CN113504226A - Functional paper-based micro-fluidic chip detection test paper, method for rapidly detecting pesticide residues in vegetables based on detection test paper and application of detection test paper - Google Patents
Functional paper-based micro-fluidic chip detection test paper, method for rapidly detecting pesticide residues in vegetables based on detection test paper and application of detection test paper Download PDFInfo
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Abstract
The invention discloses a functionalized paper-based microfluidic chip detection test paper, a method for rapidly detecting pesticide residues in vegetables based on the detection test paper and application of the detection test paper, wherein paper carved by a laser etching machine is placed in NATPP solution to modify the paper-based microfluidic chip so as to increase the hydrophilicity of the chip; the pesticide to be detected reacts with a specific reagent in a detection area to generate color change; after the color is stable, shooting is carried out by using a smart phone, and gray value extraction and data analysis are carried out on the picture color through software such as Image J, origin and the like, so that a novel paper-based colorimetric sensor capable of carrying out rapid nondestructive detection on various pesticide residues is established and used for synchronous quantitative detection of various pesticide residues. The invention has the characteristics of high sensitivity, good stability, simple manufacturing process, low cost, easy integration and the like. The automatic or semi-automatic process of pesticide residue detection is expected to be realized, and a new field is opened up for the on-site rapid detection of actual samples.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a functional paper-based micro-fluidic chip detection test paper, a method for rapidly detecting pesticide residues in vegetables based on the detection test paper and application of the detection test paper.
Background
In recent years, with the increasing demand of people for agricultural products, agricultural productivity in China is fully released, and pesticides serving as important chemicals for preventing diseases and pests and regulating plant growth can be classified into organic phosphorus, organic chlorine, pyrethroids, neonicotinoids and the like according to chemical structures and are widely applied to agricultural product production. In order to improve the yield and economic benefit of crops such as vegetables, pesticides are often used excessively, and the abuse and unscientific use of the pesticides not only directly causes great pollution to agricultural products, soil, water sources, air and the like, but also seriously threatens the health of human beings. The long-term exposure in the environment polluted by organophosphorus pesticide can greatly harm human health, and can cause central nervous system diseases and even death in serious cases. At present, the pesticide residue exceeding standard brings great challenges to the quality safety of agricultural products, people pay more and more attention to the detection of pesticide residue in agricultural products, and the detection technology of the pesticide residue exceeding standard is increasingly concerned by people. According to the existing inspection standard in China, food safety inspection needs to be carried out in qualified laboratories by adopting traditional inspection technologies, such as High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-tandem mass spectrometry (GC-MS/MS) and other large-scale instrument analysis methods. Therefore, in the face of severe challenges of large product inspection requirements and food safety. The technology and the method which have the advantages of rapid and nondestructive pesticide residue of agricultural products and online detection have important significance.
A microfluidic chip (also called Micro total analysis systems, μ TAS) is a Micro electromechanical system (MEMS) based on analytical chemistry, integrates sample pretreatment, separation, detection and other processes on a chip of several square centimeters, and is a Micro laboratory that controls fluid flow direction and shortens reaction time by means of a series of special effects such as surface tension, fluid resistance, energy dissipation and the like. Compared with the defects of the prior detection technology, the high-density microstructure of the microfluidic technology realizes the miniaturization, automation and integration of sample pretreatment and subsequent analysis. Compared with the traditional method, the microfluidic technology meets the requirement of instant detection, overcomes the defects of long culture time and complex pretreatment to a certain extent, has the advantages of miniaturization, high flux, rapidness, integration, less consumption and the like, is mainly prepared from silicon, glass, quartz, organic polymers and composite materials by the micromachining technology, and has the problem that the industrialization of the microfluidic chip in the fields of biology, chemistry, clean room and the like is hindered due to the high cost of the materials and the strict requirement on equipment.
In 2007, a paper-based micro-fluidic analysis system (micro-liquid paper-based analytical devices, μ PADs), namely a Lab-on-paper micro-laboratory, was proposed for the first time by Whitesids laboratories, and a research boon in the field of micro-analysis is emerging once. As a novel microfluidic platform which is rapidly developed in recent years, the paper-based microfluidic chip takes filter paper as a substrate to replace materials such as silicon, glass, high polymer and the like, the difficulties of high cost and no pollution of the traditional microfluidic chip are overcome, a certain processing technology is adopted, a hydrophilic/hydrophobic micro-channel network with a certain structure and related analytical devices are prepared on a paper base, the formed microfluidic channel controls the liquid transportation through the capillary action of the filter paper, one-step or multi-step biochemical reaction processes are carried out, and then the whole detection and diagnosis process is completed. The high-flux miniaturized device successfully combines the characteristics of low price and easy obtaining, strong biocompatibility and simple preparation of the filter paper with the advantages of automation and integration of the traditional microfluidic chip, shows strong development vigor to the detection field, and is widely applied to the fields of disease diagnosis, food quality safety detection, environmental analysis and the like at present.
In addition, the colorimetric-based method has the advantages of high cost benefit, simplicity and convenience in use, rapid detection and the like, becomes the most common detection technology in paper diagnosis, obtains a color development result which can be visually read by naked eyes for semi-quantitative analysis by utilizing the characteristic that an analyte and a fixed colorimetric reagent in a detection area generate biochemical reaction, then performs gray processing on an image by a simple scanning imaging principle, and performs quantitative analysis according to the relation between the gray value and the concentration of the analyte. For example, Ornatska et al, after silylating paper substrate with Aminopropyltrimethoxysilane (APTS), immobilized ceria nanoparticles and glucose oxidase on the paper detection zone, performed glucose detection. The color change in the detection zone was significant and had a limit of detection (LOD) of 0.5 mM. Therefore, the paper-based micro-fluidic chip based on the colorimetric method is a feasible way for detecting the rapid pesticide residue.
Disclosure of Invention
The invention provides a functional paper-based micro-fluidic chip detection test paper, a method for rapidly detecting pesticide residues in vegetables based on the detection test paper and application thereof, and mainly aims to combine a method for detecting pesticide residues based on colorimetry with the test paper to construct the test paper capable of simply and rapidly detecting chlorpyrifos, profenofos, cypermethrin and deltamethrin in a colorimetric way, and specifically comprises the following four purposes:
the first object of the present invention is: providing a colorimetric detection test paper, and dropwise adding a prepared color developing agent into each detection area of the test paper; the test paper is fork-shaped.
The second object of the present invention is: a method for preparing a colorimetric test strip is provided, which comprises the following steps:
(1) pretreatment of the test paper: cutting the tree-like part drawn by the AutoCAD software into a target shape by a laser cutting machine, washing and drying the target shape for later use
(2) Preparing colorimetric detection test paper: and dripping PVP solution on the test paper to be used to moisten the test paper, and dripping a color-developing agent in a detection area of the test paper to obtain the color-developing agent-loaded colorimetric detection test paper.
In one embodiment of the invention, the crotch-shaped detection zone has a diameter of 5mm x 5mm, and the straight channel of the sample introduction zone is 4mm wide and 6mm long.
In one embodiment of the invention, the concentration of the NaTPP solution is 1% to 1.5% (m/v); the addition amount of the NaTPP solution is 5-10 mu L.
In one embodiment of the present invention, the method specifically comprises the following steps:
(1) cutting Whatman No. 1 qualitative filter paper by using a laser engraving machine, soaking the cut filter paper in ultrapure water, soaking the filter paper in absolute ethyl alcohol, and drying the filter paper in an oven;
(2) dripping a NaTPP solution to moisten the test paper, dripping a color developing agent in a detection area of the test paper, and placing the test paper at room temperature until the test paper is completely dried; the concentration (m/v) of the NaTPP solution is 1-1.5%, iodothio-acetylcholine is 2mmol/L, 5, 5' -dithiobis (2-nitrobenzoic acid) is 2mmol/L, acetylcholinesterase is 6 mu g/mL, 4-aminoantipyrine is 1.5%, potassium ferricyanide is 2%, ammonium acetate is 3%, ninhydrin is 3.5%, and the particle size of AuNPs is 12-25 nm.
A third object of the invention is: provides an application of the colorimetric test paper in detecting pesticide residue surface.
A fourth object of the invention is: the method comprises the steps of adopting the colorimetric detection test paper for detection, dripping a solution to be detected into a sample solution dripping area of the test paper, reacting for 15-20min, and directly identifying the color through a mobile phone APP and computer software.
(1) Chlorpyrifos: under the condition that iodothio-acetylcholine exists, acetylcholinesterase catalyzes iodothio-acetylcholine to hydrolyze to generate thiocholine, and the thiocholine reacts with a color developing agent to form yellow. Chlorpyrifos has strong inhibition effect on acetylcholinesterase activity, so that the yield of thiocholine is reduced, and the yellow intensity is reduced.
(2) Profenofos: the profenofos is hydrolyzed under the alkaline condition to generate 2-chloro-4-bromophenol, and the 2-chloro-4-bromophenol further reacts with potassium ferricyanide and 4-aminoantipyrine to generate a special red chelate.
(3) Cypermethrin is hydrolyzed to cyanide under alkaline conditions, the cyanide is further decomposed to free cyanide ions, and then the cyanide reacts with ninhydrin and ammonium acetate to form a purple compound.
(4) AuNPs-MNBT was synthesized using 2-Mercapto-6-nitrobenzothiazole (2-Mercapto-6-nitrobenzothiazole, MNBT) as a ligand, and formed strong Au-S bonds with AuNPs by a self-assembly method. After deltamethrin is added, the benzene ring on MNBT and the benzene ring on deltamethrin form a core-shell structure through a pi-pi stacking effect. The reaction changes the surface plasmon effect of AuNPs, and the color of the solution changes from red to purple.
The invention has the beneficial effects that:
(1) the invention combines the test paper with the colorimetric method to realize the rapid detection of 4 pesticides, and the detection can be finished within 20min
(2) The invention can realize qualitative and semi-quantitative detection of four pesticides directly through mobile phone APP and computer software, and the concentration of the lowest detection limit is chlorpyrifos: 0.042 mg/kg; profenofos: 0.519mg/kg, cypermethrin: 0.674 mg/kg; deltamethrin: 0.173 mg/kg.
Drawings
FIG. 1 shows a design pattern of the test paper;
FIG. 2 is a detection mechanism for four pesticides;
FIG. 3 is the degree of color development before and after modification of the paper substrate;
FIG. 4 is a graph of the color change of the paper substrate before and after pesticide addition;
FIG. 5 shows the effect of different profenofos concentrations on the gray value detection results.
Detailed Description
The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.
Example 1: colorimetric test paper for modifying color-developing agent
A colorimetric test paper for modifying a color developing agent comprises the following steps:
(1) preparation of dendritic test paper
Firstly, a graph is made by software AutoCAD (shown in figure 1), and then Whatman No. 1 qualitative filter paper is cut according to the graph by a laser engraving machine. And soaking the cut paper in ultrapure water for 12h, then fishing out, and drying in a drying oven at 37 ℃ to obtain the test paper for later use.
(2) Preparation of functionalized gold nanoparticles
A20 m L concentration 5m M aqueous solution of HAu Cl4 was mixed with 200mL Milli-Q water. Then, a newly prepared 0.1M solution of Na BH4 of 5.0M L was added dropwise to the mixed solution under vigorous stirring at room temperature. The reaction lasted for 30 minutes and changed color from pink to wine-red during stirring. Finally, the Au NPs solution was stored in a refrigerator at 4 ℃. After 40. mu.L of Au NPs colloid of 1m M concentration and 40m L was mixed and stirred rapidly for about 2 hours, functionalized Au NPs-MNBT was obtained.
(3) Preparation of color developer
As shown in figure 2, four different color development mechanisms are adopted to independently detect corresponding pesticides, and the change of the paper base color can be obviously observed after the color development agents and the pesticides are completely reacted. The detection method comprises the steps of mixing an ATCH solution for chlorpyrifos detection and a DTNB solution for preparation, mixing a potassium ferricyanide solution for profenofos detection and a 4-aminoantipyrine solution for preparation, mixing a ninhydrin solution for cypermethrin detection and an ammonium acetate solution for preparation, and mixing AuNPs-MNBT for deltamethrin detection.
(4) Preparation of color-developing agent modified colorimetric detection pesticide residue test paper
Dripping 10 mu L of NaTPP solution to moisten the test paper, dripping 2 mu L of color developing agent in a detection area of the test paper, and placing the test paper at room temperature until complete drying to obtain the color developing agent-loaded test paper for colorimetric detection of pesticides, wherein the concentration (m/v) of the NaTPP solution is 1%, the concentration of iodothio-acetylcholine is 2mmol/L, the concentration of 5, 5' -dithiobis (2-nitrobenzoic acid) is 2mmol/L, the concentration of acetylcholinesterase is 6 mu g/mL, 1.5% of 4-aminoantipyrine, 2% of potassium ferricyanide, 3% of ammonium acetate, 3.5% of ninhydrin, and the particle size of AuNPs is 12-25 nm.
Example 2: optimization of pretreatment of filter paper
Test strips were prepared according to the method of example 1 except that the test strips were wetted with ultrapure water and 1% NaTPP solution, respectively. The method comprises the following specific operations: taking the washed and dried test paper, wetting the test paper by 10 mu L of solution, then dropwise adding 2 mu L of developer solution into the detection area of the test paper, and then placing the test paper at room temperature until the test paper is completely dried; comparing fig. 3 before and after the treatment, it can be seen from fig. 3 that the untreated filter paper can generate caffeine reaction, and the treated filter paper can reduce the capillary transport effect of the filter paper and protect the enzyme, so that the hydrophilicity is improved and the color can be uniformly distributed on the test paper.
Example 3: detection of four pesticides by functionalized paper base
The functionalized paper base prepared in example 2 is utilized, required color developing agents for detecting each pesticide are successfully prepared and dripped into a reaction area, ATCH solution for chlorpyrifos detection and DTNB solution are mixed and prepared, potassium ferricyanide solution for profenofos detection and 4-aminoantipyrine solution are mixed and prepared, ninhydrin solution for detecting cypermethrin is mixed and prepared with ammonium acetate solution, and AuNPs-MNBT for detecting deltamethrin is prepared, wherein iodothioacetyl choline is 2mmol/L, 5, 5' -dithiobis (2-nitrobenzoic acid) is 2mmol/L, acetylcholinesterase is 6 mu g/mL, 4-aminoantipyrine is 1.5%, potassium ferricyanide is 2%, ammonium acetate is 3%, ninhydrin is 3.5%, and the particle size of AuNPs is 12-25 nm. Standing and naturally drying the mixture, dropwise adding the mixture to a straight channel of the functionalized paper-based microfluidic chip at different concentrations of pesticide, and allowing the liquid to flow into a detection area to perform color reaction. As shown in fig. 4, the color changes before and after the pesticide is added are compared, and the color changes in stages from low to high.
Example 4: drawing a profenofos standard curve
The standard substance of profenofos with different concentrations is added on the functional paper-based micro-fluidic chip prepared in the example 2, two solutions of potassium ferricyanide and 4-aminoantipyrine are mixed, 2 mu L of mixed solution is added into a micro-fluidic reaction hole for color development, and a mobile phone is used for taking a picture in time after color development. And (4) performing data processing and gray level analysis by using Excel and Image J software, and drawing a standard curve. Obviously, the colors corresponding to the standards with different concentrations are also different, and as can be seen from fig. 5, the concentrations of the standards and the gray values have a certain correlation, and the fitting degree is high. According to the method, a standard library with the one-to-one correspondence of the relative gray value and the profenofos concentration can be established, so that the concentration of an unknown sample can be detected.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. The functionalized paper-based micro-fluidic chip detection test paper is characterized in that cellulose paper is used as a substrate, a laser etching machine is used for cutting the cellulose paper into a crotch-shaped paper chip according to the shape designed by AutoCAD software, the crotch-shaped paper chip is washed and dried and serves as a paper-based micro-fluidic chip detection area, the head of the crotch-shaped paper chip serves as the detection area, and the bottom of the crotch-shaped paper chip serves as a sample injection area.
2. The functionalized paper-based microfluidic chip test paper according to claim 1, wherein the cellulose paper is whatman No. 1 filter paper.
3. The functionalized paper-based microfluidic chip test paper according to claim 1, wherein the diameter of the detection area is 5mm x 5mm, and the sample injection area is a straight channel with a length of 6mm and a width of 4 mm.
4. The method for rapidly detecting pesticide residues in vegetables based on the functionalized paper-based microfluidic chip detection test paper in claim 1 is characterized by comprising the following steps:
(1) soaking cellulose paper into NATPP solution and drying to realize modification treatment on the surface of the paper and form a super-hydrophilic channel;
(2) taking the head of the crotch-shaped paper chip as a detection area to add a specific color developing material, and taking the bottom as a mixed pesticide sample adding area;
(3) taking the final reaction color displayed by the reaction of the mixed sample and the color developing agent in the detection area as a standard, shooting the image by using a smart phone, extracting the gray value of the developed image, and establishing a linear detection model about three pesticides and above according to the obtained gray value.
5. The method for rapidly detecting pesticide residues in vegetables based on the functionalized paper-based microfluidic chip detection test paper according to claim 3, wherein the concentration of the NATPP solution in the step (1) is 1-1.5%, and the modification time is 10-15 min.
6. The method for rapidly detecting pesticide residues in vegetables based on the functionalized paper-based microfluidic chip detection test paper according to claim 3, wherein the drying temperature in the step (1) is 37 ℃.
7. The method for rapidly detecting pesticide residues in vegetables based on the functionalized paper-based microfluidic chip detection test paper according to claim 3, wherein the mixed pesticides in the step (2) are chlorpyrifos, deltamethrin, cypermethrin and profenofos, and the specific chromogenic material is DTNB, AuNPs, ninhydrin and potassium ferricyanide.
8. The method for rapidly detecting pesticide residues in vegetables based on the functionalized paper-based microfluidic chip detection test paper according to claim 3, wherein the step (3) of rapidly determining the detection of pesticide residues by using a paper-based microfluidic chip method comprises the following steps:
(1) color developing agent: mixing the substrate A and the substrate B, and depositing 2 mu L of mixed solution to a detection area;
(2) color development: dropping a mixed standard containing chlorpyrifos, profenofos, deltamethrin and cypermethrin to a hydrophilic micro-channel, and fully reacting with a color development reagent along with the capillary force of paper to a detection area to generate color change;
(3) and (3) analysis: after the color is stable, shooting is carried out at a position 10cm away from the paper-based micro-fluidic chip by using a smart phone, and gray processing and data analysis are carried out on the obtained picture through Image J and origin software to obtain the linear relation between the gray value and the concentrations of chlorpyrifos, profenofos, cypermethrin and deltamethrin.
9. Use of the test strip of claim 1 for detecting pesticide residue.
10. The method for rapidly detecting chlorpyrifos, profenofos, deltamethrin and cypermethrin by using the functional paper-based microfluidic chip detection test paper in claim 1 is characterized in that a solution to be detected is dripped into a sample solution dripping area of detection test paper, reaction is carried out for 15-20min, and the color is directly identified by using a mobile phone APP and computer software.
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