CN103940868B - A kind of rapid detector for pesticide residue based on enzyme immunosensor - Google Patents
A kind of rapid detector for pesticide residue based on enzyme immunosensor Download PDFInfo
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Abstract
The invention discloses a kind of rapid detector for pesticide residue based on enzyme immunosensor, belong to agricultural product security detection technique field.Rapid detector for pesticide residue based on enzyme immunosensor provided by the present invention is by three-electrode system, and input and disposal system, display is formed with printing storage system and electric power system.Whether whether three-electrode system, first by enzyme sensor, measures the change of electric current before and after contact sample, know in sample and exceed standard containing agricultural chemicals.If there is agricultural chemicals to exceed standard, re-use immunosensor, measure the change of electric current before and after contact sample, know the information such as the concentration containing which kind of agricultural chemicals and agricultural chemicals in sample.Detector provided by the present invention has simple to operate, cheap, highly sensitive, reproducible and can realize on-line quick detection, is applicable to the field quick detection of agricultural product.
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, play very important effect to the control of disease, worm, grass, mouse harm.But the hazard analysis and HACCP of pollution by pesticides and generation thereof is serious, and 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 air, the impact on environmental organism and the harm etc. to health.At present because user generally lacks scientifical use pesticide knowldgde, covet output, causes the agricultural products circulated in the market to there is Pesticide Residue all to some extent, and its harm also causes the concern of the public day by day.China is a large agricultural country, and along with our people's living standard improves 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 kind of pesticide of use is Organophosphorus and carbamate pesticides class agricultural chemicals (accounting for 70%), and wherein highly toxic Organophosphorus and carbamate pesticides insecticides is about 70%, therefore easily causes food pesticide to remain poisoning.Visible, strengthening to the detection of Residual Pesticides in Farm Produce 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 pesticides class agricultural chemicals.
The main method of current pesticide residue analysis is gas chromatograph, liquid chromatograph, gas chromatograph-mass spectrometer, LC-MS instrument etc., although these method analysis precisions are high, quantitatively accurately, but the complex pretreatment of its sample, detect length consuming time, cost high, need the operating personnel that are skilled in technique.The fast determining method of China's residues of pesticides is enzyme level test paper method and enzyme level spectrophotometric method (the residual fast detector of agriculture), can realize the field quick detection of organophosphorus pesticide and carbamate chemicals for agriculture, have good practical value.Quick measuring card is changed by the 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 level spectrophotometric method is also relatively more extensive, and it is all based on this principle that domestic existing Multiple Pesticides remains tacheometer.Light splitting light ratio juris carries out detecting based on the change of absorbance, but pigments a large amount of in vegetable and fruit 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 the shortcomings such as low-residual and 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 that is simple to operate, cheap, highly sensitive, that realize on-line quick detection.Accurately can know in sample and whether exceed standard containing residues of pesticides and which kind of information such as residues of pesticides and concentration of energy.The technical scheme taked is as follows:
Based on a rapid detector for pesticide residue for enzyme immunosensor, be by three-electrode system, input and disposal system, display is formed with printing storage system and electric power system;
Described three-electrode system is by enzyme electrode and immunosensor, three electrode detection circuit, potentiostatic circuit and analog multichannel switch are formed, enzyme electrode and immunosensor gather current signal from detection sample, circuit signal is transmitted to input and disposal system by three electrode detection circuit, potentiostatic circuit provides constant voltage for electrode, and the current signal collection that immunosensor produces successively also is sent into input and disposal system by analog multichannel switch respectively;
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 operation electrode, and array operation electrode is fixed with pesticide antibody;
Described input and disposal system are by TLC272CP chip, A/D conversion chip and single-chip microcomputer are formed, the multichannel discharge circuit of TLC272CP chip carries out I/V conversion to the current signal sent from enzyme electrode and immunosensor, amplify, filtering process, digital signal after conversion is sent into single-chip microcomputer by A/D conversion chip, and single-chip microcomputer has microcontroller, carries out routine processes to the digital signal after conversion;
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 the using method of instrument fast and carry out data-printing and storage;
Described display and printing storage system are by display circuit, print circuit and memory circuitry are formed, display, for showing data and being controlled by operating key, to be printed and memory circuit is respectively mini-printer and storer and is connected and carries out data-printing and storage;
Described electric power system electric routing switch power supply and voltage conversion circuit are formed, alternating current is converted to direct current and powers respectively to each circuit system and mini-printer through voltage conversion circuit by Switching Power Supply, Switching Power Supply is+12V, 1A ,-12V, 1A ,+5V, 5A tri-tunnel exports, and power supply is connected with the Switching Power Supply in electric power system.
Described potentiostatic circuit, for providing the constant voltage of 500mV between working electrode and contrast electrode.
The STC89C52RC+ microcontroller of described microcontroller to be kernel be MCS-51.
Described input and the storage unit of disposal system insert the acetylcholine ester enzyme sensor pesticide concentration of 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 that utilizes is as follows:
1) enzyme electrode being fixed with acetylcholinesterase is put into not containing the substrate solution of agricultural chemicals, detect not containing electric current I during agricultural chemicals
1, or adopt the default value of default;
2) take out after enzyme electrode, with distilled water flushing, then enzyme electrode is put into sample liquid hatch 10min;
3) after taking out 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, repeat step 1) to step 4) process again detecting step 4) institute survey inhibiting rate higher than 20% sample, and acquisition exceeds standard inhibiting rate;
6) inhibiting rate that exceeded standard by step 5) gained contrasts with the inhibiting rate and pesticide concentration typical curve database of inserting detector in advance, obtains 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: whether whether three-electrode system, first by enzyme sensor, measures the change of electric current before and after contact sample, know in sample and exceed standard containing agricultural chemicals.If truly have agricultural chemicals to exceed standard, re-use immunosensor, measure the change of electric current before and after contact sample, know the information such as the concentration containing 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, filtering, and routine processes is carried out in A/D conversion the most at last digital quantity signal feeding single-chip microcomputer.Data by the residual parameter display such as pesticide concentration, degree of suppression out, and store so that user checks in time and carries out data analysis by display and printing storage system.Display includes mini-printer with printing storage system, data can be carried out corresponding printout.
In order to realize above-mentioned functions, described enzyme sensor has a working electrode to form, and fixes acetylcholinesterase at working electrode surface, preparation acetylcholine ester enzyme 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, equal with the distance of each working electrode to guarantee electrode, contrast electrode, wherein array operation electrode is used for fixing different types of pesticide antibody.Detector storage unit inserts the acetylcholine ester enzyme sensor pesticide concentration of detection residues of pesticides corresponding to immunosensor and the typical curve database of current relationship.First fixing acetylcholine ester enzyme sensor is connected with detector during use, according to the signal of curent change before and after the contact sample that acetylcholine ester enzyme sensor obtains, determines whether containing agricultural chemicals and whether exceed standard.If there is the display that exceeds standard, again the Immuno-sensor arrays of sessile antibody is connected on detector, successively the current signal collection that array immunization sensor produces is sent in detector respectively by analog multichannel switch, according to the variable signal of electric current before and after the contact sample that immunosensor obtains, determine which kind of agricultural chemicals concrete exceeds standard and concentration is how many.Therefore whether this detector can obtain agricultural chemicals simultaneously exceed standard in a sample, the information such as the agricultural chemicals concrete containing which kind of and concentration.
Beneficial effect of the present invention: the rapid detector for pesticide residue based on enzyme immunosensor given provided by the present invention, can make up the defect of acetylcholinesterasebiosensor biosensor and immunosensor existence.Acetylcholine ester enzyme sensor can only detect Organophosphorus and carbamate pesticides class, whether containing this class agricultural chemicals, and deterministicly cannot 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 costly, if the information not having residues of pesticides to exceed standard, just there is no need the measurement carrying out second step.Therefore a kind of rapid detector for pesticide residue based on enzyme immunosensor of the present invention, had both saved testing cost, deterministicly can know again and the information that concrete any residues of pesticides exceed standard be suitable for the quick detection of residues of pesticides.1ug/L is limited to the detection that the method can reach.Accuracy rate because use two kinds of sensors to detect, so accuracy is higher than the accuracy of simple enzyme sensor and immunosensor.
Accompanying drawing explanation
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 that the present invention three electrode and potentiostatic circuit and A/D change treatment circuit in early stage.
Embodiment
First, structural schematic block diagram of the present invention is illustrated; Secondly, the potentiostatic circuit included by input and disposal system, A/D conversion treatment circuit in early stage are described in detail; Finally, the application software of 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, display and printing storage system, electric power system forms.Three-electrode system gathers low current signal by enzyme sensor and immunosensor; The low current signal that three electrodes collect by input and disposal system carries out I/V conversion, amplifies, filtering, and routine processes is carried out in A/D conversion the most at last digital quantity signal feeding single-chip microcomputer; Data by the residual parameter display such as pesticide concentration, degree of suppression out, and store so that user checks in time and carries out data analysis by display and printing storage system.Data can be carried out corresponding printout by display and printing storage system built-in miniature printer.The single-chip microcomputer of input and disposal system inside carries out programming Control to showing with printing storage system is unified, also responds other system simultaneously.It is that each circuit system and mini-printer are powered that alternating current is converted to three road direct currents by electric power system.Described microcontroller employing kernel is the STC89C52RC+ microcontroller of MCS-51, and it loads trace routine.
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 two-way operational amplifier U10TLC272CP and R44, R46, R4, C35, C16, R48 are formed.The pin 6 of amplifier B is connected with 7 and is connected with 3 pin of A, 2 pin of A are connected with the working electrode of three-electrode system and have connected C16, strengthen antijamming capability, R48 be as feedback resistance on amplifier A, the external potentiometer R46 of 5 pins of B, by positive 5V for electric excitation, R44, R46, R4 is formed potentiostatic circuit by voltage supply 500mv voltage with 5 pins of B, initial voltage be 500mv. amplifier A and pin 8 connect+5V power supply, pin 4 meets-5V, the amplifier B pin 7 of U9TLC272CP 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, load-carrying ability can be improved simultaneously, therefore U9 and U10 together constitutes the three-electrode system of biology sensor, and test side used is drawn by U9 and U10, gathers the low current signal produced with detection electrochemical reaction.
A kind of testing process of the rapid detector for pesticide residue based on enzyme immunosensor is: first, is put into by the sensor of fixing acetylcholinesterase not containing the substrate solution of agricultural chemicals, records not containing electric current I during agricultural chemicals
1, electric current (default value that before suppressing, electric current also can set in employing system) before namely suppressing; Afterwards the working electrode of enzyme sensor is taken out, after distilled water flushing, the working electrode of fixing acetylcholinesterase is put into sample liquid and hatches, wait for taking-up distilled water flushing after 10 minutes, again put into substrate solution, record the electric current I after suppression
2; Automatically calculate inhibiting rate finally by the trace routine that microcontroller runs, computing formula is as follows:
Inhibiting rate=(I
1-I
2)/I
1% × 100%.
If inhibiting rate is more than 20%, array immunization sensor is just used to repeat above operation, obtain and detect inhibiting rate, measure the result of curent change according to each passage of sensor array, then the inhibiting rate of inserting according to detector is corresponding with the typical curve database of pesticide concentration knows that the concentration of which kind of agricultural chemicals and agricultural chemicals in sample is how many.According to the national standard of the Pesticide maximum residue limit specified in GB2763-2012, determine whether this kind of agricultural chemicals detected exceeds standard.
Microcontroller makes qualitative conclusions according to the inhibiting rate of the mensuration obtained, and result is delivered to display screen display, and by end product printout, data carry out storing for later the searching and studying of related personnel simultaneously in the lump.
The trace routine that microcontroller of the present invention runs also comprises screen redirect demonstration and dialog box, print prompt to important step prompting, and user can be helped to grasp the using method of instrument fast.
Embodiment below with regard to testing process illustrates: first, crocodile clip is stepped up respectively three columnar electrodes that three-electrode system is corresponding, three electrodes of harden monitoring are immersed in Pen-down detection key in the phosphate buffered solution containing 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 is tested: enzyme sensor is put into the sample solution extracted, about 10min, immersed in the phosphate buffered solution containing acetylthiocholine iodide afterwards again, and second time observes prompting after clicking and detecting, display result I
2and inhibiting rate; If inhibiting rate is more than 20%, screen prompt enters second and takes turns test.The array immunization sensor of various pesticide antibody is put into test end liquid, successively the current signal collection that array immunization sensor produces is sent in detector respectively by analog multichannel switch, 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 is more than 20% in test result, then the typical curve database of result with the electric current of inserting and agricultural chemicals is compared, finally show kind and the concentration of the agricultural chemicals exceeded standard.
Attention: 1. to measure next testing sample, clicks and resets.
2. instrument zeroing and standard model test after shutdown, must be re-started.
A kind of rapid detector for pesticide residue based on enzyme immunosensor that the present invention uses, the qualitative, quantitative that can realize residues of pesticides is measured, 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 by the restriction of embodiment.
Claims (7)
1., based on a rapid detector for pesticide residue for enzyme immunosensor, it is characterized in that, by three-electrode system, input and disposal system, display is formed with printing storage system and electric power system;
Described three-electrode system is by enzyme electrode and immunosensor, three electrode detection circuit, potentiostatic circuit and analog multichannel switch are formed, enzyme electrode and immunosensor gather current signal from detection sample, circuit signal is transmitted to input and disposal system by three electrode detection circuit, potentiostatic circuit provides constant voltage for electrode, and the current signal collection that immunosensor produces successively also is sent into input and disposal system by analog multichannel switch respectively;
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 operation electrode, and array operation electrode is fixed with pesticide antibody;
Described input and disposal system are by TLC272CP chip, the multichannel discharge circuit that A/D conversion chip and single-chip microcomputer form TLC272CP chip carries out I/V conversion to the current signal sent from enzyme electrode and immunosensor, amplify, filtering process, digital signal after conversion is sent into single-chip microcomputer by A/D conversion chip, single-chip microcomputer has microcontroller, carries out routine processes to the digital signal after conversion;
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 the using method of instrument fast and carry out data-printing and storage;
Described display and printing storage system are by display circuit, print circuit and memory circuitry are formed, display is for showing data and being controlled by operating key, and printing is connected with storer with mini-printer respectively with memory circuit and carries out data-printing and storage;
Described electric power system electric routing switch power supply and voltage conversion circuit are formed, alternating current is converted to direct current and powers respectively to each circuit system and mini-printer through voltage conversion circuit by Switching Power Supply, Switching Power Supply is+12V, 1A ,-12V, 1A ,+5V, 5A tri-tunnel exports, and 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 constant voltage of 500mV between working electrode and contrast electrode.
3. detector described in claim 1, is characterized in that, the STC89C52RC+ microcontroller of described microcontroller to be kernel be MCS-51.
4. detector described in claim 1, is characterized in that, described input and the storage unit of disposal system implant the acetylcholine ester enzyme sensor pesticide concentration of 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 being fixed with acetylcholinesterase is put into not containing the substrate solution of agricultural chemicals by step 1), detects not containing the electric current I 1 during agricultural chemicals, or adopts the default value of default;
Step 2) take out after enzyme electrode, with distilled water flushing, then enzyme electrode is put into sample liquid hatch 10min;
Step 3) with distilled water flushing, puts into substrate solution after taking out enzyme electrode again, detects electric current I 2;
Step 4) according to inhibiting rate formula, calculates inhibiting rate by the trace routine on microcontroller;
Step 5) utilizes immunosensor to substitute enzyme electrode, repeats step 1) to step 4) process again detecting step 4) institute survey inhibiting rate higher than 20% sample, and acquisition exceeds standard inhibiting rate;
The step 6) inhibiting rate that exceeded standard by step 5) gained contrasts with pesticide concentration typical curve database with the inhibiting rate implanting detector in advance, and acquisition exceeds 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=(I1-I2)/I1 × 100%.
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| CN105424770B (en) * | 2015-11-10 | 2018-11-09 | 中国科学院电子学研究所 | A kind of Electrochemical detector for the detection of Direct Electrochemistry immunosensor |
| CN106706731A (en) * | 2017-02-15 | 2017-05-24 | 黑龙江省农业科学院农产品质量安全研究所 | Quick household fruit and vegetable pesticide residue detector |
| 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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