CN103940868A - Fast detector of pesticide residues based on enzyme immunosensor - Google Patents
Fast detector of pesticide residues based on enzyme immunosensor Download PDFInfo
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- CN103940868A CN103940868A CN201410150101.7A CN201410150101A CN103940868A CN 103940868 A CN103940868 A CN 103940868A CN 201410150101 A CN201410150101 A CN 201410150101A CN 103940868 A CN103940868 A CN 103940868A
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Abstract
The invention discloses a fast detector of pesticide residues based on an enzyme immunosensor, belonging to the technical field of safe detection of agricultural products. The fast detector of pesticide residues based on the enzyme immunosensor comprises a three-electrode system, a signal detecting and processing system, a displaying, printing and storing system and a power supply system, wherein the three-electrode system is used for measuring change of current before and after touching a sample through an enzyme sensor to know whether the sample contains pesticide and whether the content of the pesticide exceeds standard; if the pesticide exceeds standard, an immunosensor is used for detecting change of current before and after touching the sample to know information about the type of the pesticide and the concentration of the pesticide in the sample. The detector is simple to operate, low in cost, high in sensitivity, excellent in repeatability, capable of realizing online fast detection and applicable to the field fast detection of the agricultural products.
Description
Technical field
The present invention relates to a kind of rapid detector for pesticide residue based on enzyme immunosensor, belong to agricultural product security detection technique field.
Technical background
Agricultural chemicals is important capital goods in the agricultural sector, in agricultural product are produced, the control of disease, worm, grass, mouse harm is played to very important effect.But the harm consequence of pollution by pesticides and generation thereof is serious, the loss that agricultural chemicals environmental pollution causes is many-sided, comprise the pollution to water environment, to the pollution of soil, to the pollution of atmosphere, the impact on environmental organism and the harm to health etc.Due to generally shortage science use agricultural chemicals knowledge of user, the output that covets, causes the agricultural products of circulation in the market all to have to some extent Pesticide Residue at present, and its harm also causes the public's concern day by day.China is a large agricultural country, and along with China's living standards of the people improve constantly, the quality security problem of agricultural product more and more receives publicity, and especially Pesticide Residues in Vegetables problem has become the focus of public attention.China produces and the most kinds of pesticide of use are organophosphorus and carbamate chemicals for agriculture (accounting for 70%), and wherein highly toxic organophosphorus and carbamate insecticides are 70% left and right, therefore easily cause that food pesticide is residual poisoning.Visible, reinforcement to preserving the ecological environment, especially ensure that human health has very profound significance, and the emphasis of Detecting Pesticide should be placed on organophosphorus and carbamate chemicals for agriculture to the detection of Residual Pesticides in Farm Produce.
The main method of pesticide residue analysis is gas chromatograph, liquid chromatograph, gas chromatograph-mass spectrometer, LC-MS instrument etc. at present, although these method analysis precisions are high, quantitatively accurately, but the complex pretreatment of its sample, detect the operating personnel that length consuming time, cost are high, need to be skilled in technique.The fast determining method of China's residues of pesticides is that enzyme suppresses test paper method and enzyme inhibition-spectrophotometry (the residual fast detector of agriculture), can realize the field quick detection of organophosphorus pesticide and carbamate chemicals for agriculture, has good practical value.Quick measuring card is by the change color of visual inspection card, and the vegetable sample that therefore generally can only be used for severe overweight carries out observational measurement.The application of enzyme inhibition-spectrophotometry is also more extensive, and the residual tacheometer of domestic existing Multiple Pesticides is all based on this principle.Light splitting light ratio juris is that the variation based on absorbance detects, but in vegetable and fruit, a large amount of pigments can cause very large impact to spectrophotometric method, causes the inaccurate of testing result.And said method exists, and the recovery is low, false retrieval, undetected ratio are higher, poor repeatability, be difficult to meet low-residual and the shortcoming such as the requirement that quantitatively detects.
Summary of the invention
The object of the present invention is to provide and a kind ofly can overcome above-mentioned defect and rapid detector for pesticide residue simple to operate, cheap, highly sensitive, that realize on-line quick detection.Can accurately know and in sample, contain whether residues of pesticides exceed standard and can which kind of residues of pesticides and the information such as concentration.The technical scheme of taking is as follows:
Based on a rapid detector for pesticide residue for enzyme immunosensor, be by three-electrode system, input and disposal system, show and print storage system and electric power system formation;
Described three-electrode system is by enzyme electrode and immunosensor, three electrode detection circuit, potentiostatic circuit and analog multichannel switch form, enzyme electrode and immunosensor gather current signal from detect sample, circuit signal is transmitted to input and disposal system by three electrode detection circuit, potentiostatic circuit provides steady voltage for electrode, and the current signal collection that analog multichannel switch successively produces immunosensor respectively is also sent into input and disposal system;
Described enzyme electrode is made up of a working electrode, is fixed with acetylcholinesterase at working electrode surface; Described immunosensor is made up of a circular electrode disk, and the centre bit of electrode disk is equipped with electrode and contrast electrode, and periphery is equally distributed array working electrode, and array working electrode is fixed with pesticide antibody;
Described input and disposal system are by TLC272CP chip, A/D conversion chip and single-chip microcomputer form, the multichannel discharge circuit of TLC272CP chip carries out I/V conversion to the current signal sending from enzyme electrode and immunosensor, amplify, filtering processing, A/D conversion chip is sent the digital signal after conversion into single-chip microcomputer, and single-chip microcomputer has microcontroller, and the digital signal after conversion is carried out to routine processes;
The microcontroller of described single-chip microcomputer is STC89C52 microcontroller, the routine package of single-chip microcomputer is containing data-printing and storage, time showing, operation instruction and the dialog box to important step prompting, help user grasp fast the using method of instrument and carry out data-printing and storage;
Described demonstration and printing storage system are by display circuit, print circuit and memory circuitry form, display is used for showing data and controls by operating key, prints to be respectively mini-printer and storer with memory circuit and to be connected and to carry out data-printing and storage;
Described electric power system electric routing switch power supply and voltage conversion circuit form, Switching Power Supply converts alternating current to direct current and powers to each circuit system and mini-printer respectively through voltage conversion circuit, Switching Power Supply is+12V, 1A ,-12V, 1A ,+5V, the output of 5ASan road, power supply is connected with the Switching Power Supply in electric power system.
Described potentiostatic circuit, for providing the steady voltage of 500mV between working electrode and contrast electrode.
Described microcontroller is that kernel is the STC89C52RC+ microcontroller of MCS-51.
The storage unit of described input and disposal system is inserted the pesticide concentration of acetylcholinesterase sensor detection residues of pesticides corresponding to immunosensor and the typical curve database of current relationship.
Described display adopts 3.5 cun of display screens; Described mini-printer adopts micro embedded printer.
A kind of detection method step of described detector of utilizing is as follows:
1) enzyme electrode that is fixed with acetylcholinesterase is put into the not substrate solution containing agricultural chemicals, the electric current I while detecting not containing agricultural chemicals
1, or the default value of employing default;
2) take out after enzyme electrode, with distilled water flushing, then enzyme electrode is put into sample liquid hatch 10min;
3) take out after enzyme electrode, with distilled water flushing, again put into substrate solution, detect electric current I
2;
4) by the trace routine on microcontroller according to inhibiting rate formula, calculate inhibiting rate;
5) utilize immunosensor to substitute enzyme electrode, repeating step 1) to step 4) process again detecting step 4) the inhibiting rate of surveying higher than 20% sample, and obtain the inhibiting rate that exceeds standard;
6) step 5) gained is exceeded standard inhibiting rate contrasts with inhibiting rate and the pesticide concentration typical curve database of inserting in advance detector, acquisition exceed standard pesticide variety and concentration;
7) method described in claim 6, is characterized in that, inhibiting rate formula described in step 4), is specially:
Inhibiting rate=(I
1-I
2)/I
1% × 100%.
The ultimate principle of detector provided by the present invention is: three-electrode system, first by enzyme sensor, is measured the variation of contact sample front and back electric current, knows whether in sample, whether contain agricultural chemicals exceeds standard.If truly have agricultural chemicals to exceed standard, re-use immunosensor, measure the variation of electric current before and after contact sample, know the information such as the concentration that contains which kind of agricultural chemicals and agricultural chemicals in sample.The low current signal that instrument for detecting pesticide residue through is responsible for three electrodes to collect carries out I/V conversion, amplifies, and filtering, A/D conversion the most at last digital quantity signal is sent into single-chip microcomputer and is carried out routine processes.Show and print storage system by parameter displays such as residual pesticide concentration, inhibition degree out, and data being stored so that user checks in time and carries out data analysis.Show with printing storage system and include mini-printer, data can be carried out to corresponding printout.
In order to realize above-mentioned functions, described enzyme sensor has a working electrode to form, and at the fixing acetylcholinesterase of working electrode surface, prepares acetylcholinesterase sensor.Described immunosensor is made up of a circular electrode disk, centre bit is equipped with electrode and contrast electrode, periphery is equally distributed working electrode, electrode, contrast electrode are equated with the distance of each working electrode guaranteeing, wherein array working electrode is used for fixing different types of pesticide antibody.Detector storage unit is inserted the pesticide concentration of acetylcholinesterase sensor detection residues of pesticides corresponding to immunosensor and the typical curve database of current relationship.When use, first will fix acetylcholinesterase sensor and be connected with detector, before and after the contact sample obtaining according to acetylcholinesterase sensor, whether the signal of curent change, determine whether to contain agricultural chemicals and exceed standard.If there is the demonstration that exceeds standard, again the immunosensor array of sessile antibody is connected on detector, the current signal collection successively array immunization sensor being produced respectively by analog multichannel switch is sent in detector, the variable signal of electric current before and after the contact sample obtaining according to immunosensor, determines that concrete which kind of agricultural chemicals exceeds standard and concentration is how many.Therefore whether this detector can obtain agricultural chemicals simultaneously and exceed standard in the sampling of sample, contains which kind of the concrete information such as agricultural chemicals and concentration.
Beneficial effect of the present invention: the rapid detector for pesticide residue based on enzyme immunosensor of giving provided by the present invention, can make up the defect that acetylcholinesterase biology sensor and immunosensor exist.Acetylcholinesterase sensor can only detect organophosphorus and carbamates, whether contains this class agricultural chemicals, and cannot deterministicly know that any residues of pesticides exceed standard.Immunosensor is the specific recognition of antigen-antibody, therefore can know concrete any agricultural chemicals, but antibody is more expensive, if the information that does not have residues of pesticides to exceed standard just there is no need to carry out the measurement of second step.Therefore a kind of rapid detector for pesticide residue based on enzyme immunosensor of the present invention, had both saved testing cost, can deterministicly know again the information that concrete any residues of pesticides exceed standard, and was suitable for the fast detecting of residues of pesticides.Be limited to 1ug/L with the detection that the method can reach.Accuracy rate is because used two kinds of sensors to detect, so accuracy is higher than the simple accuracy with enzyme sensor and immunosensor.
Brief description of the drawings
Fig. 1 is the structured flowchart of a kind of rapid detector for pesticide residue based on enzyme immunosensor of the present invention.
Fig. 2 is the present invention's three electrodes and potentiostatic circuit and A/D conversion treatment circuit in early stage.
Embodiment
First, structural representation block diagram of the present invention is illustrated; Secondly, input and included potentiostatic circuit, the A/D conversion treatment circuit in early stage of disposal system are described in detail; Finally, the application software to whole system and testing process are illustrated.
Fig. 1 is the structured flowchart of a kind of rapid detector for pesticide residue based on enzyme immunosensor of the present invention.As shown in Figure 1, the present invention is by three-electrode system, and input and disposal system, show and print storage system, electric power system composition.Three-electrode system gathers low current signal by enzyme sensor and immunosensor; The low current signal that input and disposal system collect three electrodes carries out I/V conversion, amplifies, and filtering, A/D conversion the most at last digital quantity signal is sent into single-chip microcomputer and is carried out routine processes; Show and print storage system by parameter displays such as residual pesticide concentration, inhibition degree out, and data being stored so that user checks in time and carries out data analysis.Show and print storage system built-in miniature printer, data can be carried out to corresponding printout.The single-chip microcomputer of input and disposal system inside carries out programming Control to showing with the unification of printing storage system, also other system is responded simultaneously.It is each circuit system and mini-printer power supply that electric power system converts alternating current to three road direct currents.Described microcontroller adopts the STC89C52RC+ microcontroller that kernel is MCS-51, loads trace routine on it.
In the present invention, potentiostatic circuit in testing circuit provides stable operating voltage for three-electrode system current/voltage transforms testing circuit, Fig. 2 is potentiostat circuit and A/D conversion treatment circuit in early stage, by a two-way operational amplifier U10 TLC272CP and R44, R46, R4, C35, C16, R48 forms.The pin 6 of amplifier B is connected with 7 and is connected with 3 pin of A, 2 pin of A are connected and have connected C16 with the working electrode of three-electrode system, strengthen antijamming capability, R48 be as feedback resistance on amplifier A, the external potentiometer R46 of 5 pins of B, supply electric excitation by positive 5V, R44, R46, R4 supplies with 500mv voltage to forming potentiostatic circuit with 5 pins of B by voltage dividing potential drop, initial voltage be 500mv. amplifier A and connect+5V of pin 8 power supply, connect-5V of pin 4, the amplifier B pin 7 of U9 TLC272CP is directly connected as voltage follower with pin 6, ADE pin 3 is connected with the pin 5 of B, common ground, play jamproof effect.The pin 2 of A is directly connected with the contrast electrode of three-electrode system, pin 1 is directly connected with the auxiliary electrode of three-electrode system, between connect electric capacity 14, object has been the effect of buffer circuit, can improve load-carrying ability simultaneously, therefore U9 and U10 have formed the three-electrode system of biology sensor jointly, and test side used is drawn by U9 and U10, gather and the low current signal that detects electrochemical reaction generation.
A kind of testing process of the rapid detector for pesticide residue based on enzyme immunosensor is: first, the sensor of fixing acetylcholinesterase is put into the not substrate solution containing agricultural chemicals, the electric current I while recording not containing agricultural chemicals
1, electric current before suppressing (default value that before suppressing, electric current is set in also can employing system); Afterwards the working electrode of enzyme sensor is taken out, with after distilled water flushing, the working electrode of fixing acetylcholinesterase is put into sample liquid and hatch, wait for taking-up distilled water flushing after 10 minutes, again put into substrate solution, record the electric current I after inhibition
2; Finally automatically calculate inhibiting rate by the trace routine of moving on microcontroller, computing formula is as follows:
Inhibiting rate=(I
1-I
2)/I
1% × 100%.
If inhibiting rate exceedes 20%, just use array immunization sensor to repeat above operation, obtain detection inhibiting rate, measure the result of curent change according to the each passage of sensor array, the more corresponding concentration of knowing which kind of agricultural chemicals in sample and agricultural chemicals of the typical curve database of the inhibiting rate of inserting according to detector and pesticide concentration is how many.According to the national standard of the Pesticide maximum residue limit specifying in GB2763-2012, determine whether this kind of agricultural chemicals detecting exceeds standard.
Microcontroller is made qualitative conclusion according to the inhibiting rate of the mensuration obtaining, and result is delivered to display screen and show, and by end product printout, data is stored later the searching and studying for related personnel simultaneously in the lump.
The trace routine of moving on microcontroller of the present invention also comprises screen redirect demonstration and dialog box, print prompt to important step prompting, can help user to grasp fast the using method of instrument.
Illustrate with regard to the embodiment of testing process below: first, crocodile clip is stepped up respectively to three columnar electrodes that three-electrode system is corresponding, three electrodes that solidify enzyme are immersed in to Pen-down detection key in the phosphate buffered solution that contains acetylthiocholine iodide, screen display starts to detect, after about 1min, baseline reaches maintenance level, after about 30s, obtain first complete current data, be designated as I
1.Testing sample test: enzyme sensor is put into the sample solution having extracted, about 10min, in the phosphate buffered solution that again its immersion is contained to acetylthiocholine iodide afterwards, after clicking detection for the second time, observes prompting, shows result I
2and inhibiting rate; If inhibiting rate exceedes 20%, screen prompt enters second and takes turns test.The array immunization sensor of various pesticide antibodies is put into test end liquid, the current signal collection successively array immunization sensor being produced respectively by analog multichannel switch is sent in detector, carry out the second test (at the bottom of test now, liquid changes potassium ferricyanide solution into) of above-mentioned repetition, obtain test result, if the curent change inhibiting rate of some immunosensors exceedes 20% in test result, result and the electric current of inserting and the typical curve database of agricultural chemicals are compared, final kind and the concentration that shows the agricultural chemicals exceeding standard.
Attention: if 1. will measure next testing sample, click and reset.
2. after shutdown, must re-start instrument zeroing and standard model test.
A kind of rapid detector for pesticide residue based on enzyme immunosensor that the present invention uses, can realize the qualitative, quantitative of residues of pesticides and measure, and the present invention simultaneously goes for detecting the residual detection occasion of Multiple Pesticides simultaneously.Above-described embodiment is that the present invention will be further described, and the present invention is not subject to the restriction of embodiment.
Claims (7)
1. the rapid detector for pesticide residue based on enzyme immunosensor, is characterized in that, by three-electrode system, input and disposal system, show and print storage system and electric power system formation;
Described three-electrode system is by enzyme electrode and immunosensor, three electrode detection circuit, potentiostatic circuit and analog multichannel switch form, enzyme electrode and immunosensor gather current signal from detect sample, circuit signal is transmitted to input and disposal system by three electrode detection circuit, potentiostatic circuit provides steady voltage for electrode, and the current signal collection that analog multichannel switch successively produces immunosensor respectively is also sent into input and disposal system;
Described enzyme electrode is made up of a working electrode, is fixed with acetylcholinesterase at working electrode surface; Described immunosensor is made up of a circular electrode disk, and the centre bit of electrode disk is equipped with electrode and contrast electrode, and periphery is equally distributed array working electrode, and array working electrode is fixed with pesticide antibody;
Described input and disposal system are by TLC272CP chip, A/D conversion chip and single-chip microcomputer form, the multichannel discharge circuit of TLC272CP chip carries out I/V conversion to the current signal sending from enzyme electrode and immunosensor, amplify, filtering processing, A/D conversion chip is sent the digital signal after conversion into single-chip microcomputer, and single-chip microcomputer has microcontroller, and the digital signal after conversion is carried out to routine processes;
The microcontroller of described single-chip microcomputer is STC89C52 microcontroller, the routine package of single-chip microcomputer is containing data-printing and storage, time showing, operation instruction and the dialog box to important step prompting, help user grasp fast the using method of instrument and carry out data-printing and storage;
Described demonstration and printing storage system are by display circuit, print circuit and memory circuitry form, display is used for showing data and controls by operating key, prints and is connected with storer and carries out data-printing and storage with mini-printer respectively with memory circuit;
Described electric power system electric routing switch power supply and voltage conversion circuit form, Switching Power Supply converts alternating current to direct current and powers to each circuit system and mini-printer respectively through voltage conversion circuit, Switching Power Supply is+12V, 1A ,-12V, 1A ,+5V, the output of 5ASan road, power supply is connected with the Switching Power Supply in electric power system.
2. detector described in claim 1, is characterized in that, described potentiostatic circuit, for providing the steady voltage of 500mV between working electrode and contrast electrode.
3. detector described in claim 1, is characterized in that, described microcontroller is that kernel is the STC89C52RC+ microcontroller of MCS-51.
4. detector described in claim 1, is characterized in that, the storage unit of described input and disposal system is implanted the pesticide concentration of acetylcholinesterase sensor detection residues of pesticides corresponding to immunosensor and the typical curve database of current relationship.
5. detector described in claim 1, is characterized in that, described display adopts 3.5 cun of display screens; Described mini-printer adopts micro embedded printer.
6. a detection method for detector described in claim 1, is characterized in that, step is as follows:
The enzyme electrode that is fixed with acetylcholinesterase is put into the not substrate solution containing agricultural chemicals, the electric current I while detecting not containing agricultural chemicals
1, or the default value of employing default;
Take out after enzyme electrode, with distilled water flushing, then enzyme electrode is put into sample liquid hatch 10min;
Take out after enzyme electrode, with distilled water flushing, again put into substrate solution, detect electric current I
2;
According to inhibiting rate formula, calculate inhibiting rate by the trace routine on microcontroller;
Utilize immunosensor to substitute enzyme electrode, repeating step 1) to step 4) process again detecting step 4) the inhibiting rate of surveying higher than 20% sample, and obtain the inhibiting rate that exceeds standard;
The step 5) gained inhibiting rate that exceeds standard is contrasted to acquisition exceed standard pesticide variety and concentration with inhibiting rate and the pesticide concentration typical curve database of implanting in advance detector.
7. method described in claim 6, is characterized in that, inhibiting rate formula described in step 4), is specially: inhibiting rate=(I
1-I
2)/I
1% × 100%.
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| CN106942973A (en) * | 2017-02-25 | 2017-07-14 | 深圳市赛亿科技开发有限公司 | A kind of intelligent chopsticks of quick detection fruits and vegetables Pesticide Residues |
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| CN103940868B (en) | 2016-02-24 |
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