CN103543180B - For immune impedance biosensor detection method and the pick-up unit of rapid detection for pesticide residue - Google Patents
For immune impedance biosensor detection method and the pick-up unit of rapid detection for pesticide residue Download PDFInfo
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- CN103543180B CN103543180B CN201310520942.8A CN201310520942A CN103543180B CN 103543180 B CN103543180 B CN 103543180B CN 201310520942 A CN201310520942 A CN 201310520942A CN 103543180 B CN103543180 B CN 103543180B
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Abstract
The invention provides a kind of accuracy good, the detection method of the highly sensitive immune impedance biosensor for the detection of liquid Pesticide Residues and device, the method is on the working electrode (s antibody linked; Measure resistance value after sessile antibody and the effect of variable concentrations standard pesticide, formulation typical curve, then measure resistance value after agricultural chemicals to be measured and sessile antibody effect, compare with typical curve and draw the pesticide concentration in agricultural chemicals to be measured.This device comprise syringe pump, rotating disk, electrolytic tank, extraction pump, working electrode, to electrode and contrast electrode; Electrolyte passage, phosphate buffer passage, sample to be tested passage, electrode clean passage, electrode pre-processing channel, antibody injection channel is had in rotating disk; It is circumferentially same that each feeder connection and outlet lay respectively at rotating disk; Rotating disk is when center rotating, and a feeder connection and outlet export with syringe pump simultaneously respectively, electrolytic tank entrance communicates; Electrolyzer outlet communicates with extraction pump entrance.This device can Reusability, reproducible, can ensure the consistance detected.
Description
Technical field
The present invention relates to a kind of immune impedance biosensor detection method to rapid detection for pesticide residue and pick-up unit.
Background technology
National governments constantly strengthen the monitoring of persticide residue, and the standard of formulation is more and more stricter.The conventional method of Detecting Pesticide and quantitative test is chromatogram, chromatograph-mass spectrometer coupling etc.The measurement result of these methods accurately and reliably, is the mainstream technology of current farming residual analysis.But the deficiencies such as these methods also exist instrument costly, and the pre-treatment time is long, process is loaded down with trivial details, are suitable only for and use in specialized laboratory.At present, agricultural chemicals fast determining method on home market is more with inhibiting AChE application, it utilizes agricultural chemicals to some certain enzyme, the particularly inhibiting effect of cholinesterase, the substrate after hydrolysis or hydrolysate is detected by contrast, judge that whether enzyme is suppressed with the method for colorimetric or spectrophotometric analysis, thus measure residues of pesticides.This method fast, simply, conveniently, but also exists following not enough:
1, the total residue of agricultural chemicals can only be detected, the kind of concrete agricultural chemicals can not be distinguished;
2, colour developing is unstable, easily affects by surrounding environment, temperature etc., brings error to judgement;
3, detect precision not high, easily occur false positive, false negative result;
4, enzyme reaction condition is harsh, high to operation requirements;
Chinese patent ZL01253887.6 discloses the calorimetric biosensor of detection residues of pesticides, and it detects pesticide concentration in testing liquid by measuring the temperature variation flowing through the testing liquid of immobilized enzyme column.Its weak point is: complex structure, and immobilized enzyme column used requires high.The quick biology sensor paper detecting agricultural chemicals of research is also being delivered in the world, main still based on enzyme biologic sensor, there are no the immune impedance biosensor for detecting agricultural chemicals.
Summary of the invention
The object of the present invention is to provide a kind of detection method of immune impedance biosensor detected for liquid Pesticide Residues, it can Fast Measurement target agricultural chemicals residual quantity in the sample to which, and accuracy is good, highly sensitive.
Immune impedance biosensor detection method for rapid detection for pesticide residue of the present invention, use immune impedance biosensor pick-up unit, immune impedance biosensor pick-up unit comprise be electrically connected with electric impedance analyzer working electrode, extend in electrolytic tank to electrode and contrast electrode; Electric impedance analyzer is connected with computing machine; This detection method comprises the steps:
A. electrode clean
With cleaning materials confrontation working electrode, electrode and contrast electrode are cleaned;
B. electrode pre-service
With solution of silane to Electrode treatment; Non-specific adsorption is removed again with first alcohol and water cleaning electrode;
C. antibody is fixed
Pesticide antibody solution is injected in electrolytic tank, makes antibody pass through crosslinked fixing on the working electrode (s; With phosphate buffer and deionized water, electrode is cleaned;
D. testing impedance
Testing liquid sample is injected electrolytic tank, makes the sessile antibody generation specific reaction on testing liquid sample and electrode; Then cleaning electrode is led to phosphate buffer, then containing [Fe (CN)
6]
3-or [Fe (CN)
6]
4-electrolytic solution inject in electrolytic tank, then calculate the Faradaic impedance of electrolytic solution with electric impedance analyzer and COMPUTER DETECTION;
E. data analysis
To the pesticide standard solution of variable concentrations, respectively according to above-mentioned a-d step, obtain the Faradaic impedance corresponding to the agricultural chemicals of each concentration, formulate pesticide concentration and the typical curve drawing impedance; By the Faradaic impedance that testing liquid sample obtains according to above-mentioned a-d step, judge the pesticide concentration in testing liquid sample according to typical curve.
Above-mentioned detection method, in step a, during to electrode clean, cleans with acetone, ethanol and deionized water respectively, then with 1:1:5 (v/v) H
2o
2/ NH
4oH/H
2o solution clean, after 10 minutes, then with deionized water rinsing, after pass into nitrogen drying;
In step b, solution of silane is 3-aminopropyl triethoxysilane;
In step c, be the bovine serum albumin(BSA) of 1% containing containing volume ratio in the phosphate buffer of bovine serum albumin(BSA), the pH value of phosphate buffer is 7.4;
In steps d, in electrolytic solution, [Fe (CN)
6]
3-with [Fe (CN)
6]
4-concentration ratio 1:1; Faradaic impedance test frequency 1Hz-100kHz, amplitude is ± 5mV.
Beneficial effect of the present invention: detection method of the present invention is the mode with flow injection, fixes pesticide antibody on the working electrode (s, as the sensitive element of biology sensor.After agricultural chemicals antigen generation specific binding in sessile antibody and solution, form the compound of " antigen-antibody " at electrode surface, change the Charger transfer of electrostatic double layer and the resistance of superficial layer.By the change of the resistance value of system after mensuration sessile antibody and the effect of variable concentrations standard pesticide, formulate typical curve, measure the resistance value after agricultural chemicals to be measured and sessile antibody effect again, and compare with typical curve, thus draw the content of the target agricultural chemicals in agricultural chemicals to be measured.Detection method of the present invention fast, simple, conveniently, accuracy is simultaneously high, highly sensitive, reproducible.
It is the immune impedance biosensor pick-up unit that uses in above-mentioned detection method that the present invention provides a kind of simultaneously, and its structure is simple, easy to make, with low cost, can Reusability, reproducible, can ensure the consistance detected.
The immune impedance biosensor pick-up unit used in above-mentioned detection method of the present invention, comprises syringe pump, rotating disk, electrolytic tank, extraction pump; The working electrode be electrically connected with electric impedance analyzer, extend in electrolytic tank to electrode and contrast electrode; Electric impedance analyzer is connected with computing machine; There are 6 passages radially extended: electrolyte passage, phosphate buffer passage, sample to be tested passage, electrode clean passage, electrode pre-processing channel, antibody injection channel in rotating disk; Article 6, the entrance and exit of passage lays respectively at the circumferentially same of rotating disk; When rotating disk is around center rotating, the entrance and exit of a passage communicates with the entrance of injection delivery side of pump, electrolytic tank simultaneously respectively; The outlet of electrolytic tank communicates with the entrance of extraction pump.
Described immune impedance biosensor pick-up unit, also comprises the waste liquid pool communicated with discharge delivery side of pump.
Described immune impedance biosensor pick-up unit, working electrode is dish battle array microelectrode or interdigital band row microelectrode.
The beneficial effect of this immune impedance biosensor pick-up unit: during use, all uses syringe pump to pump into various material; And by the rotation of rotating disk, different passages can be made to communicate with syringe pump and electrolytic tank respectively, to inject different materials by different passages, ensure consistance and the repeatability of detecting step, use the result accuracy detected high.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of immune impedance biosensor pick-up unit;
Fig. 2 is the theory diagram of immune impedance biosensor pick-up unit.
Fig. 3 is the equivalent circuit diagram of electrolytic tank.
Fig. 4 is parathion pesticide concentration-impedance standard curve.
Embodiment
See the immune impedance biosensor pick-up unit for rapid detection for pesticide residue shown in Fig. 1,2, be made up of drive system, sampling system, reactive system, detection system, control system.Drive system is by syringe pump 1, extraction pump 10 forms, sampling system is made up of rotating disk 2, conduit 3, the reactive system of electrolytic tank 4, detection system by working electrode 5, to electrode 6, contrast electrode 7 forms, control system is by electric impedance analyzer 8, and computing machine 9 forms, and the waste liquid flowed out from the outlet of electrolytic tank enters waste liquid pool 11 through extraction pump 10.Working electrode can be interdigital band row microelectrode or dish battle array microelectrode (disk electrode), and be platinum electrode to electrode, contrast electrode is Ag/AgCl electrode.
The working electrode 5 be electrically connected with electric impedance analyzer, extend in electrolytic tank to electrode 6 and contrast electrode 7; Electric impedance analyzer is connected with computing machine.There is in rotating disk 26 passage: the electrolyte passage A, phosphate buffer channel B, sample to be tested channel C, electrode clean passage D, electrode pre-processing channel E, the antibody injection channel F that radially extend; Article 6, the entrance of passage is positioned at the same inner circle in close center of turntable portion circumferentially; Injection delivery side of pump contacts with the circumferential sealing of described 6 feeder connection place inner circles.Article 6, the outlet of passage is positioned at the circumferentially same of rotating disk periphery.The import of conduit contacts with the circumferential sealing of described 6 channel outlet place cylindricals.The outlet of conduit communicates with the entrance of electrolytic tank; The outlet of electrolytic tank is communicated with waste liquid pool by positive displacement pump.Mouth is when rotating disk is around center rotating, and in 6 passages, the entrance and exit of arbitrary passage can communicate with the entrance of injection delivery side of pump, conduit simultaneously respectively.
To the immune impedance biosensor detection method of rapid detection for pesticide residue, be to electrode after pre-service, fixing pesticide antibody, application of sample detects and obtains ac impedance spectroscopy, carrying out Equivalent Circuit Fitting, by comparing the charge transfer resistance Ret value of variable concentrations action of agricultural chemicals, formulating typical curve; Record the charge transfer resistance Ret value under action of agricultural chemicals to be measured, and compare with typical curve, judge the target residues of pesticides concentration in determinand.Its concrete grammar is as follows:
1, the concentration-impedance standard curve of parathion agricultural chemicals is set up.
A. electrode clean
Respectively 4mL acetone, ethanol and deionized water to be injected in electrolytic tank by electrode clean passage D under the speed of 2mL/min with syringe pump and discharged by extraction pump, then with syringe pump the 1:1:5 of 4mL (v/v) H
2o
2/ NH
4oH/H
2o mixed solution to be injected in electrolytic tank by electrode clean passage D and discharges, and after 10 minutes, then injects deionized water cleaning down with syringe pump and discharges, after pass into nitrogen drying; To working electrode, cleaning is carried out and drying to electrode and contrast electrode.
B. electrode pre-service
With syringe pump 4mL2%3-aminopropyl triethoxysilane solution to be injected in electrolytic tank by electrode pre-processing channel E and discharge, the work of process electrode; Respectively the cleaning of first alcohol and water to be injected in electrolytic tank by electrode clean passage with syringe pump again and discharge, cleaning electrode, removing non-specific adsorption;
C. antibody is fixed
With syringe pump the parathion Anti-TNF-α liquid solution 4mL of 1mg/mL concentration, under the speed of 0.5mL/min, to be injected in electrolytic tank by antibody injection channel and discharge, make antibody pass through crosslinked fixing on the working electrode (s; After 20 minutes with syringe pump to be injected in electrolytic tank by phosphate buffer passage containing 1% sero-abluminous phosphate buffer (pH value is for 7.4) and discharge, with syringe pump deionized water to be injected in electrolytic tank by electrode clean passage and discharge, electrode is cleaned, removes the antibody do not bound.
D. testing impedance
With syringe pump, the pesticide standard solution of variable concentrations is injected electrolytic tank discharging by sample to be tested passage, make the sessile antibody generation specific reaction on pesticide standard solution and electrode; With syringe pump phosphate buffer to be injected in electrolytic tank by phosphate buffer passage after 30 minutes and discharge, then with syringe pump the [Fe (CN) of 0.01mol/L
6]
3-/4-(1:1) phosphate electrolytic solution pumps in electrolytic tank by electrolyte passage A, and then with the ac impedance spectroscopy of electric impedance analyzer and COMPUTER DETECTION electrolytic solution, the sample frequency scope 1Hz-100kHz of electric impedance analyzer, amplitude is ± 5mV.Equivalent Circuit Fitting shown in Fig. 3 is adopted to ac impedance spectroscopy, calculates the Faradaic impedance corresponding to pesticide concentration.In Fig. 3, Ret represents charge transfer resistance, R
trepresent solution resistance, C
dlrepresent double-layer capacitance, CPE represents normal phasing degree element, represents the attribute of redox probe.
E. data analysis
According to the Faradaic impedance corresponding to the agricultural chemicals of each concentration obtained, formulate pesticide concentration and the typical curve drawing impedance, as shown in Figure 4.In Fig. 4, ordinate represents charge transfer resistance, unit ohm; Horizontal ordinate represents the concentration of parathion agricultural chemicals, unit nanograms/milliliter.
2, the quick detection of parathion agricultural chemicals
Step a, b, c, d are same as described above, only replace pesticide standard solution with parathion agricultural chemicals to be measured, count out the Faradaic impedance corresponding to the pesticide concentration of parathion agricultural chemicals to be measured.Then so that the Faradaic impedance of parathion agricultural chemicals to be measured is compared with typical curve, the pesticide concentration in parathion agricultural chemicals to be measured is calculated.
In order to reach the reusable object of sensor, after testing, being pumped into phosphate buffer flushing by phosphate buffer passage, then pumping into protein scaling agent by electrode clean passage, removing the antigen antibody complex that electrode surface combines.
The present invention can Fast Measurement target agricultural chemicals residual quantity in the sample to which, and accuracy is good, highly sensitive, can reuse.
Finally, it is also to be noted that what enumerate above is only specific embodiments of the invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.
Claims (3)
1., for the immune impedance biosensor pick-up unit of rapid detection for pesticide residue, it is characterized in that: it comprises syringe pump, rotating disk, electrolytic tank, extraction pump; The working electrode be electrically connected with electric impedance analyzer, extend in electrolytic tank to electrode and contrast electrode; Electric impedance analyzer is connected with computing machine; There are 6 passages radially extended: electrolyte passage, phosphate buffer passage, sample to be tested passage, electrode clean passage, electrode pre-processing channel, antibody injection channel in rotating disk; Article 6, the entrance and exit of passage lays respectively at the circumferentially same of rotating disk; When rotating disk is around center rotating, the entrance and exit of a passage communicates with the entrance of injection delivery side of pump, electrolytic tank simultaneously respectively; The outlet of electrolytic tank communicates with the entrance of extraction pump;
This pick-up unit is used to comprise the steps: for the immune impedance biosensor detection method of rapid detection for pesticide residue
A. electrode clean:
With cleaning materials confrontation working electrode, electrode and contrast electrode are cleaned;
B. electrode pre-service:
With solution of silane to Electrode treatment; Non-specific adsorption is removed again with first alcohol and water cleaning electrode;
C. antibody is fixed:
Pesticide antibody solution is injected in electrolytic tank, makes antibody pass through crosslinked fixing on the working electrode (s; With phosphate buffer and deionized water, electrode is cleaned;
D. testing impedance:
Testing liquid sample is injected electrolytic tank, makes the sessile antibody generation specific reaction on testing liquid sample and electrode; Then cleaning electrode is led to phosphate buffer, then containing [Fe (CN)
6]
3-or [Fe (CN)
6]
4-electrolytic solution inject in electrolytic tank, then calculate the Faradaic impedance of electrolytic solution with electric impedance analyzer and COMPUTER DETECTION;
E. data analysis:
To the pesticide standard solution of variable concentrations, respectively according to above-mentioned a, b, c, Step d, obtain the Faradaic impedance corresponding to the agricultural chemicals of each concentration, formulate pesticide concentration and the typical curve drawing impedance; By the Faradaic impedance that testing liquid sample obtains according to above-mentioned a, b, c, Step d, judge the pesticide concentration in testing liquid sample according to typical curve.
2. immune impedance biosensor pick-up unit as claimed in claim 1, is characterized in that: it also comprises the waste liquid pool communicated with discharge delivery side of pump.
3. immune impedance biosensor pick-up unit as claimed in claim 1, is characterized in that: working electrode is dish battle array microelectrode or interdigital band row microelectrode.
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| CN103940868B (en) * | 2014-04-15 | 2016-02-24 | 山东理工大学 | A kind of rapid detector for pesticide residue based on enzyme immunosensor |
| CN108072621B (en) | 2016-11-18 | 2021-02-26 | 财团法人工业技术研究院 | Residual poison detection system and method |
| CN106860972B (en) * | 2017-01-22 | 2022-10-21 | 杭州电子科技大学 | Intelligent insulin pen |
| CN110609064A (en) * | 2018-06-14 | 2019-12-24 | 深圳碳森科技有限公司 | Differential impedance potential type biosensor and manufacturing method thereof |
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| CN101233408A (en) * | 2005-10-28 | 2008-07-30 | 松下电器产业株式会社 | Measuring device, measuring apparatus and method of measuring |
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Application publication date: 20140129 Assignee: Nantong Bolin Mechanical and Electrical Technology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: 2018320000372 Denomination of invention: Immune impedance biosensor detection method and device for quick pesticide residue detection Granted publication date: 20151111 License type: Common License Record date: 20181210 Application publication date: 20140129 Assignee: Nantong Guangyi Electromechanical Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: 2018320000368 Denomination of invention: Immune impedance biosensor detection method and device for quick pesticide residue detection Granted publication date: 20151111 License type: Common License Record date: 20181210 |
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Application publication date: 20140129 Assignee: Chihe (Shanghai) Machinery Technology Co., Ltd Assignor: Nanjing Forestry University Contract record no.: X2019980000910 Denomination of invention: Immune impedance biosensor detection method and device for quick pesticide residue detection Granted publication date: 20151111 License type: Common License Record date: 20191206 |
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