oven for heat curing treatment, after natural cooling to room temperature, the printed filter paper is taken out from the oven and cut, to obtain a paper-based microfluidic chip composed of a hydrophobic barrier zone and a hydrophilic flow zone;

[0027] Wherein, the printer ink is solidified into the fiber pores of the chromatography filter paper, forming a hydrophobic barrier zone where the solution cannot flow; on one side of the hydrophobic barrier zone, a microfluidic detection area is surrounded by ink; the microfluidic detection area is composed of detection area one, detection area two, detection area three, sample addition area and flow channel, the detection area one, detection area two and detection area three are independent of each other and are connected to the sample addition area through the flow channel;

[0028] Finally, the nano probe solution A prepared in step seven is added dropwise to the detection area one of the microfluidic paper-based chip, the nano probe solution B is added dropwise to the detection area two of the microfluidic paper-based chip, and the nano probe solution C is added dropwise to the detection area three of the microfluidic paper-based chip, to obtain a multi-channel paper-based microfluidic chip for detection.

[0029] Step nine, detection of the content of hazards in food:

[0030] (a) Establishment of standard curve: First, prepare food hazard A standard solution, food hazard B standard solution and food hazard C standard solution with different concentrations, and add them dropwise to the sample addition area of the multi-channel paper-based microfluidic chip obtained in step eight, and then through the flow channel one, flow channel two and flow channel three to the detection area one, detection area two and detection area three, incubate at room temperature, so that the food hazard A, food hazard B and food hazard C fully react with the up-conversion fluorescent probe, to obtain multi-channel paper-based microfluidic chips with different concentrations; after incubation, the fluorescence signal value of the detection area one is collected, and then the food hazard A concentration and fluorescence intensity are linearly fitted to establish the standard curve for detecting the content of food hazard A;

[0031] Similarly, the fluorescence signal value of the detection area two is collected, and the standard curve for detecting the content of the food hazard B is established by linear fitting of the food hazard B concentration and the fluorescence intensity; the fluorescence signal value of the detection area three is collected, and the standard curve for detecting the content of the food hazard C is established by linear fitting of the food hazard C concentration and the fluorescence intensity.

[0032] Meanwhile, the image of the detection area one is recorded, and the color characteristic value A is extracted to establish the standard curve of the color characteristic value A and the food hazard A concentration; then, the image of the detection area two is recorded, and the color characteristic value B is extracted to establish the standard curve of the color characteristic value B and the food hazard B concentration; finally, the image of the detection area three is recorded, and the color characteristic value C is extracted to establish the standard curve of the color characteristic value C and the food hazard C concentration.

[0033] (b) Actual sample detection: the actual sample is pretreated to extract a solution containing food hazards; the solution is added to the sample adding area of the multi-channel microfluidic paper-based chip, and after incubation at room temperature, the fluorescence intensity of the detection area one is measured, the corresponding image is taken to extract the color characteristic value, and the content of the food hazard A in the actual sample is calculated by using the food hazard A content detection standard curve obtained in step nine; the fluorescence intensity of the detection area two is measured, the corresponding image is taken to extract the color characteristic value, and the content of the food hazard B in the actual sample is calculated by using the food hazard B content detection standard curve obtained in step nine; the fluorescence intensity of the detection area three is measured, the corresponding image is taken to extract the color characteristic value, and the content of the food hazard C in the actual sample is calculated by using the food hazard C content detection standard curve obtained in step nine;

[0034] Preferably, in step one, the amount of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, thulium chloride hexahydrate, methanol A, oleic acid, 1-octadecene, sodium hydroxide, ammonium fluoride and methanol B is 118.3 mg:38.8 mg:0.7 mg:10 mL:4.0 mL:9.0 mL:50.0 mg:74.1 mg:10 mL; the mixture of cyclohexane and ethanol is used for cleaning, and the volume ratio of cyclohexane to ethanol is 1:3; the first heating and stirring time is 20-30 min, and the heating temperature is 160°C; the second heating and stirring temperature is 300°C, and the stirring time is 1.0-1.5 h; the centrifugal separation condition is that the rotation speed is 8000-10000 rpm / min, and the time is 5-10 min.

[0035] Preferably, in step two, the ratio of the upconversion nanomaterial, hydrochloric acid solution, ethanol, ultrapure water, ammonia, tetraethyl orthosilicate, 3-aminopropyl triethoxysilane is 20 mg: 1 mL: 60 mL: 10 mL: 2.5 mL: 20 uL: 50 uL; the first reaction time is 4-6 h, and the second reaction time is 2-3 h; the concentration of the hydrochloric acid solution is 0.1 mol / L; the temperature condition is 65℃; the centrifugal separation condition is: the speed is 8000-10000 rpm / min, and the time is 5-10 min; and the concentration of the water-soluble upconversion nanomaterial solution is 1 mg / mL.

[0036] Preferably, in step three, the ratio of the water-soluble upconversion nanomaterial solution, glutaraldehyde aqueous solution, phosphate buffer A, and target aptamer complementary chain solution A is 10 mg: 1.0 mL: 5.0 mL: 60 uL; the pH of the phosphate buffer is 7.4; the concentration of the upconversion fluorescent probe solution A is 1 mg / mL; the concentration of the upconversion fluorescent probe solution B is 1 mg / mL; the concentration of the upconversion fluorescent probe solution C is 1 mg / mL; the activation reaction temperature is 25℃, the time is 1-2 h, and the shaking speed is 200-300 rpm / min; the incubation temperature is 25℃, the time is 10-12 h, and the shaking speed is 200-300 rpm / min; and the centrifugal separation condition is: the speed is 6000 rpm / min, and the time is 5-10 min.

[0037] Preferably, in step four, the ratio of the ferric chloride hexahydrate, poly(4-styrenesulfonic acid-co-maleic acid) sodium salt, sodium acetate, ethylene glycol, and sodium hydroxide is 1.08 g: 1.0 g: 2.5 g: 40.0 mL: 0.6 g; the cleaning is performed using a mixed solution of ethanol and pure water, and the volume ratio of ethanol to pure water is 1:1; the first heating and stirring time is 30 min, and the heating temperature is 50℃; the second stirring time is 90 min, and the temperature is 50℃; and the hydrothermal reaction time is 9 h, and the heating temperature is 200℃.

[0038] Preferably, in step five, the ratio of the magnetic nanomaterial and sodium borohydride is 0.1 g: 1.0 g; the concentration of the copper sulfate solution is 0.2 mol / mL; the cleaning is performed using a mixed solution of ethanol and pure water, and the volume ratio of ethanol to pure water is 1:1; the first heating and stirring time is 30 min, and the heating temperature is 50℃; and the second heating and stirring time is 2 h, and the heating temperature is 50℃.

[0039] Preferably, in step six, the amount of Fe3O4@Cu, phosphate buffer A, 1-ethyl-(3- dimethylaminopropyl) carbodiimide solution, N-hydroxysuccinimide solution, target A aptamer solution, Fe3O4@Cu solution A is 10.0 mg: 1.0 mL: 1.0 mL: 10.0 mL: 60 uL; the concentration of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution is 2 mg / mL, and the concentration of the N-hydroxysuccinimide solution is 1 mg / mL; the pH of the phosphate buffer is 7.4; the concentration of the Fe3O4@Cu solution A is 1 mg / mL; the concentration of the Fe3O4@Cu solution B is 1 mg / mL; the concentration of the Fe3O4@Cu solution C is 1 mg / mL; the temperature of the activation reaction is 25°C, and the time is 1-2 h, with a shaking speed of 200-300 rpm / min; the temperature of the incubation is 25°C, and the time is 8-10 h, with a shaking speed of 200-300 rpm / min; the centrifugal separation conditions are a speed of 6000 rpm / min and a time of 5-10 min.

[0040] Preferably, in step seven, the amount of the upconversion fluorescent probe solution A modified with the target aptamer complementary strand, the target aptamer modified Fe3O4@Cu solution A, and the phosphate buffer A is 1.0 mL: 0.5 mL: 1.0 mL; the pH of the phosphate buffer is 7.4; the concentration of the nanoprobe solution A is 1.0 mg / mL, the concentration of the nanoprobe solution B is 1.0 mg / mL, and the concentration of the nanoprobe solution C is 1.0 mg / mL; the temperature of the incubation reaction is 25°C, and the time is 1-2 h, with a shaking speed of 200-300 rpm / min.

[0041] Preferably, in step eight, the filter paper is Whatman No. 1 chromatography filter paper, with a size of 15 x 20 mm; the microfluidic pattern includes a sample loading area 6, which is a semicircular area with a diameter of 10 mm; detection area one, detection area two, and detection area three, which are circular areas with a diameter of 5 mm; sample flow channel one, sample flow channel two, and sample flow channel three, which have a length of 3 mm and a width of 1 mm; wherein sample flow channel one is connected to the sample loading area and detection area one, sample flow channel two is connected to the sample loading area and detection area two, and sample flow channel three is connected to the sample loading area and detection area three; the temperature of the heat curing treatment is 200°C, and the curing time is 3-4 h; the amount of the nanoprobe solution A, the nanofluorescent probe solution B, and the nanoprobe solution C is 4 uL: 4 uL: 4 uL.

[0042] Preferably, in (a) of step nine, the food hazards A, food hazards B and food hazards C are different food hazards, and different letters only distinguish different food hazards in name; wherein the food hazards include but are not limited to glyphosate, acetamiprid and malathion; the concentration of the food hazard standard solution ranges from 1 to 5000 ng / mL; the dosages of the hazard A standard solution, the hazard B standard solution and the hazard C standard solution are 30 uL: 30 uL: 30 uL.

[0043] The characteristic values of the color characteristic value A, the color characteristic value B and the color characteristic value C are B: R: G in RGB respectively; the incubation time at room temperature is 30 min; and the fluorescence signal value is a fluorescence intensity value at 450 nm under excitation of 980 nm excitation light.

[0044] The step of extracting the image color characteristic value of the microfluidic paper-based chip is as follows: a fluorescence image of the solid-phase biosensor under excitation of 980 nm excitation light at 450 nm is photographed by using a smart phone, and corresponding RGB values are extracted, that is, the required color characteristic value.

[0045] Preferably, in (b), the dosages of the to-be-detected solutions are all 30 uL; and the incubation times are all 30 min.

[0046] Methanol A and methanol B are both methanol, and phosphate buffer A and phosphate buffer B are both phosphate buffer, and different letters only distinguish different names; similarly, the food hazards A, the food hazards B and the food hazards C refer to different food hazards, and target A, target B and target C also refer to different food hazards to be detected, and different letters only distinguish different names.

[0047] The present application discloses the following technical effects:

[0048] 1. The present application discloses a food hazard dual-mode detection method based on a multi-channel microfluidic paper-based chip, which uses upconversion fluorescent nanoparticles as a fluorescent donor, uses Fe3O4@Cu nanocomposites with high quenching activity as a fluorescent acceptor, deposits the prepared fluorescent nanoprobe in a microfluidic paper-based detection area, realizes accurate quantitative detection of a target based on specific recognition of the target by an aptamer to cause a change in a characteristic fluorescence signal, and only needs a very small amount of sample liquid (30 muL) to realize high-sensitivity real-time detection of food hazards.

[0049] 2.The application discloses a dual-mode detection method for food hazards, and can realize simultaneous detection of three food hazards based on a prepared multi-channel microfluidic paper-based chip, and specifically comprises the following steps: three independent detection zones are prepared on a paper base through laser toner printing combined with a heat curing technology; and fluorescent nano probes are deposited in the microfluidic paper-based detection zones, specific recognition of targets based on aptamers in each detection zone leads to changes in characteristic fluorescence signals and image characteristic values, simultaneous quantitative detection of multiple hazards is realized, and the diet health and safety of the public are ensured.

[0050] 3.The paper-based sensing detection method for food hazards established by the application has a linear concentration range of 1ng / mL-5*10 3 ng / mL for glyphosate, 1ng / mL-5*10 3 ng / mL for acetamiprid, and 1ng / mL-5*10 3 ng / mL for malathion, and a detection limit LOD of 0.16ng / mL for glyphosate, 0.77ng / mL for acetamiprid, and 0.34ng / mL for malathion. 3 ng / mL for glyphosate, 1ng / mL-5*10 3 ng / mL for acetamiprid, and 1ng / mL-5*10 3 ng / mL for malathion, and a detection limit LOD of 0.37ng / mL for glyphosate, 0.59ng / mL for acetamiprid, and 0.28ng / mL for malathion. The designed paper-based sensing method can meet the requirements of high-sensitivity simultaneous detection of food hazards, has good universality, and can provide a theoretical basis for real-time monitoring of food hazards in actual detection processes.

[0051] 4.The specific detection system constructed by the application has a strong fluorescence response to different food hazards, can effectively eliminate background fluorescence and interference of other molecules, has high specificity for detection of hazards, can realize high-sensitivity detection of the content of hazards, and can detect different hazards by replacing different aptamer sequences, thereby overcoming the shortcomings of traditional methods and being crucial for ensuring food and environmental safety. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 It is a schematic diagram of a multi-channel microfluidic paper-based chip for food hazard detection.

[0053] Figure 2The characterization figure of the upconversion nanomaterial prepared in Example 1; wherein A is the transmission electron microscope figure of the water-soluble upconversion nanomaterial, and B is the scanning electron microscope figure of the Fe3O4@Cu material;

[0054] Figure 3 The characterization data of the aptamer sequence modified nanomaterial; wherein A is the ultraviolet absorption figure before and after the upconversion nanomaterial modified aptamer complementary chain, and B is the ultraviolet absorption figure before and after the Fe3O4@Cu material modified aptamer.

[0055] Figure 4 It is a structure diagram of a portable device for detecting food hazards based on a multi-channel paper-based microfluidic chip, and the reference signs are as follows: 1-hydrophobic barrier region, 2-detection region one, 3-flow channel one, 4-detection region two, 5-flow channel two, 6-sample adding region, 7-flow channel three, and 8-detection region three.

[0056] Figure 5 It is a fluorescence standard curve established by detecting different concentrations of glyphosate, acetamiprid and malathion in Example 1; wherein A is the sensor fluorescence signal figure for detecting different concentrations of glyphosate; B is a standard curve established by the glyphosate concentration and the fluorescence intensity signal characteristic value of the sensor at 450 nm; C is the sensor fluorescence signal figure for detecting different concentrations of acetamiprid; D is a standard curve established by the acetamiprid concentration and the fluorescence intensity signal characteristic value of the sensor at 450 nm; E is the sensor fluorescence signal figure for detecting different concentrations of malathion; and F is a standard curve established by the malathion concentration and the fluorescence intensity signal characteristic value of the sensor at 450 nm.

[0057] Figure 6 It is a fluorescence standard curve for establishing image color characteristic values for detecting different concentrations of glyphosate, acetamiprid and malathion in Example 1.

[0058] Figure 7 It is the specificity and anti-interference analysis of the multi-channel microfluidic paper-based chip. DETAILED DESCRIPTION

[0059] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be 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 intermediate value in the stated range and any other stated value or intermediate value in the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0060] Unless defined otherwise, 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 any 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 patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference for the disclosure and the description of the procedures and / or materials in connection with which the disclosure was made. In the case of conflict, the present specification will control.

[0061] Many modifications and variations of this application of the application described herein will be apparent to those of ordinary skill in the art without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in scope by the specific embodiments described herein. Rather, the intent is to embrace all changes and modifications that fall within the spirit and scope of the claims.

[0062] The method of the present application is a general detection method for food hazards, including any one of pesticides, veterinary drugs, pathogenic bacteria and toxins; wherein the pesticides include glyphosate, acetamiprid, carbendazim, paraquat, etc. organophosphorus pesticides; veterinary drugs include quinolones, malachite green, tetracycline antibiotics; pathogenic bacteria include escherichia coli, staphylococcus aureus; toxins include aflatoxin, zearalenone, fumonisin. The corresponding target aptamer and target aptamer complementary chain can be directly purchased, and the aptamer and aptamer complementary chain corresponding to the replacement of different hazards in the method described in the present application can realize the detection of the corresponding hazards.

[0063] The food hazards of the present application are exemplified by glyphosate, acetamiprid and malathion. The chlorpyrifos is purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., and the target aptamer and target aptamer complementary chain are purchased from Shengong Bioengineering (Shanghai) Co., Ltd. The specific sequences are as follows: glyphosate aptamer, 5'-TGC TAG ACG ATATTC GTCCAT CCG AGC CCG TGG CGG GTC TTA GGA CTC TGC GGC GGG CTT CGC GGC GCT GTC AGACTG AAT ATG TCA-3'; acetamiprid aptamer, 5'-CTGAC ACCAT ATTAT GAAGA-3'; malathion aptamer, 5'-AGC TTG CTG CAG CGA TTC ATC GCC ACAGAG CT-3'; glyphosate aptamer complementary chain, 5'-TGC TAGACG ATATTC GTC CAT CCG AGC CCG TGG CGG GTC TTA GGA CTC TGC GGC GGG CTT CGCGGC GCT GTC AGA CTG AAT ATG TCA-3'; acetamiprid aptamer complementary chain, 5'-TCTTC ATAAT ATGGTGTCAG-3'; malathion aptamer complementary chain, 5'-AG CTC TGT GGC GAT GAA TCG CTG CAG CAAGCT-3'. The present application does not involve a sequence listing application, and the provided sequences are used as conventional primer instructions.

[0064] Figure 1 To construct a schematic diagram of a microfluidic paper-based chip for food hazard detection; see the specific steps in the examples.

[0065] Example 1:

[0066] Step one, preparation of oil-soluble up-conversion nanomaterial:

[0067] Accurately weigh 118.3 mg yttrium chloride hexahydrate, 38.8 mg ytterbium chloride hexahydrate, 0.7 mg thulium chloride hexahydrate, and ultrasonically disperse them in 10 mL of methanol solvent. After adding 4.0 mL of oleic acid and 9.0 mL of 1-octadecene, the mixture is stirred magnetically at 160°C for 30 min under an argon atmosphere to obtain a transparent solution. After cooling to room temperature, a mixed solution containing 50 mg of sodium hydroxide and 74.1 mg of ammonium fluoride dissolved in 10 mL of methanol is added dropwise. The flask is sealed and heated at 70°C for 40 min to evaporate the methanol, and then heated to 100°C under an argon stream. After 10 min, the excess methanol and air in the device are removed. Then, the mixture is stirred magnetically at 300°C for 1 h. After cooling to room temperature, the upconversion nanoparticle precipitate is separated by centrifugation at 8000 rpm for 5 min. The precipitate is washed three times with a 1:3 mixture of ultrapure water and ethanol, and dried to obtain pure oleic acid-coated upconversion nanoparticles.

[0068] Step two, preparation of water-soluble upconversion nanomaterials:

[0069] Accurately weigh 118.3 mg yttrium chloride hexahydrate, 38.8 mg ytterbium chloride hexahydrate, 0.7 mg thulium chloride hexahydrate, and ultrasonically disperse them in 10 mL of methanol solvent. After adding 4.0 mL of oleic acid and 9.0 mL of 1-octadecene, the mixture is stirred magnetically at 160°C for 30 min under an argon atmosphere to obtain a transparent solution. After cooling to room temperature, a mixed solution containing 50 mg of sodium hydroxide and 74.1 mg of ammonium fluoride dissolved in 10 mL of methanol is added dropwise. The flask is sealed and heated at 70°C for 40 min to evaporate the methanol, and then heated to 100°C under an argon stream. After 10 min, the excess methanol and air in the device are removed. Then, the mixture is stirred magnetically at 300°C for 1 h. After cooling to room temperature, the upconversion nanoparticle precipitate is separated by centrifugation at 8000 rpm for 5 min. The precipitate is washed three times with a 1:3 mixture of ultrapure water and ethanol, and dried to obtain pure oleic acid-coated upconversion nanoparticles.

[0070] Step three, preparation of upconversion fluorescent probes:

[0071] The 1.0 mL of water-soluble upconversion nanomaterial solution is activated with 1.0 mL of glutaraldehyde aqueous solution. The activated upconversion nanomaterial is separated and dispersed in 5.0 mL of phosphate buffer. Then, 60 uL of glyphosate aptamer complementary chain solution, 60 uL of acetamiprid aptamer complementary chain solution, and 60 uL of malathion aptamer complementary chain solution are added, respectively. Incubation is carried out at 25°C for 2 h on a shaker with a rotation speed of 250 rpm / min. After incubation, the precipitate is collected by centrifugation at 6000 rpm for 5 min. The precipitate is washed with phosphate buffer and then centrifuged again to collect the precipitate. The precipitate is then re-dissolved in 10.0 mL of phosphate buffer to obtain upconversion fluorescent probe solution A (glyphosate upconversion fluorescent probe solution), upconversion fluorescent probe solution B (acetamiprid upconversion fluorescent probe solution), and upconversion fluorescent probe solution C (malathion upconversion fluorescent probe solution), each with a concentration of 1 mg / mL.

[0072] Step four, preparation of magnetic nanomaterial (Fe3O4):

[0073] Take 1.08 g of ferric chloride hexahydrate, 1.0 g of sodium polystyrene sulfonate, 2.5 g of anhydrous sodium acetate and 40.0 mL of ethylene glycol in a round-bottom flask, heated and stirred at 50°C for 30 min, then 0.6 g of sodium hydroxide was added to the mixture, and maintained at 50°C for 90 min stirring; the resulting solution was transferred to a stainless steel autoclave lined with polytetrafluoroethylene, and heated at 200°C for 9h, after the reaction, cooled to room temperature, the reaction product was collected, and washed with 30 mL of a mixture of ethanol and pure water and centrifuged; the precipitate obtained by centrifugation was dried in a vacuum oven at 50°C overnight to obtain magnetic nanomaterial (Fe3O4);

[0074] Step five, preparation of magnetic nanocomposite material (Fe3O4@Cu) with high quenching activity:

[0075] Take 100 mg of magnetic nanomaterial (Fe3O4) into 150 mL of 0.2 mM copper sulfate solution and heat and stir at 220 rpm for 30 min, the heating temperature is 50°C; then add 1.0 g of sodium borohydride, and stir at 220 rpm at 50°C for 2h. After magnetic separation of the reaction product, wash it with 30 mL of 50% ethanol aqueous solution for 3 times, finally, dry the washed product in a vacuum oven at 50°C overnight to obtain the magnetic nanocomposite material (Fe3O4@Cu).

[0076] Figure 2 Figure A is a transmission electron micrograph of water-soluble upconversion nanoparticles, and Figure B is a transmission electron micrograph of Fe3O4@Cu material; it can be seen that the prepared water-soluble upconversion nanoparticles have a silica shell modified with amino groups, indicating that the prepared nanoparticles have been successfully surface water-soluble modified.

[0077] Step six, preparation of target aptamer modified Fe3O4@Cu:

[0078] Take 10 mg Fe3O4@Cu respectively dissolved in 10 mL, pH = 7.4 phosphate buffer, then add 1 mL of N-hydroxysuccinimide (2 mg / mL) and 1 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (1 mg / mL) solution respectively to react, and stir the mixture at 25℃ for 2h to activate the carboxyl on the surface of Fe3O4@Cu; then add 60 uL glyphosate aptamer solution, 60 uL acetamiprid aptamer solution, 60 uL malathion aptamer solution respectively, incubate at 25℃ for 10h, incubation is carried out on the shaking bed, the rotation speed of the shaking bed is 250 rpm / min, after incubation, centrifuge at 6000 rpm for 5 min, the obtained precipitate is washed with phosphate buffer and then centrifuged to collect the precipitate again, and then the precipitate is respectively redissolved in 10.0 mL of phosphate buffer, to obtain Fe3O4@Cu solution A (glyphosate aptamer modified Fe3O4@Cu solution, concentration 1 mg / mL), Fe3O4@Cu solution B (acetamiprid aptamer modified Fe3O4@Cu solution, concentration 1 mg / mL) and Fe3O4@Cu solution C (malathion aptamer modified Fe3O4@Cu solution, concentration 1 mg / mL).

[0079] Step seven, preparation of upconversion nanomaterials coupled with Fe3O4@Cu to form nanoprobes:

[0080] Take 2 mL of upconversion nanomaterials surface modified with target glyphosate aptamer complementary chain, acetamiprid aptamer complementary chain, malathion aptamer complementary chain respectively in step three, then add 1 mL of glyphosate aptamer modified Fe3O4@Cu solution, acetamiprid aptamer modified Fe3O4@Cu solution, malathion aptamer modified Fe3O4@Cu solution respectively prepared in step six, incubate at 25℃ for 2h in a shaking bed; after the reaction is completed, centrifuge, and suspend the precipitate in 2 mL of phosphate buffer to obtain glyphosate nanoprobe solution (concentration 1 mg / mL), acetamiprid nanoprobe solution (concentration 1 mg / mL), malathion nanoprobe solution (concentration 1 mg / mL) of upconversion nanomaterials coupled with Fe3O4@Cu;

[0081] Step eight, preparation of a multi-channel paper-based microfluidic chip:

[0082] Cut Whatman No. 1 chromatographic filter paper (15 mm x 20 mm), print the designed microfluidic pattern on the filter paper using a laser printer, and then place the filter paper in a forced air drying oven for heat curing treatment at 200℃ for 4h to obtain a paper-based microfluidic chip composed of a hydrophobic barrier zone 1 and a hydrophilic flow zone. Figure 4The structure diagram of the portable device for detecting food hazards based on the multi-channel paper-based microfluidic chip; the hydrophilic flow area is composed of a detection area one 2, a detection area two 4, a detection area three 8, a sample adding area 6 and a flow channel, the detection area one 2, the detection area two 4 and the detection area three 8 are independent of each other and are connected with the sample adding area 6 through the flow channel;

[0083] The glyphosate nanoprobes solution prepared in step seven is added dropwise to the detection area one 2 of the paper-based microfluidic chip, the acetamiprid nanoprobes solution is added dropwise to the detection area two 4 of the paper-based microfluidic chip, and the malathion nanoprobes solution is added dropwise to the detection area three 8 of the paper-based microfluidic chip, to prepare a multi-channel paper-based microfluidic chip.

[0084] Step nine, detection of the content of hazards in food, taking glyphosate, acetamiprid and malathion as examples:

[0085] (a) Establishment of standard curve: first prepare glyphosate, acetamiprid and malathion standard solutions with different concentrations (concentrations of 1, 10, 100, 500, 1000, 5000 ng / mL), first add the glyphosate standard solution to the sample adding area of the multi-channel microfluidic paper-based device obtained in step eight, and then through the flow channel one, the flow channel two and the flow channel three, the glyphosate standard solution reaches the detection area one, the detection area two and the detection area three respectively, incubates at room temperature, so that the glyphosate standard solution fully reacts with the nanoprobes, and after 30 minutes of reaction at room temperature, a 980 nm laser is used as an excitation source, the fluorescence signal value of the detection area is collected and an image is taken; the concentration of glyphosate is linearly fitted with the fluorescence signal value and the color feature signal extracted by taking a photo of the microfluidic paper-based chip with a smart phone, and the standard curve of glyphosate with different concentrations in the fluorescence and image detection modes is established;

[0086] Similarly, the glyphosate standard solution is replaced by acetamiprid and malathion standard solutions respectively, and the acetamiprid standard curve and the malathion standard curve in the fluorescence and image detection modes are established correspondingly;

[0087] Figure 5 The standard curve for detecting different concentrations of glyphosate, acetamiprid and malathion by fluorescence detection: as can be seen from Fig. A and Fig. B, the fluorescence intensity at 450 nm gradually increases with the increase of the concentration of glyphosate, which shows a positive correlation between the concentration of glyphosate and the increased fluorescence intensity; a good linear regression equation y = 2978.61x + 2274.90 is obtained by linear fitting, the correlation coefficient R 2 is 0.9911, and the range is 1 ng / mL-1x10 5ng / mL, the detection limit is 0.16 ng / mL, which can meet the requirements of glyphosate detection. Among them, y represents the up-conversion fluorescence intensity at 450 nm and x represents the logarithmic concentration of glyphosate. As can be seen from FIG. C and FIG. D, the fluorescence intensity at 450 nm gradually increases with the increase of acetamiprid concentration, which shows a positive correlation between acetamiprid concentration and increased fluorescence intensity; a good linear regression equation y = 2484.91x + 2446.08 is obtained by linear fitting, the correlation coefficient R 2 = 0.9926, the linear range is 1 ng / mL-1×10 5 ng / mL, the detection limit is 0.77 ng / mL, which can meet the requirements of acetamiprid detection. Among them, y represents the up-conversion fluorescence intensity at 450 nm and x represents the logarithmic concentration of acetamiprid. As can be seen from FIG. E and FIG. F, the fluorescence intensity at 450 nm gradually increases with the increase of malathion concentration, which shows a positive correlation between malathion concentration and increased fluorescence intensity; a good linear regression equation y = 2649.76x + 2342.17 is obtained by linear fitting, the correlation coefficient R 2 = 0.9901, the linear range is 1 ng / mL-1×10 5 ng / mL, the detection limit is 0.34 ng / mL, which can meet the requirements of malathion detection. Among them, y represents the up-conversion fluorescence intensity at 450 nm and x represents the logarithmic concentration of malathion;

[0088] Figure 6 is a standard curve established based on image color feature value detection of different concentrations of glyphosate, acetamiprid and malathion. As can be seen from FIG. A, the image in the microfluidic paper-based chip also has a corresponding color change with the increase of glyphosate concentration; taking the B value in the color feature value as an example, the standard curve y = 84.72 + 28.42x for detection is obtained by linear fitting with the content of glyphosate, the correlation coefficient R 2 = 0.9760, the detection limit LOD is 0.37 ng / mL, the linear range is 1 ng / mL-1×10 5 ng / mL; As can be seen from FIG. B, the image in the microfluidic paper-based chip also has a corresponding color change with the increase of acetamiprid concentration; taking the R value in the color feature value as an example, the standard curve y = 9.268 + 5.467x for detection is obtained by linear fitting with the content of acetamiprid, the correlation coefficient R 2 = 0.9824, the detection limit LOD is 0.59 ng / mL, the linear range is 1 ng / mL-1×10 5ng / mL; as can be seen from Figure C, the image in the microfluidic paper-based chip also has a corresponding color change with the increase of malathion concentration; taking the G value in the color characteristic value as an example, the standard curve y = 9.476 + 9.111x for detection is obtained by linear fitting of the malathion content, the correlation coefficient R 2 = 0.9827, the detection limit LOD is 0.28 ng / mL, and the linear range is 1 ng / mL-1x10 5 ng / mL

[0089] (b) Actual sample detection: the actual sample is pretreated to obtain a test solution containing glyphosate, acetamiprid and malathion; the test solution is added dropwise to the sample addition area of the multi-channel microfluidic paper-based chip, and after incubation at room temperature, the fluorescence signal value of the detection area is measured, and the fluorescence standard curve and the image standard curve of the glyphosate, acetamiprid and malathion content detection obtained in step (a) are respectively brought in, and the content of glyphosate, acetamiprid and malathion in the actual sample is calculated;

[0090] (1) Detection of the contents of glyphosate, acetamiprid and malathion in matcha;

[0091] 5g of matcha powder is weighed, and then 1mL of glyphosate, acetamiprid and malathion with concentrations of 100, 500 and 1000 ng / mL respectively is added to the matcha sample, and after homogenization, 50mL of acetonitrile solution is added, and high-speed homogenization is performed for 5min, and centrifugation is performed at 6000rpm for 10min to collect the first supernatant; then the precipitate is collected again, 50mL of acetonitrile solution is added, and the above steps are repeated to obtain the second supernatant, and finally the supernatants are combined.

[0092] The combined supernatant is heated in a rotary evaporator at 80°C to evaporate the acetonitrile solvent to near dryness to obtain a dry residue. The dry residue is made up to 2mL of methanol solution, and vortex mixed as a test solution.

[0093] 30uL of the test solution is added dropwise to the sample addition area of the multi-channel microfluidic paper-based chip, and after incubation at room temperature, the fluorescence signal value and image data (RGB in color characteristic value) of the detection area are measured, and the fluorescence standard curve and the image standard curve of glyphosate, acetamiprid and malathion constructed in step (1) are respectively brought in, and the recovery rate is calculated. The specific is shown in Table 1;

[0094] Table 1 Detection of glyphosate, acetamiprid and malathion in matcha sample

[0095]

[0096]

[0097] aRSD: relative standard deviation;

[0098] b1 P: Two-tailed t-test results between fluorescence and HPLC methods;

[0099] b2 P: Two-tailed t-test results between image and HPLC methods;

[0100] (2) Detection of glyphosate, acetamiprid and malathion in black tea;

[0101] Take 5g of black tea powder, then add 1mL of glyphosate, acetamiprid and malathion with concentrations of 100, 500 and 1000ng / mL respectively into the matcha sample, add 50mL of acetonitrile solution after homogenization, high-speed homogenization for 5min, centrifugation at 6000rpm for 10min to collect the first supernatant; then collect the precipitate and add 50mL of acetonitrile solution again, repeat the above steps to obtain the second supernatant, and finally combine the supernatants.

[0102] Heat the combined supernatant in a rotary evaporator at 80℃ to evaporate the acetonitrile solvent to near dryness to obtain a dry residue. Dilute the dry residue to 2mL of methanol solution, vortex mix, as the test solution.

[0103] Take 30uL of the test solution and add it to the sample addition area of the multi-channel microfluidic paper-based chip, incubate at room temperature; measure the fluorescence signal value and image data (RGB in color feature value) of the detection area, respectively, into the fluorescence standard curve and image standard curve of glyphosate, acetamiprid and malathion constructed in step nine (1), and calculate the standard addition recovery rate. As shown in Table 2;

[0104] Table 2 Detection of glyphosate, acetamiprid and malathion in black tea samples

[0105]

[0106]

[0107] a RSD: relative standard deviation;

[0108] b1 P: Two-tailed t-test results between fluorescence and HPLC methods;

[0109] b2 P: Two-tailed t-test results between image and HPLC methods;

[0110] Comparative Example 1:

[0111] The difference from Example 1 is that the detection object is 250ng / mL benomyl.

[0112] Comparative Example 2:

[0113] The difference from Example 1 is that the detection object is 250 ng / mL paraquat.

[0114] Comparative Example 3:

[0115] The difference from Example 1 is that the detection object is 250 ng / mL carbendazim.

[0116] Comparative Example 4:

[0117] The difference from Example 1 is that the detection object is 250 ng / mL diazinon.

[0118] The results are shown in Table 1. Figure 7 As shown in Table 1, when the constructed detection method is used for detection of the remaining pesticides benomyl, paraquat, carbendazim, and diazinon, no obvious fluorescence change of the system is caused, and only when glyphosate, acetamiprid, and malathion solutions are added again, the fluorescence intensity of the corresponding detection zone changes obviously, indicating that the constructed multi-channel paper-based microfluidic chip has good specificity.

[0119] Note: The above examples are only used to illustrate the present application and not to limit the technical solutions described in the present application; therefore, although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the present application can still be modified or replaced equivalently; 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 method for detecting hazardous substances in food based on a multi-channel microfluidic paper-based chip, characterized in that, Includes the following steps: Step 1: Preparation of oil-soluble upconversion nanomaterials: Yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and thulium chloride hexahydrate were dissolved by ultrasonication in a certain volume of methanol A. Then, oleic acid and 1-octadecene were added and thoroughly mixed. The mixture was heated and stirred under closed conditions for the first time, and then cooled to room temperature. The closed conditions were then removed, and methanol B containing sodium hydroxide and ammonium fluoride was added dropwise. The mixture was heated and stirred a second time under closed conditions, and then cooled to room temperature. The precipitate was obtained by centrifugation, washed a first time with a mixture of ultrapure water and ethanol, and then washed a second time with a cyclohexane solution. After drying, oleic acid-coated upconversion nanomaterials were obtained for later use. Step 2: Preparation of water-soluble upconversion nanomaterials: The oleic acid-coated upconversion nanoparticles prepared in step one were weighed and dispersed in hydrochloric acid solution and ultrasonically treated. After treatment, ethanol was added and ultrasonically dispersed evenly. Then, ultrapure water and ammonia were added and stirred evenly under a certain temperature. After stirring evenly, tetraethyl orthosilicate was added for the first reaction, and then 3-aminopropyltriethoxysilane was added for the second reaction. After the reaction was completed, the product was centrifuged and washed with ultrapure water. After washing, the product was dispersed in ultrapure water to obtain a water-soluble upconversion nanomaterial solution. Step 3: Prepare upconversion nanomaterial fluorescent probes: S1. The upconversion nanomaterial solution prepared in step 2 is added to glutaraldehyde aqueous solution and activated on a shaker. After activation, the upconversion nanomaterial is obtained by centrifugation and dispersed in phosphate buffer A. Then, target aptamer complementary chain solution A is added and incubated at a constant temperature on a shaker. After incubation, the precipitate is centrifuged again. The precipitate is washed with phosphate buffer and collected by centrifugation again. The precipitate is then redissolved in phosphate buffer. The resulting dispersion is denoted as upconversion fluorescent probe solution A. S2 is the same as S1, except that the target aptamer complementary chain solution A is replaced with target aptamer complementary chain solution B and target aptamer complementary chain solution C; all other operations are the same, and the final result is upconversion fluorescent probe solution B and upconversion fluorescent probe solution C. Step 4: Preparation of magnetic nanomaterials: First, ferric chloride hexahydrate, sodium poly(4-styrenesulfonic acid-copoly-maleic acid), anhydrous sodium acetate, and ethylene glycol were weighed and mixed, and then subjected to a first heating and stirring reaction. After the reaction, sodium hydroxide was added, and a second stirring was carried out until dissolved. Then, the resulting solution was transferred to a stainless steel high-pressure reactor lined with polytetrafluoroethylene for hydrothermal reaction. After the reaction was completed, it was cooled to room temperature, the reaction product was collected, and washed with a mixed solution of ethanol and pure water and centrifuged. The collected precipitate was dried overnight in a vacuum oven and labeled as Fe3O4 for later use. Step 5: Prepare nanocomposite materials with high quenching activity: The magnetic nanomaterials prepared in step four were weighed and dispersed in a copper sulfate solution for a first heating and stirring reaction. Then, sodium borohydride was added for a second stirring reaction. The precipitate was obtained by magnetic separation and washed with a mixed solution of ethanol and pure water by centrifugation. The precipitate obtained by centrifugation was collected and dried under vacuum to obtain a nanocomposite material with high quenching activity, denoted as Fe3O4@Cu. Step 6: Preparation of target aptamer-modified Fe3O4@Cu: S1. Weigh the Fe3O4@Cu material prepared in step five and dissolve it in phosphate buffer A. Then, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution and carry out the activation reaction on a shaker. After activation, add the target A aptamer solution and carry out a second isothermal incubation on a shaker. After incubation, wash and centrifuge with phosphate buffer, and then redissolve the precipitate in phosphate buffer B. The resulting dispersion is denoted as Fe3O4@Cu solution A. S2 is the same as S1, except that the target A aptamer solution is replaced with the target B aptamer solution and the target C aptamer solution; all other operations are the same, and the final results are Fe3O4@Cu solution B and Fe3O4@Cu solution C. Step 7: Prepare nanoprobes formed by coupling upconversion nanomaterials with Fe3O4@Cu: S1. Take the upconversion fluorescent probe solution A with the target aptamer complementary chain prepared in step three, add the target aptamer modified Fe3O4@Cu solution A prepared in step six, and then place it in a shaker for incubation. After the reaction is completed, centrifuge and collect the precipitate, which is then suspended in phosphate buffer A to obtain nanoprobe solution A formed by coupling upconversion fluorescent nanomaterials with Fe3O4@Cu. S2 is the same as S1, except that upconversion fluorescent probe solution A is replaced with upconversion fluorescent probe solution B and upconversion fluorescent probe solution C; target aptamer modified Fe3O4@Cu solution A is replaced with target aptamer modified Fe3O4@Cu solution B and target aptamer modified Fe3O4@Cu solution C; all other operations are the same, and finally nanoprobe solution B and nanoprobe solution C are obtained. Step 8: Fabrication of a multi-channel microfluidic paper-based chip: Cut the filter paper and use a laser toner printer to print the drawn paper pattern on the filter paper. Then place the filter paper in a drying oven for heat curing. After naturally cooling to room temperature, take the printed filter paper out of the oven and cut it to obtain a paper-based microfluidic chip composed of a hydrophobic barrier region and a hydrophilic flow region. In this process, printer ink is solidified into the pores of the chromatography filter paper, forming a hydrophobic barrier region where the solution cannot flow. On one side of the hydrophobic barrier region, a microfluidic detection area is formed by the ink. The microfluidic detection area consists of detection area one, detection area two, detection area three, sample dispensing area, and flow channel. Detection area one, detection area two, and detection area three are independent of each other and are connected to the sample dispensing area through flow channels. Finally, the nanoprobe solution A prepared in step seven is added to detection area one of the microfluidic paper-based chip, nanoprobe solution B is added to detection area two of the microfluidic paper-based chip, and nanoprobe solution C is added to detection area three of the microfluidic paper-based chip to obtain a multi-channel paper-based microfluidic chip for detection. Step nine: Testing the content of harmful substances in food: (a) Establishment of standard curve: First, prepare standard solutions of food hazard A, food hazard B, and food hazard C at different concentrations, and add them dropwise to the sample application area of ​​the multi-channel paper-based microfluidic chip obtained in step eight. The samples pass through flow channel one, flow channel two, and flow channel three to reach detection area one, detection area two, and detection area three, respectively. Incubate at room temperature to allow food hazard A, food hazard B, and food hazard C to fully react with the upconversion fluorescent probe, thus obtaining multi-channel paper-based microfluidic chips of different concentrations. After incubation, collect the fluorescence signal value of detection area one, and then perform linear fitting between the concentration of food hazard A and the fluorescence intensity to establish a standard curve for the detection of food hazard A content. Similarly, the fluorescence signal values ​​of detection area 2 are linearly fitted with the concentration of food hazard B and the fluorescence intensity to establish a standard curve for the detection of food hazard B content; the fluorescence signal values ​​of detection area 3 are linearly fitted with the concentration of food hazard C and the fluorescence intensity to establish a standard curve for the detection of food hazard C content. Simultaneously, the image of detection area one is recorded, and the color feature value A is extracted to establish a standard curve between the color feature value A and the concentration of food hazard A; then, the image of detection area two is recorded, and the color feature value B is extracted to establish a standard curve between the color feature value B and the concentration of food hazard B; finally, the image of detection area three is recorded, and the color feature value C is extracted to establish a standard curve between the color feature value C and the concentration of food hazard C. (b) Detection of actual samples: The actual sample is pretreated to extract a test solution containing food hazards; the test solution is added to the sample application area of ​​the multi-channel microfluidic paper-based chip and incubated at room temperature; the fluorescence intensity of detection area one is measured and the corresponding image is captured to extract the color feature value, which is then input into the food hazard A content detection standard curve obtained in step nine to calculate the content of food hazard A in the actual sample; the fluorescence intensity of detection area two is measured and the corresponding image is captured to extract the color feature value, which is then input into the food hazard B content detection standard curve obtained in step nine to calculate the content of food hazard B in the actual sample; the fluorescence intensity of detection area three is measured and the corresponding image is captured to extract the color feature value, which is then input into the food hazard C content detection standard curve obtained in step nine to calculate the content of food hazard C in the actual sample.

2. The method for detecting hazardous substances in food based on a multi-channel microfluidic paper-based chip according to claim 1, characterized in that, In step one, the amounts of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, thulium chloride hexahydrate, methanol A, oleic acid, 1-octadecene, sodium hydroxide, ammonium fluoride, and methanol B are in the following ratios: 118.3 mg: 38.8 mg: 0.7 mg: 10 mL: 4.0 mL: 9.0 mL: 50.0 mg: 74.1 mg: 10 mL; the washing process uses a mixture of cyclohexane and ethanol, wherein the volume ratio of cyclohexane to ethanol is 1:

3. The first heating and stirring time is 20-30 min, and the heating temperature is 160 ℃; the second heating and stirring temperature is 300 ℃, and the stirring time is 1.0-1.5 h; the centrifugation conditions are: rotation speed of 8000-10000 rpm / min, and time of 5-10 min.

3. The method for detecting hazardous substances in food based on a multi-channel microfluidic paper-based chip according to claim 1, characterized in that, In step two, the ratio of upconversion nanomaterials, hydrochloric acid solution, ethanol, ultrapure water, ammonia, tetraethyl orthosilicate, and 3-aminopropyltriethoxysilane is 20 mg: 1 mL: 60 mL: 10 mL: 2.5 mL: 20 uL: 50 uL; the first reaction time is 4-6 h, and the second reaction time is 2-3 h; the concentration of the hydrochloric acid solution is 0.1 mol / L; the specific temperature condition is 65 ℃; the centrifugation conditions are: rotation speed of 8000-10000 rpm / min, time of 5-10 min; and the concentration of the water-soluble upconversion nanomaterial solution is 1 mg / mL.

4. The method for detecting hazardous substances in food based on a multi-channel microfluidic paper-based chip according to claim 1, characterized in that, In step three, the amounts of the water-soluble upconversion nanomaterial solution, glutaraldehyde aqueous solution, phosphate buffer A, and target aptamer complementary chain solution A are in the following ratio: 10 mg: 1.0 mL: 5.0 mL: 60 μL; the pH of the phosphate buffer is 7.4; the concentration of the upconversion fluorescent probe solution A is 1 mg / mL; the concentration of the upconversion fluorescent probe solution B is 1 mg / mL; and the concentration of the upconversion fluorescent probe solution C is 1 mg / mL. The activation reaction is carried out at a temperature of 25°C for 1-2 hours, with a shaking speed of 200-300 rpm / min; the incubation is carried out at a temperature of 25°C for 10-12 hours, with a shaking speed of 200-300 rpm / min; the centrifugation conditions are: a speed of 6000 rpm / min for 5-10 minutes.

5. A method for detecting hazardous substances in food based on a multi-channel microfluidic paper-based chip according to claim 1, characterized in that, In step four, the amounts of ferric chloride hexahydrate, sodium poly(4-styrenesulfonic acid-copoly-maleic acid), sodium acetate, ethylene glycol, and sodium hydroxide are in the following ratio: 1.08 g: 1.0 g: 2.5 g: 40.0 mL: 0.6 g; the cleaning is performed using a mixed solution of ethanol and pure water, wherein the volume ratio of ethanol to pure water is 1:

1. The first heating and stirring time is 30 min, and the heating temperature is 50℃; the second stirring time is 90 min, and the temperature is 50℃; the hydrothermal reaction time is 9 h, and the heating temperature is 200℃.

6. A method for detecting hazardous substances in food based on a multi-channel microfluidic paper-based chip according to claim 1, characterized in that, In step five, the ratio of the magnetic nanomaterial to sodium borohydride is 0.1 g: 1.0 g; the concentration of the copper sulfate solution is 0.2 mol / mL; the washing is performed using a mixed solution of ethanol and pure water, wherein the volume ratio of ethanol to pure water is 1:1; the first heating and stirring time is 30 min, and the heating temperature is 50℃; the second heating and stirring time is 2 h, and the heating temperature is 50℃.

7. A method for detecting hazardous substances in food based on a multi-channel microfluidic paper-based chip according to claim 1, characterized in that, In step six, the amounts of Fe3O4@Cu, phosphate buffer A, 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution, N-hydroxysuccinimide solution, target A aptamer solution, and Fe3O4@Cu solution A are in the following ratio: 10.0 mg: 1.0 mL: 1.0 mL: 10.0 mL: 60 μL; the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 2 mg / mL, and the concentration of the N-hydroxysuccinimide solution is 1 mg / mL; the pH of the phosphate buffer is 7.4; the concentration of Fe3O4@Cu solution A is 1 mg / mL; the concentration of Fe3O4@Cu solution B is 1 mg / mL; and the concentration of Fe3O4@Cu solution C is 1 mg / mL. The activation reaction is carried out at a temperature of 25 ℃ for 1-2 h with a shaking speed of 200-300 rpm / min; the incubation is carried out at a temperature of 25 ℃ for 8-10 h with a shaking speed of 200-300 rpm / min; the centrifugation is carried out at a speed of 6000 rpm / min for 5-10 min.

8. A method for detecting hazardous substances in food based on a multi-channel microfluidic paper-based chip according to claim 1, characterized in that, In step seven, the amounts of the upconversion fluorescent probe solution A with the target aptamer complementary chain, the Fe3O4@Cu solution A modified with the target aptamer, and the phosphate buffer A are in the following ratio: 1.0 mL: 0.5 mL: 1.0 mL; the pH of the phosphate buffer is 7.4; the concentrations of the nanoprobe solution A, nanoprobe solution B, and nanoprobe solution C are 1.0 mg / mL; the shaking incubation reaction is carried out at a temperature of 25 °C for 1-2 h, and the shaking speed is 200-300 rpm / min.

9. A method for detecting hazardous substances in food based on a multi-channel microfluidic paper-based chip according to claim 1, characterized in that, In step eight, the filter paper is Whatman No. 1 chromatography filter paper with a size of 15×20 mm; the microfluidic chip includes a sample loading area, which is a semi-circular region with a diameter of 10 mm; detection areas one, two, and three, which are circular regions with a diameter of 5 mm; sample flow channels one, two, and three, each with a length of 3 mm and a width of 1 mm; wherein, sample flow channel one connects the sample loading area and detection area one, sample flow channel two connects the sample loading area and detection area two, and sample flow channel three connects the sample loading area and detection area three; the temperature of the thermosetting treatment is 200 ℃, and the curing time is 3-4 h; the volume relationship of the nanoprobe solution A, nanofluorescent probe solution B, and nanoprobe solution C is 4 uL:4 uL:4 uL.

10. A method for detecting hazardous substances in food based on a multi-channel microfluidic paper-based chip according to claim 1, characterized in that, In step nine (a), the food hazards include, but are not limited to, glyphosate, acetamiprid, and malathion; the amount of the food hazard standard solution added is 30 μL, and the concentration range of the food hazard standard solution is 1-5000 ng / mL; the incubation time at room temperature is 30 min; in step (b), the amount of the test solution added is 30 μL; the incubation time is 30 min in all cases.

Images