CN114199860B - Gel film for detecting organophosphorus residues, preparation method and detection method thereof - Google Patents
Gel film for detecting organophosphorus residues, preparation method and detection method thereof Download PDFInfo
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- CN114199860B CN114199860B CN202111391073.4A CN202111391073A CN114199860B CN 114199860 B CN114199860 B CN 114199860B CN 202111391073 A CN202111391073 A CN 202111391073A CN 114199860 B CN114199860 B CN 114199860B
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- 102000012440 Acetylcholinesterase Human genes 0.000 claims abstract description 77
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7756—Sensor type
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides an organophosphorus residue detection gel film, a preparation method and a detection method thereof, comprising the following steps: step S1, preparing MnO 2 A nanoparticle; step S2, preparing MnO 2 Nanoparticle-loaded gel films; step S3, preparing acetylcholinesterase MnO 2 Nanometer sub-loaded gel film: by MnO 2 The swelling property of the gel film loaded by the nano particles is used for fixing acetylcholinesterase and leading the acetylcholinesterase to each MnO film 2 Dripping acetylcholinesterase solution into nanoparticle-loaded gel soft film, fully swelling, and lyophilizing to obtain self-priming instant acetylcholinesterase MnO 2 Nanoparticle-loaded gel films. The invention utilizes acetylcholinesterase MnO 2 The nano particle loaded gel film colorimetric detection method has the advantages of few types of required reagents, simple and convenient operation steps and mild conditions, realizes on-site rapid detection of cereal OPs on the operation site of a harvester, and is low in cost and simple and convenient to operate.
Description
Technical Field
The invention belongs to the field of simulated enzyme and agricultural product safety detection, and particularly relates to a ready-to-use organophosphorus pesticide residue gel film facing a harvester operation site and a preparation method thereof, and a method for detecting organophosphorus pesticide residues in grains by using the gel film.
Background
The pesticide is an important production material, plays an important role in increasing yield and income of crops and reducing plant diseases and insect pests, and is a large country for pesticide production and use in China, so that the problem of pesticide residue caused by misuse is serious. The organophosphorus pesticides occupy 70% of the current pesticide consumption because of the characteristics of high efficiency and low toxicity, various diseases caused by organophosphorus pesticides remained in agricultural products, especially main grain crops, such as Parkinson's disease, pulmonary edema, cancer and the like, have seriously threatened the life health of people, and the current methods for realizing the pesticide residue detection of the grains on the operation site of a harvester are less, so that it is very important to construct a pesticide detection platform with high sensitivity and high accuracy for the harvesting site.
The traditional pesticide residue detection is usually carried out in a laboratory by using large-scale instruments such as high performance liquid chromatography, mass spectrometry, electrochemical workstations and the like, and has the defects of complex operation, expensive equipment, inconvenient carrying, adverse field detection and the like, so that the application of the method in field detection is greatly limited. Therefore, in order to meet the requirement of on-site detection of organophosphorus pesticide residues, chinese patent CN 111518113A (fluorescent probe for detecting glyphosate, detection test paper and preparation method thereof) discloses a method for preparing a fluorescent probe by using 3-aminophenol, 2-chloromethylpyridine and triethylamine to reflux, dripping the fluorescent probe on the surface of the test paper, and drying to obtain the detection test paper carrying the fluorescent probe for fluorescence detection of glyphosate. Chinese patent CN 106501250B, a rapid test strip and a method for detecting organophosphorus and carbamate pesticide residues by using the same, discloses a method for preparing a rapid test strip for detecting organophosphorus and carbamate pesticide residues by using a coupling reaction principle, wherein a substrate pad, an enzyme sheet and a sample pad are respectively stuck on the surface of a support back plate, and the organophosphorus pesticide is utilized to quantitatively detect the organophosphorus and carbamate pesticide by using a colorimetric method under the inhibition effect of the organophosphorus pesticide on enzymes. Chinese patent CN 109164100B "test paper strip for rapid detection of pesticide" discloses a preparation method of test paper strip for in situ generation of ferroferric oxide nano particles with imitated peroxidase activity and modification of acetylcholinesterase and choline oxidase, and organophosphorus pesticide can irreversibly inhibit acetylcholinesterase on the test paper strip, thereby reducing hydrolysis amount of acetylcholinesterase and lightening color development of color development substrate. The test paper can detect the organophosphorus pesticide by a colorimetric method and can eliminate the influence of an interfering substance.
The disclosed method for detecting organophosphorus pesticide residues has certain detection efficiency, can meet the requirements of on-site detection, but still has the following disadvantages and shortcomings: 1) The preparation of the detection test papers is complex, and a plurality of steps of operation or a plurality of modules are needed to obtain the final detection test papers; 2) Some detection methods have harsh environmental requirements, relatively high cost and high reagent preservation conditions.
Disclosure of Invention
Aiming at the problems and the defects existing in the prior art, the invention aims to provide a self-priming type instant organophosphorus pesticide residue detection gel film for the site, a preparation method thereof and a method for detecting organophosphorus pesticide residues in grains by adopting the gel film.
The method uses MnO 2 Meanwhile, as stable particles of the Peak Lin Gaona emulsion and the oxidase-like mimics, mnO is obtained after the emulsion is polymerized by thermal initiation and the oil phase is washed off 2 The loaded hydrogel with communicated pores is then self-absorbed by the hydrogel, acetylcholinesterase is fixed in the pores of the gel by swelling, and the gel film for detecting the organophosphorus pesticide is obtained after freeze drying. After the organophosphorus pesticide is added, the activity of acetylcholinesterase (AChE) is inhibited, the amount of thiocholine generated by hydrolysis is reduced, and the thiocholine is added to MnO under neutral conditions 2 The process of oxidizing the chromogenic substrate TMB has an effect and the gel film will turn blue. Therefore, the invention can realize the rapid detection of the organophosphorus pesticide by observing the color change of the self-priming instant organophosphorus pesticide residue detection gel film caused by organophosphorus pesticides with different concentrations in a certain time, and can utilize a smart phone to combine RGB analysis software to perform operation processing through the linear relation between the R value of a developed photo and the organophosphorus concentration, thereby obtaining more stable and reliable detection data, and accordingly, organophosphorus pesticide residues in grains can be determined.
The organophosphorus pesticide residue detection method is simple and convenient to prepare, simple to operate and low in cost, and the nanoenzyme has the activity equivalent to that of the natural enzyme, low cost and high stability; the porous hydrogel is simple and convenient to prepare, stable in structure, self-absorption and high in mass transfer rate, is an ideal carrier, combines nano enzyme with the porous hydrogel, detects organophosphorus pesticide residues by using a colorimetric method, does not need a complex instrument, and is very suitable for quantitative analysis of organophosphorus pesticide residues in complex field grains.
The invention is realized by the following technical scheme: a preparation method of a gel film for detecting organophosphorus pesticide residues in grains comprises the following steps:
step S1, preparing MnO 2 A nanoparticle;
step S2, preparing MnO 2 Nanoparticle-loaded gel films;
step S3, preparing acetylcholinesterase MnO 2 Nanometer sub-loaded gel film: by MnO 2 The swelling property of the gel film loaded by the nano particles is used for fixing acetylcholinesterase and leading the acetylcholinesterase to each MnO film 2 Dripping acetylcholinesterase solution into nanoparticle-loaded gel soft film, fully swelling, and lyophilizing to obtain self-priming instant acetylcholinesterase MnO 2 Nanoparticle-loaded gel films.
In the above scheme, the step S2 specifically includes:
MnO is added to 2 Dispersing the nano particles in deionized water by ultrasonic waves, then adding acrylamide serving as a monomer, N, N-methylene bisacrylamide serving as a cross-linking agent and potassium persulfate serving as an initiator, and obtaining a water phase after complete dissolution; then liquid paraffin is added as an oil phase, and stable high-internal emulsion is obtained after high-speed stirring; injecting the high-internal emulsion into a film-making mould, perforating after polymerization, washing with ethanol and deionized water, and freeze-drying to obtain MnO 2 Nanometer sub-loaded gel film.
Further, the high-speed stirring speed in the step S2 is 11000-18000 rpm, and the time is 1-5 min; the polymerization temperature is 40-60 ℃ and the time is 10-14 h.
Further, mnO in the step S2 2 The dosage is 1-10 mg; the dosage of the acrylamide is 0.5-2.0 g, the dosage of the N, N-methylene bisacrylamide is 0.1-0.5 g, and the dosage of the potassium persulfate is 0.1-0.15 g.
In the scheme, the volume of the acetylcholinesterase added into each gel in the step S3 is 15-25 mu L, and the concentration is 0-200U/L.
The cereal organophosphorus pesticide residue detection gel film is prepared according to the preparation method of the cereal organophosphorus pesticide residue detection gel film.
The detection method for detecting the gel film according to the organophosphorus pesticide residues of the grains comprises the following steps:
step S1) directed to acetylcholinesterase MnO 2 Dripping standard solution into the nano sub-loaded gel film for reaction, and preparing an organophosphorus concentration-R value standard curve;
step S2) re-directing to acetylcholinesterase MnO 2 Adding the nano sub-loaded gel film into a thio-acetylcholine ATCH solution for reaction;
step S3) final reaction towards acetylcholinesterase MnO 2 Adding 3,3', 5' -tetramethyl benzidine TMB solution into the nanometer sub-loaded gel film for reaction;
s4) recording the color RGB value of each gel film, drawing an organophosphorus concentration-R value standard curve with TMB as a developer, and/or extracting each developed gel film image to prepare an organophosphorus concentration standard color comparison card;
step S5) repeating the steps S1) to S4) on the organophosphorus sample to be detected, and measuring acetylcholinesterase MnO using TMB as a substrate 2 And (3) comparing the R value of the nano sub-loaded gel film with an organophosphorus concentration-R value standard curve to obtain the concentration of organophosphorus pesticide residues in the organophosphorus sample to be detected, and/or extracting a developed gel film image of each piece, and comparing the developed gel film image with an organophosphorus concentration standard colorimetric card to obtain the concentration of organophosphorus pesticide residues in the organophosphorus sample to be detected.
In the scheme, the standard solution volume in the step S1) is 10-30 mu L, the reaction is carried out for 10-25 min, and the standard curve concentration values are 400ng/mL, 200ng/mL, 100ng/mL, 40ng/mL, 20ng/mL, 10ng/mL and 4ng/mL respectively.
In the scheme, the volume of the thiocholine ATCH solution in the step S2) is 5-15 mu L, the concentration is 15-25 mM, and the reaction time is 20-40 min.
In the scheme, the TMB solution in the step S3) has the volume of 10-30 mu L and the concentration of 3-8 mM; the reaction time is 1-3 min.
Compared with the prior art, the invention has the beneficial effects that:
the invention is oriented to the fieldPreparation of self-priming instant organophosphorus residue detection gel film and method for rapid detection of OPs by using acetylcholinesterase MnO 2 The nano particle loaded gel film colorimetrically detects the content of organophosphorus pesticide residues, the required reagent types are few, the operation steps are simple and convenient, the condition is mild, the on-site rapid detection of the grain OPs on the operation site of the harvester is realized, the cost is low, and the operation is simple and convenient; acetylcholinesterase MnO according to the present invention 2 The detection limit of the nanoparticle-loaded gel film on OPs is as low as 0.63ng/mL, and the detection range is as wide as 4-400 ng/mL; mnO utilizing acetylcholinesterase 2 The nano particle loaded gel film detects the content of organophosphorus pesticide residues in grains, has accurate detection results on actual samples, sensitive response and good stability, and can realize the selective detection of organophosphorus pesticide residues in crop grains.
Drawings
FIG. 1 shows TMB and MnO 2 Ultraviolet-visible absorption spectrum of the system when the system reacts for 2min in different environmental atmospheres;
FIG. 2 shows acetylcholinesterase MnO 2 RGB plot of nanoparticle loaded gel film response to phosphorus-sulfur doubling;
FIG. 3A is MnO 2 An addition amount optimization schematic diagram, and fig. 3B is an AChE usage amount optimization schematic diagram;
FIG. 4 shows the use of acetylcholinesterase MnO 2 The detection effect diagram of detecting the phosphorus-sulfur by the nanoparticle-loaded gel film, wherein fig. 4A is a response diagram of the gel film to the concentration of the phosphorus-sulfur; FIG. 4B is a straight line fit of the phosphorus-sulfur-R values;
FIG. 5 shows acetylcholinesterase MnO 2 A selectivity graph of the nano particle loaded gel film on the detection of the phosphorus-sulfur doubling;
FIG. 6 shows acetylcholinesterase MnO 2 Stability diagram of nanoparticle-loaded gel film against detection of phosphorus-sulfur.
Detailed Description
The embodiments of the present invention are described in detail below with reference to the accompanying drawings, which are exemplary and intended to be illustrative of the present invention and not to be construed as limiting the invention.
The preparation method of the organophosphorus residue detection gel film comprises the following steps:
step S1, preparing MnO 2 A nanoparticle;
step S2, preparing MnO 2 Nanoparticle-loaded gel films;
step S3, preparing acetylcholinesterase MnO 2 Nanometer sub-loaded gel film: by MnO 2 The swelling property of the gel film loaded by the nano particles is used for fixing acetylcholinesterase and leading the acetylcholinesterase to each MnO film 2 Dripping acetylcholinesterase solution into nanoparticle-loaded gel soft film, fully swelling, and lyophilizing to obtain self-priming instant acetylcholinesterase MnO 2 Nanoparticle-loaded gel films.
The step S1 prepares MnO 2 The nano particles are specifically as follows:
0.2 g to 0.7g KMnO in a 500mL beaker 4 Dissolved in 250mL H 2 O, preferably 0.5g, is sonicated for 15min, followed by dropwise addition of 5mL of oleic acid with stirring, and reaction at 20-45℃for 8-12 h, preferably 38℃for 10h. Centrifugal separation, eluting with deionized water and ethanol respectively to obtain MnO 2 Drying the precursor at 60 ℃, and then pyrolyzing the dried precursor at 180-250 ℃ for 0.5-3 h in an air atmosphere to obtain MnO 2 The nanoparticles are pyrolyzed for 2 hours, preferably at 200 ℃.
The step S2 prepares MnO 2 The nanoparticle-loaded gel film comprises the following components:
1 to 10mg of MnO obtained in step S1 2 The nanoparticles are dispersed in 4mL deionized water, preferably 4mg, followed by the addition of 0.5-2.0 g Acrylamide (AM) as monomer, 0.1-0.5 g n, n-methylenebisacrylamide (Bis) as crosslinker, 0.1-0.15 g potassium persulfate (APS) as initiator, preferably AM1.42 g, bis 0.35g, APS 0.12g, after complete dissolution to give the aqueous phase. 16mL of liquid paraffin was added as an oil phase, and the mixture was stirred at a high speed of 11000 to 18000rpm for 1 to 5 minutes to obtain a stable high internal emulsion, preferably at 14000rpm for 2 minutes.
Injecting the high internal emulsion into a film-making mould, polymerizing for 10-14 h at 40-60 ℃ in an oven, preferably polymerizing for 12h at 50 ℃,punching with 8mm diameter puncher, washing with ethanol and deionized water, and freeze drying to obtain MnO 2 Nano-sub-loaded porous hydrogels.
The step S3 prepares acetylcholinesterase MnO 2 The nanometer sub-loaded gel film comprises the following concrete components:
by MnO 2 The inherent swelling property of the nanoparticle-loaded gel film is used for fixing AChE, and each MnO piece 2 Dripping 15-25 mu L of AChE solution with the concentration of 0-200U/L, preferably 20 mu L and 80U/L, and freeze-drying to obtain self-priming instant acetylcholinesterase MnO 2 Nanometer sub-loaded gel film.
The detection method for detecting the gel film according to the organophosphorus residue specifically comprises the following steps:
step S1) separate reaction of the step S1) with acetylcholinesterase MnO 2 Dripping 10-30 mu L parathion solution with different concentrations into the nanometer sub-loaded gel film, and reacting for 10-25 min, preferably 20 mu L for 15min; the concentration of parathion added dropwise is 400ng/mL, 200ng/mL, 100ng/mL, 40ng/mL, 20ng/mL, 10ng/mL, 4ng/mL, and an organophosphorus concentration-R value standard curve is prepared.
Step S2) re-directing to acetylcholinesterase MnO 2 The nano-sized loaded gel film is added with 5 to 15 mu L of 15 to 25mM of thiocholine ATCH solution (dissolved in 0.1M PBS buffer solution, pH 7.4) and reacted for 20 to 40min, preferably 10 mu L of 20mM ATCH and reacted for 30min.
Step S3) final reaction towards acetylcholinesterase MnO 2 The nano-sized sub-loaded gel film is added with 10-30. Mu.L of 3-8 mM TMB (0.1M PBS buffer, pH 7.4), and reacted for 1-3 min, preferably 20. Mu.L of 5mM TMB, and reacted for 2min.
S4) recording the color RGB value of each gel film by combining the smart phone with IambeJ color-taking software, and drawing an organophosphorus concentration-R value standard working curve with TMB as a color developing agent; photographing each developed gel sheet by using a smart phone, and preparing an organophosphorus concentration standard colorimetric card for visual observation;
step S5) repeating the step S1) to the step S4) on the organophosphorus sample to be detected, and respectively measuring the organophosphorus sample with a smart phone based on TMBAcetylcholinesterase MnO in the case of the material 2 And (3) calculating the R value of the nano sub-loaded gel film and comparing the R value with a standard working curve to obtain the concentration of organophosphorus pesticide residues in the sample.
In a preferred embodiment of the present invention, the PBS buffer has a pH of 7.4 and a concentration of 0.1M.
In the preferred disclosed embodiment of the invention, the detectable concentration range of the organophosphorus sample to be detected is 4-400 ng/mL, and the detection limit is as low as 0.63ng/mL.
In the present specification, the term "oxidase-like" refers to a material having oxidase catalytic activity. Specifically, the oxidase of the invention uses oxygen as an electron acceptor, and generates colored substances through catalytic oxidation of corresponding substrates, so as to be used for colorimetric detection.
In this specification, the term "TMB" is an abbreviated name of the compound "3,3', 5' -tetramethylbenzidine", which are used interchangeably.
In this specification, the term "TMBox" is an abbreviated name for the oxidation product of the compound "3,3', 5' -tetramethylbenzidine", which are used interchangeably.
In the present specification, the term "AChE" refers to acetylcholinesterase, which is used interchangeably.
In the present specification, the term "ATCh" refers to acetylcholine, both of which are used interchangeably.
The reactants and reagents used in the invention are all commercially available.
EXAMPLE 1 preparation of MnO 2 Nanoparticles
Step 1. 0.5g KMnO was placed in a 500mL beaker 4 Dissolved in 250mL H 2 O, sonicated for 15min, then 5mL oleic acid was added dropwise with stirring and reacted at 38℃for 10h.
Step 2, centrifugally separating the product in the step 1, and respectively eluting with deionized water and ethanol to obtain MnO 2 The precursor was dried at 60 ℃.
Step 3, after the precursor in the step 2 is completely dried, pyrolyzing the precursor for 2 hours at 200 ℃ in an air atmosphere to obtain MnO 2 And (3) nanoparticles. MnO (MnO) 2 Nanoparticle as oxidases to catalyze TMB reactionThe application test is carried out as follows:
step 1) 2.8mL of 0.1M phosphate buffer (pH 7.4) was added to each of the two test tubes, one of which was continuously purged with nitrogen for 30 minutes, followed by sequential addition of 0.1mL of 5mM TMB and 0.1mL of 1mg/mL MnO thereto 2 Dispersing the solution, and then uniformly mixing the solution;
step 2) reacting the system at room temperature for 3min, wherein the color of the liquid changes from colorless to blue along with the time;
step 3) measuring the ultraviolet-visible absorption spectrum of the mixed solution by an ultraviolet-visible absorption spectrophotometer. FIG. 1 records the prepared MnO 2 UV-Vis diagram of catalytic oxidation of TMB reactions in different systems. As can be seen from FIG. 1, when TMB and MnO 2 In the presence of the same, a color reaction occurs and in the absence of oxygen, the reaction is weaker; when TMB and MnO 2 Only one of them is present, no color reaction occurs. The reaction is carried out by TMB at MnO 2 Is caused by oxidation reduction under the catalysis of (a) and proves that the prepared MnO 2 Has good oxidase activity.
Example 2 gel film preparation:
step 1.4 mg MnO 2 The nanoparticles were dispersed in 4mL of deionized water with ultrasound, followed by the addition of 1.42g of Acrylamide (AM) as monomer, 0.35g of N, N-methylenebisacrylamide (Bis) as crosslinker, 0.12g of potassium persulfate (APS) as initiator, and the dissolution was complete to give the aqueous phase. Subsequently, 16mL of liquid paraffin was added as an oil phase, and stirred at a high speed of 14000rpm for 2 minutes to obtain a stable high internal emulsion.
Step 2, injecting the high inward emulsion obtained in the step 1 into a film-making mould, polymerizing for 12 hours at 50 ℃ in an oven, punching by using a puncher with diameter of 8mm, washing by ethanol and deionized water, and freeze-drying to obtain MnO 2 Nano-sub-loaded porous hydrogels.
Step 3, utilizing MnO 2 The inherent swelling property of the nanoparticle-loaded gel film is used for immobilization of acetylcholinesterase (AChE), each MnO 2 Dripping 20 mu L of 80U/LAChE solution into the nanoparticle-loaded gel film, and lyophilizing to obtain self-priming filmInstant acetylcholinesterase MnO 2 Nanometer sub-loaded gel film.
Acetylcholinesterase MnO 2 The feasibility test of detecting the phosphorus and the sulfur by taking the nanometer sub-loaded gel film as a platform comprises the following steps:
step 1) taking two final acetylcholinesterase MnO sheets 2 The nanometer loaded gel film is respectively dripped with 20 mu L of mixed solution containing 40ng/mL and 0ng/mL of phosphorus sesquisulfate (0.1M PBS contains 5 percent of methanol by volume, pH 7.4) and reacts for 15 minutes at room temperature;
step 2) respectively dripping 10 mu L of 20mM ATCH solution into the gel film in the step 1), and reacting for 30min at room temperature;
and 3) finally, dropwise adding 20 mu L of 5mM TMB solution into the gel sheet for reaction for 2min, and then performing color analysis on the gel film by using a smart phone set ImageJ color-taking software. As shown in FIG. 2, the gel film exhibited blue color only in the presence of phosphorus sesquioxide, and the R value was significantly reduced, so that acetylcholinesterase MnO was produced 2 The nanometer sub-loaded gel film can sensitively respond to organophosphorus pesticides and is used for detecting pesticide residues.
FIG. 3A shows MnO 2 The influence of the usage amount of 1-10 mg on the apparent color and the catalytic performance of the gel film is changed, so as to obtain the optimal detection effect MnO 2 The optimized use amount is 4mg; FIG. 3B shows the effect of AChE solution concentration from 0 to 200U/L on the catalytic performance of the gel film, with an AChE optimum usage of 80U/L for best detection.
EXAMPLE 3 utilization of acetylcholinesterase MnO 2 Colorimetric detection of phoxim by nano sub-loaded gel film
Step 1) respectively directing acetylcholinesterase MnO 2 Dropwise adding 20 mu L of parathion solutions with different concentrations into the nanometer sub-loaded gel film, and reacting for 15min at room temperature, wherein the concentration of the drop doubling parathion is 400ng/mL, 200ng/mL, 100ng/mL, 40ng/mL, 20ng/mL, 10ng/mL and 4ng/mL;
step 2) further directing to acetylcholinesterase MnO 2 The nano-sized loaded gel film was added with 10. Mu.L of 20mM of a solution of thioacetylcholine ATCH (dissolved in 0.1M PBS buffer, pH7.4 Reaction for 30min;
step 3) final reaction with acetylcholinesterase MnO 2 Adding 20 mu L of 5mM TMB (0.1M PBS buffer solution, pH 7.4) into the nanometer sub-loaded gel film, and reacting for 2min after uniformly mixing the system;
step 4) recording the color RGB value of each gel film by combining the smart phone with the IambeJ color-taking software, and drawing an organophosphorus concentration-R value standard working curve with TMB as a color developing agent; photographing each developed gel sheet by using a smart phone, and preparing an organophosphorus concentration standard colorimetric card for visual observation;
the result of detecting the content of the phosphorus-sulfur-doubling compound by combining the gel film of the invention with a color comparator of a smart phone is shown in figure 4. Among them, fig. 4A illustrates that the color of the gel film gradually deepens as the content of phosphorus-sulfur increases. FIG. 4B is a straight line fitted with R value-phosphorus-sulfur concentration, which shows that the method has excellent detection effect on the detectable range of phosphorus-sulfur content of 4-400 ng/mL.
EXAMPLE 4 utilization of acetylcholinesterase MnO 2 Nanometer sub-loaded gel film detection of phosphorus-sulfur-doubling selectivity
Step 1) 20 mu L of each of the samples containing K + 、Ca 2+ 、Mg 2+ 、Cd 2+ 、Br - 、Cl - 、CO 3 2- 、SO 4 2- The solution of glucose, glucose oxidase (GOx), bovine Serum Albumin (BSA), glutamic acid, malathion and carbaryl is added dropwise to acetylcholinesterase MnO 2 In the nano-sized loaded gel film, the reaction is carried out for 15min at room temperature, then 10 mu L of 20mM of thioacetylcholine ATCH solution (dissolved in 0.1M of PBS buffer solution, pH 7.4) is added for 30min; finally, 20 mu L of 5mM TMB (0.1M PBS buffer solution, pH 7.4) is added, and the mixture is reacted for 2min after uniform mixing; recording the color R value of each piece of gel film by combining the smart phone with IambeJ color-taking software;
step 2) respectively taking 20 mu L of phosphorus-sulfur compound K + 、Ca 2+ 、Mg 2+ 、Cd 2+ 、Br - 、Cl - 、CO 3 2- 、SO 4 2- Glucose, glucose oxidase (GOx), bovine serum albumin(BSA), glutamic acid, malathion and carbaryl are added dropwise to acetylcholinesterase MnO 2 In the nano-sized loaded gel film, the reaction is carried out for 15min at room temperature, then 10 mu L of 20mM of thioacetylcholine ATCH solution (dissolved in 0.1M of PBS buffer solution, pH 7.4) is added for 30min; finally, 20 mu L of 5mM TMB (0.1M PBS buffer solution, pH 7.4) is added, and the mixture is reacted for 2min after uniform mixing; recording the color R value of each piece of gel film by combining the smart phone with IambeJ color-taking software;
the results are shown in FIG. 4. FIG. 5 shows the use of acetylcholinesterase MnO 2 The nanometer sub-loaded gel film detects the selective bar graph of the phoxim, which is a single interferent mode and a target-interferent mixed mode respectively, and the bar graph is blank and K in sequence from left to right + 、Ca 2+ 、Mg 2+ 、Cd 2+ 、Br - 、Cl - 、CO 3 2- 、SO 4 2- The sensor can be used for detecting pesticide residues with high selectivity and stability, and the graph shows that only the parathion, the malathion and the carbaryl can obviously promote the color reaction of TMB, and other interferents can not influence the detection of the gel film when coexisting.
EXAMPLE 5 acetylcholinesterase MnO 2 Stability of nano sub-loaded gel film for detecting phoxim
60 pieces of acetylcholinesterase MnO are taken 2 Storing the nanometer sub-loaded gel film at room temperature, taking the gel film on days 0, 2, 4, 6, 8, 10, 20 and 30, respectively dripping 20 mu L of blank solution and 20ng/mL of phosphorus-sulfur doubling solution, reacting for 15min at room temperature, then adding 10 mu L of 20mM of thiocholine ATCH solution (dissolved in 0.1M of PBS buffer solution, pH of 7.4) and reacting for 30min; finally, 20 mu L of 5mM TMB (0.1M PBS buffer solution, pH 7.4) is added, and the mixture is reacted for 2min after uniform mixing; the result of recording the R value of each gel film by combining the smart phone with the IambeJ color-taking software is shown in figure 6, and the acetylcholinesterase MnO is stored at room temperature for 30 days 2 The detection effect of the nano sub-loaded gel film on the phosphorus-sulfur is not obviously changed,indicating that acetylcholinesterase MnO 2 The nanometer sub-loaded gel film has excellent room temperature stability.
EXAMPLE 6 acetylcholinesterase MnO 2 Nanometer sub-loaded gel film for detecting phoxim in wheat and rice
Step 1) respectively grinding wheat and rice samples, extracting with methanol and freeze-drying;
step 2) re-dissolving the freeze-dried extract liquid of the wheat and the rice by using 20 mu L of buffer solution containing the phosphorus-sulfur with different concentrations;
step 3) dripping the redissolved solution in the step 2) into acetylcholinesterase MnO 2 In the nanometer sub-loaded gel film, reacting for 15min at room temperature, then adding 10 mu L of 20mM of thioacetylcholine ATCH solution (dissolved in 0.1M of PBS buffer solution, pH 7.4) and reacting for 30min; finally, 20 mu L of 5mM TMB (0.1M PBS buffer solution, pH 7.4) is added, and the mixture is reacted for 2min after uniform mixing;
step 4) recording the color R value of each piece of gel film by combining the smart phone with the IambeJ color taking software; r value-phosphorus concentration fitting straight line is entered to convert into phosphorus-sulfur content;
the measurement results are shown in the following table 1:
TABLE 1 comparison of results of the present method and national standard method
As is clear from the above table, acetylcholinesterase MnO 2 The nanometer sub-loaded gel film has sensitive response to the change of the content of the phosphorus and the sulfur in the actual sample, and the result is similar to the actual measurement value of the national standard method.
The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.
Claims (9)
1. The preparation method of the organophosphorus residue detection gel film is characterized by comprising the following steps of:
step S1, preparing MnO 2 A nanoparticle;
step S2, preparing MnO 2 The nanoparticle-loaded gel film comprises the following concrete components: mnO is added to 2 Dispersing the nano particles in deionized water by ultrasonic waves, then adding acrylamide serving as a monomer, N, N-methylene bisacrylamide serving as a cross-linking agent and potassium persulfate serving as an initiator, and obtaining a water phase after complete dissolution; then liquid paraffin is added as an oil phase, and stable high-internal emulsion is obtained after high-speed stirring; injecting the high-internal emulsion into a film-making mould, perforating after polymerization, washing with ethanol and deionized water, and freeze-drying to obtain MnO 2 Nanometer sub-loaded gel film;
step S3, preparing acetylcholinesterase MnO 2 Nanometer sub-loaded gel film: by MnO 2 The swelling property of the gel film loaded by the nano particles is used for fixing acetylcholinesterase and leading the acetylcholinesterase to each MnO film 2 Dripping acetylcholinesterase solution into nanoparticle-loaded gel soft film, fully swelling, and lyophilizing to obtain self-priming instant acetylcholinesterase MnO 2 Nanoparticle-loaded gel films.
2. The method for producing a gel film for detecting residual organic phosphorus according to claim 1, wherein the high-speed stirring speed in the step S2 is 11000 to 18000rpm for 1 to 5 minutes; the polymerization temperature is 40-60 ℃ and the polymerization time is 10-14 h.
3. The method for producing an organic phosphorus residue detection gel film according to claim 1, wherein MnO in step S2 2 The dosage is 1-10 mg; the dosage of acrylamide is 0.5-2.0 g, the dosage of N, N-methylene bisacrylamide is 0.1-0.5 g, and the dosage of potassium persulfate is 0.1-0.15 g.
4. The method for preparing a gel film for detecting residual organic phosphorus according to claim 1, wherein the volume of acetylcholinesterase added to each gel in the step S3 is 15-25 mu L, and the concentration is 0-200U/L.
5. An organic phosphorus residue detection gel film prepared by the method of any one of claims 1 to 4.
6. A method for detecting an organophosphorus residue detecting gel film according to claim 5, comprising the steps of:
step S1) directed to acetylcholinesterase MnO 2 Dripping standard solution into the nano sub-loaded gel film for reaction, and preparing an organophosphorus concentration-R value standard curve;
step S2) re-directing to acetylcholinesterase MnO 2 Adding the nano sub-loaded gel film into a thio-acetylcholine ATCH solution for reaction;
step S3) final reaction towards acetylcholinesterase MnO 2 Adding 3,3', 5' -tetramethyl benzidine TMB solution into the nanometer sub-loaded gel film for reaction;
s4) recording the color RGB value of each gel film, drawing an organophosphorus concentration-R value standard curve with TMB as a developer, and/or extracting each developed gel film image to prepare an organophosphorus concentration standard color comparison card;
step S5) repeating the steps S1) to S4) on the organophosphorus sample to be detected, and measuring acetylcholinesterase MnO using TMB as a substrate 2 And (3) comparing the R value of the nano sub-loaded gel film with an organophosphorus concentration-R value standard curve to obtain the concentration of organophosphorus pesticide residues in the organophosphorus sample to be detected, and/or extracting a developed gel film image of each piece, and comparing the developed gel film image with an organophosphorus concentration standard colorimetric card to obtain the concentration of organophosphorus pesticide residues in the organophosphorus sample to be detected.
7. The method for detecting the residual organic phosphorus detecting gel film according to claim 6, wherein the standard solution in the step S1) has a volume of 10-30 mu L and is reacted for 10-25 min, and the standard curve concentration values are 400ng/mL, 200ng/mL, 100ng/mL, 40ng/mL, 20ng/mL, 10ng/mL and 4ng/mL, respectively.
8. The method for detecting a gel film for detecting residual organic phosphorus according to claim 6, wherein the volume of the solution of the thiocholine ATCH in the step S2) is 5-15 mu L, the concentration is 15-25 mM, and the reaction time is 20-40 min.
9. The method for detecting an organic phosphorus residue detecting gel film according to claim 6, wherein the TMB solution in the step S3) has a volume of 10-30. Mu.L and a concentration of 3-8 mM; the reaction time is 1-3 min.
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