CN105806922A - Preparation method of parathion-methyl immunosensor based on combination of two electrochemical methods and application - Google Patents

Preparation method of parathion-methyl immunosensor based on combination of two electrochemical methods and application Download PDF

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CN105806922A
CN105806922A CN201610148509.XA CN201610148509A CN105806922A CN 105806922 A CN105806922 A CN 105806922A CN 201610148509 A CN201610148509 A CN 201610148509A CN 105806922 A CN105806922 A CN 105806922A
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parathion
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CN105806922B (en
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张勇
邓保军
庞雪辉
任祥
魏琴
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University of Jinan
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Abstract

The invention discloses a preparation method of a biological immunosensor which has an electrochemiluminescence and photoelectrochemistry dual signal developed strategy and is constructed on the basis of a functional nano material.The prepared immunosensor is easy to operate, convenient to carry, rapid in detection, low in cost and capable of being used for rapid and sensitive detection on parathion-methyl in the fields of daily production and life and the like.

Description

The preparation method of a kind of parathion-methyl immunosensor based on two kinds of electrochemical method couplings and application
Technical field
The present invention relates to the preparation method of a kind of immunosensor for detecting parathion-methyl and application, belong to Nano-function thin films and electrochemical biosensor analysis technical field.
Background technology
Parathion-methyl is a kind of broad spectrum activity height poison insecticide, have tag, stomach toxicity, fumigation action, and can infiltrate through in plant.Parathion-methyl to insecticide effect quickly, can be used for preventing and treating fruit tree pest insect and the wheat red mites etc. such as Cotton Gossypii, Fructus Mali pumilae, mandarin orange, pears, Fructus Persicae.In soil, parathion-methyl slightly can be migrated to deep subsoil by the eluviation of water.Parathion-methyl in soil can be absorbed by plant root and enter in plant.People eat this kind of plant by mistake or containing after the plant of its residue, parathion-methyl can pass through digestive tract, respiratory tract and complete skin and mucosa enters human body, there will be Nausea and vomiting, have a headache, have loose bowels, the poisoning preliminary symptom such as whole body weakness, long-term edible or excessive edible meeting causes canceration.
At present, the method detecting parathion-methyl mainly has chromatography, mass spectrography etc..This type of method instrument is valuable, complicated operation, and laboratory personnel just can detect after needing professional training.Therefore, R&D costs are low, it is fast, highly sensitive to detect, the parathion-methyl sensor of high specificity is significant.
Electrochemical biosensor analytical technology, due to easy and simple to handle, the detection advantages such as speed is fast, obtains the attention of people day by day.The electrochemical biosensor analytical technology being presently used for detection parathion-methyl is divided according to detection means and is mainly had electrochemical sensor, Electrochemiluminescsensor sensor and Optical Electro-Chemistry sensor three kinds.Wherein, Electrochemiluminescsensor sensor and Optical Electro-Chemistry sensor, relative to electrochemical sensor, have the features such as background signal noise is few, highly sensitive, testing cost is low, were paid close attention to by increasing researcher in recent years.
Electrogenerated chemiluminescence is also referred to as electrochemiluminescence, refer to and produce some special materials by electrochemical method at electrode surface, between these materials or and system in form excited state by electron transmission between other components, excited state return to ground state and produce luminescence phenomenon.Namely Electrochemiluminescsensor sensor by changing the decorative material of electrode surface, produces electrochemiluminescence with analyte, in optimal conditions, realizes the qualitative and quantitative analysis to analyte according to the associated change of analyte concentration with electrochemiluminescence intensity.
Optical Electro-Chemistry sensor is based on additional light source activation Electrophotosensitivmaterial material and causes that electron-hole pair is easily separated, under suitable inclined potential condition, it is achieved the electronics quick transmission on electrode, quasiconductor and trim and analyte, and forms photoelectric current.In optimal conditions, the change of analyte concentration can directly affect the size of photoelectric current, it is possible to realizes the qualitative and quantitative analysis to analyte according to the change of photoelectric current.
But, owing to Electrochemiluminescsensor sensor needs external optical signal to catch equipment such as photodiode etc., and Optical Electro-Chemistry sensor needs peripheral hardware light source to excite Electrophotosensitivmaterial material, this have impact on the convenience of the two operation to a certain extent, limits them and is more widely applied in actual production, life.Therefore, the electrochemical biosensor analytical technology designing, preparing more simple, fast detection parathion-methyl has highly important practical value.
Summary of the invention
The preparation method that it is an object of the invention to provide a kind of simple to operate, easy to carry, detection is fast, cost is low parathion-methyl sensor, prepared sensor, can be used for quick, the Sensitive Detection to parathion-methyl in the fields such as daily production, life.Based on this purpose, the present invention, in same electrolyzer, adopts four electrode systems, namely two working electrodes, one to electrode and a reference electrode, wherein working electrode 1 adopts the molybdenum bisuphide two-dimension nano materials Cd-MoS that cadmium adulterates2Modifying with the luminol of electropolymerization, as electrogenerated chemiluminescence working electrode W1, also serve as self-emissive light source, working electrode 2 adopts titanium dioxide nano-particle colloidal sol TiO2NPs and parathion-methyl antibody are modified, as Optical Electro-Chemistry working electrode W2.When detecting, after adding fixed concentration hydrogen peroxide in electrolyzer, W1 applies step voltage, due to Cd-MoS2Catalysis, enhancing and stabilized illumination effect, luminol and hydroperoxidation, produce electrogenerated chemiluminescence, this is just equivalent to " turning on light ", and when step voltage is 0, electrogenerated chemiluminescence disappears, this is just equivalent to " turning off the light ", meanwhile applies constant voltage on W2, due to TiO always2NPs can because the luminescence that electrogenerated chemiluminescence produces excites causes that electron-hole pair is easily separated, it is marked at the horseradish peroxidase HRP catalyzing hydrogen peroxide generation oxygen that parathion-methyl two resists, hydrogen peroxide is made to become hole " donor ", thus obtaining photoelectric current on W2, when electrogenerated chemiluminescence disappears, when namely " turning off the light ", photoelectric current disappears immediately.Due under the premise of fixing concentration of hydrogen peroxide, photoelectric current size and HRP concentration positive correlation, when in measured object, parathion-methyl concentration is more big, when being attached on W2 with primary antibodie immunity, the concentration that immunity incorporation of markings has the parathion-methyl two of HRP anti-again will be more big, the photoelectric current produced is also more big, therefore can realize the detection to parathion-methyl by the size of recording light electric current.
Based on above inventive principle, the technical solution used in the present invention is as follows:
1. the preparation method based on the parathion-methyl immunosensor of two kinds of electrochemical method couplings, it is characterised in that preparation process is:
(1) preparation of electrogenerated chemiluminescence working electrode W1:
1) with ITO electro-conductive glass for working electrode, at the molybdenum bisuphide two-dimension nano materials Cd-MoS of electrode surface drop coating cadmium doping2Solution, area coverage is 1cm × 1cm, dries under room temperature；
2) by 1) working electrode that obtains, immerse in electrolyte, immersion area is Cd-MoS2The area covered, utilizes three-electrode system that working electrode is carried out electrochemical deposition, takes out working electrode after deposition, uses ultra-pure water to clean, and at 4 DEG C, lucifuge dries, and prepares electrogenerated chemiluminescence working electrode W1；
The molybdenum bisuphide two-dimension nano materials Cd-MoS of described cadmium doping2Solution is Cd-MoS2Aqueous solution, described Cd-MoS2For the molybdenum bisuphide two-dimension nano materials doped with cadmium ion, described two-dimension nano materials is scraps of paper shape, and thickness is about 10 ~ 20nm, described Cd-MoS2Preparation method be: by 0.005 ~ 0.01g Caddy (Cleary) CdCl2, the 0.1mol/L ascorbic acid solution of 2 ~ 6mL, 0.5 ~ 1.5mL sodium molybdate Na2MoO4Solution and 0.01 ~ 0.03g sodium sulfide Na2S, after being mixed and stirred for 15 minutes, puts in reactor, at 150 ~ 220 DEG C, reacts 12 ~ 16 hours；After being cooled to room temperature, use deionized water centrifuge washing, at 40 DEG C, carry out vacuum drying, namely prepare the molybdenum bisuphide two-dimension nano materials Cd-MoS of cadmium doping2, it is dissolved in deionized water and prepares Cd-MoS2Solution；
Described electrolyte is the sulfuric acid solution containing luminol, and in described electrolyte, the concentration of luminol is 1 ~ 10mmol/L, and sulfuric acid concentration is 0.1 ~ 1.0mol/L；
Described three-electrode system, including working electrode, reference electrode and to electrode, described reference electrode is saturated calomel electrode, and described is platinum electrode to electrode；
Described electrochemical deposition process, the electrochemical method of employing is cyclic voltammetry, and starting voltage is-0.2V, and final voltage is 1.5V, sweeps speed for 100mv/s, circulation 20 ~ 30 circle；
(2) preparation of Optical Electro-Chemistry working electrode W2:
1) with ITO electro-conductive glass for working electrode, at electrode surface drop coating 8 ~ 12 μ L titanium dioxide nano-particle colloidal sol TiO2NPs, dries under room temperature；
2) by 1) in the working electrode that obtains put in Muffle furnace, be annealed processing at 450 DEG C, after process, be cooled to room temperature；
3) by 2) in working electrode surface drop coating 8 ~ 12 μ L parathion-methyl antibody-solutions that obtains, dry at 4 DEG C, clean with ultra-pure water after drying, dry at 4 DEG C, prepare Optical Electro-Chemistry working electrode W2；
Described TiO2NPs is the titanium dioxide nano-particle aqueous solution of 1mg/mL；
The concentration of described parathion-methyl antibody-solutions is 300 μ g/mL；
(3) based on the preparation method of the parathion-methyl immunosensor of two kinds of electrochemical method couplings:
1) being inserted face to face in electrolyzer by W1 and W2, W1 and W2 spacing is 0.5cm ~ 1.5cm；
2) with Ag/AgCl be reference electrode RE, platinum electrode be to electrode CE, insert in electrolyzer, collectively constitute four electrode systems with W1 and W2；
3) in electrolyzer add 10mLpH value be 11 ~ 13 NaOH solution and 0.2mL concentration be the hydrogenperoxide steam generator of 1mmol/L；
4) by 1) ~ 3) obtained by four electrode systems and electrolyzer be placed in magazine, namely prepare the parathion-methyl immunosensor based on two kinds of electrochemical method couplings.
2. the parathion-methyl immunosensor prepared by the preparation method according to technical scheme 1 is applied to the detection of parathion-methyl, and its detecting step is:
(1) on W2, parathion-methyl solution to be measured is dripped, after the parathion-methyl in parathion-methyl solution to be measured parathion-methyl antibody mediated immunity on W2 is combined, rinse out all the other materials, drip that the parathion-methyl two of horseradish peroxidase HRP labelling is anti-carries out immunity combination again, the parathion-methyl two washing unnecessary HRP labelling in conjunction with backlash off resists, and prepares W2 to be measured；
(2) utilizing electrochemical workstation, in the electrolyte containing fixed concentration hydrogen peroxide, adopt the method for step voltage that W1 applies step voltage on W1, initial voltage is 0v, and step voltage is 0.7 ~ 0.9v, and snap time is 10 ~ 30s；Meanwhile, when adopting on W2 to be measured, W2 to be measured is applied constant voltage by m-current methods, and voltage is 0 ~ 0.6v；Electric current on W2 to be measured can along with in parathion-methyl solution to be measured the concentration of parathion-methyl increase and increase accordingly, increasing according to gained electric current is worth in parathion-methyl solution to be measured the concentration of parathion-methyl；
The concentration that the parathion-methyl two of described HRP labelling is anti-is 300 μ g/mL.
The useful achievement of the present invention
(1) parathion-methyl sensor of the present invention preparation is simple, easy to operate, it is not necessary to external accessory, utilize the microminiaturization of detection equipment, portability, and achieve the selective enumeration method quick, sensitive, high to parathion-methyl, there is wide market development prospect；
(2) present invention adopts four electrode system detection parathion-methyls first in same electrolyzer, and achieves electrogenerated chemiluminescence signal amplification strategy difunctional with Optical Electro-Chemistry.Along with the increase of parathion-methyl concentration in electrolyzer, the parathion-methyl two of the HRP labelling being modified on W2 is anti-will be increased, and also implies that HRP is to hydrogen peroxide catalyzed raising.Since so, on the one hand, make electrogenerated chemiluminescence intensity linearly increasing, the linear increase of photoelectric current excited；On the other hand, hydrogen peroxide is as electron donor so that the linear increase of photoelectric current in Optical Electro-Chemistry reaction.Therefore, electrogenerated chemiluminescence and two kinds of methods of Optical Electro-Chemistry are reaction jointly, interaction in same electrolyzer, under same electrochemical workstation, achieve the dual amplification that parathion-methyl is detected the signal of telecommunication, drastically increase detection sensitivity and detection limit, there is important scientific meaning and using value.
Detailed description of the invention
Embodiment 1 one kinds is based on the parathion-methyl immunosensor of two kinds of electrochemical method couplings
A kind of preparation method of the parathion-methyl immunosensor based on two kinds of electrochemical method couplings, concrete preparation process is:
(1) the molybdenum bisuphide two-dimension nano materials Cd-MoS of cadmium doping2The preparation of solution:
The molybdenum bisuphide two-dimension nano materials Cd-MoS of described cadmium doping2Solution is Cd-MoS2Aqueous solution, described Cd-MoS2For the molybdenum bisuphide two-dimension nano materials doped with cadmium ion, described two-dimension nano materials is scraps of paper shape, and thickness is about 10 ~ 20nm, described Cd-MoS2Preparation method be: by 0.005g Caddy (Cleary) CdCl2, the 0.1mol/L ascorbic acid solution of 2mL, 0.5mL sodium molybdate Na2MoO4Solution and 0.01g sodium sulfide Na2S, after being mixed and stirred for 15 minutes, puts in reactor, at 150 DEG C, reacts 16 hours；After being cooled to room temperature, use deionized water centrifuge washing, at 40 DEG C, carry out vacuum drying, namely prepare the molybdenum bisuphide two-dimension nano materials Cd-MoS of cadmium doping2, it is dissolved in deionized water and prepares Cd-MoS2Solution.
(2) preparation of electrogenerated chemiluminescence working electrode W1:
1) with ITO electro-conductive glass for working electrode, the Cd-MoS of preparation in electrode surface drop coating (1)2Solution, area coverage is 1cm × 1cm, dries under room temperature；
2) by 1) working electrode that obtains, immerse in electrolyte, immersion area is Cd-MoS2The area covered, utilizes three-electrode system that working electrode is carried out electrochemical deposition, takes out working electrode after deposition, uses ultra-pure water to clean, and at 4 DEG C, lucifuge dries, and prepares electrogenerated chemiluminescence working electrode W1；
Described electrolyte is the sulfuric acid solution containing luminol, and in described electrolyte, the concentration of luminol is 1mmol/L, and sulfuric acid concentration is 0.1mol/L；
Described three-electrode system, including working electrode, reference electrode and to electrode, described reference electrode is saturated calomel electrode, and described is platinum electrode to electrode；
Described electrochemical deposition process, the electrochemical method of employing is cyclic voltammetry, and starting voltage is-0.2V, and final voltage is 1.5V, sweeps speed for 100mv/s, circulation 20 circle.
(3) preparation of Optical Electro-Chemistry working electrode W2:
1) with ITO electro-conductive glass for working electrode, at electrode surface drop coating 8 μ L titanium dioxide nano-particle colloidal sol TiO2NPs, dries under room temperature；
2) by 1) in the working electrode that obtains put in Muffle furnace, be annealed processing at 450 DEG C, after process, be cooled to room temperature；
3) by 2) in the working electrode surface drop coating 8 μ L parathion-methyl antibody-solutions that obtains, dry at 4 DEG C, clean with ultra-pure water after drying, dry at 4 DEG C, prepare Optical Electro-Chemistry working electrode W2；
Described TiO2NPs is the titanium dioxide nano-particle aqueous solution of 1mg/mL；
The concentration of described parathion-methyl antibody-solutions is 300 μ g/mL；
(4) based on the preparation method of the parathion-methyl immunosensor of two kinds of electrochemical method couplings:
1) being inserted face to face in electrolyzer by the W1 of preparation in (2) and the W2 of (3) middle preparation, W1 and W2 spacing is 0.5cm；
2) with Ag/AgCl be reference electrode RE, platinum electrode be to electrode CE, insert in electrolyzer, collectively constitute four electrode systems with W1 and W2；
3) in electrolyzer add 10mLpH value be 11 NaOH solution and 0.2mL concentration be the hydrogenperoxide steam generator of 1mmol/L；
4) by 1) ~ 3) obtained by four electrode systems and electrolyzer be placed in magazine, namely prepare the parathion-methyl immunosensor based on two kinds of electrochemical method couplings.
Embodiment 2 one kinds is based on the parathion-methyl immunosensor of two kinds of electrochemical method couplings
A kind of preparation method of the parathion-methyl immunosensor based on two kinds of electrochemical method couplings, concrete preparation process is:
(1) Cd-MoS2The preparation of solution:
Preparation process is with Cd-MoS in embodiment 12The preparation method of solution, difference is: CdCl2Addition be 0.008g, ascorbic acid solution addition be 4mL, Na2MoO4The addition of solution is 1.0mL and Na2The addition of S is 0.02g, at 220 DEG C, reacts 12 hours.
(2) preparation of electrogenerated chemiluminescence working electrode W1:
Preparation process is with the preparation process of W1 in embodiment 1, and difference is: use the Cd-MoS obtained by (1) in the present embodiment2Modified electrode；In electrolyte, the concentration of luminol is 5mmol/L, and sulfuric acid concentration is 0.5mol/L；When cyclic voltammetry carries out electrochemical deposition, circulation 25 circle.
(3) preparation of Optical Electro-Chemistry working electrode W2:
1) with ITO electro-conductive glass for working electrode, at electrode surface drop coating 10 μ L titanium dioxide nano-particle colloidal sol TiO2NPs, dries under room temperature；
2) by 1) in the working electrode that obtains put in Muffle furnace, be annealed processing at 450 DEG C, after process, be cooled to room temperature；
3) by 2) in the working electrode surface drop coating 10 μ L parathion-methyl antibody-solutions that obtains, dry at 4 DEG C, clean with ultra-pure water after drying, dry at 4 DEG C, prepare Optical Electro-Chemistry working electrode W2；
All the other are with the preparation process of W2 in embodiment 1.
(4) based on the preparation method of the parathion-methyl immunosensor of two kinds of electrochemical method couplings:
Preparation process is with embodiment 1, and difference is W1 and W2 spacing is 1.0cm, and the pH value of the NaOH solution added in electrolyzer is 12.
Embodiment 3 one kinds is based on the parathion-methyl immunosensor of two kinds of electrochemical method couplings
A kind of preparation method of the parathion-methyl immunosensor based on two kinds of electrochemical method couplings, concrete preparation process is:
(1) Cd-MoS2The preparation of solution:
Preparation process is with Cd-MoS in embodiment 12The preparation method of solution, difference is: CdCl2Addition be 0.01g, ascorbic acid solution addition be 6mL, Na2MoO4The addition of solution is 1.5mL and Na2The addition of S is 0.03g, at 220 DEG C, reacts 12 hours.
(2) preparation of electrogenerated chemiluminescence working electrode W1:
Preparation process is with the preparation process of W1 in embodiment 1, and difference is: use the Cd-MoS obtained by (1) in the present embodiment2Modified electrode；In electrolyte, the concentration of luminol is 10mmol/L, and sulfuric acid concentration is 1.0mol/L；When cyclic voltammetry carries out electrochemical deposition, circulation 30 circle.
(3) preparation of Optical Electro-Chemistry working electrode W2:
1) with ITO electro-conductive glass for working electrode, at electrode surface drop coating 12 μ L titanium dioxide nano-particle colloidal sol TiO2NPs, dries under room temperature；
2) by 1) in the working electrode that obtains put in Muffle furnace, be annealed processing at 450 DEG C, after process, be cooled to room temperature；
3) by 2) in the working electrode surface drop coating 12 μ L parathion-methyl antibody-solutions that obtains, dry at 4 DEG C, clean with ultra-pure water after drying, dry at 4 DEG C, prepare Optical Electro-Chemistry working electrode W2；
All the other are with the preparation process of W2 in embodiment 1.
(4) based on the preparation method of the parathion-methyl immunosensor of two kinds of electrochemical method couplings:
Preparation process is with embodiment 1, and difference is W1 and W2 spacing is 1.5cm, and the pH value of the NaOH solution added in electrolyzer is 13.
Embodiment 4 one kinds is based on the application of the parathion-methyl immunosensor of two kinds of electrochemical method couplings
A kind of parathion-methyl immunosensor based on two kinds of electrochemical method couplings of embodiment 1 preparation is applied to the detection of parathion-methyl, and its detecting step is:
(1) on W2, drip the parathion-methyl standard solution of 10 μ L variable concentrations, after hatching 30min, parathion-methyl and the parathion-methyl antibody on W2 carry out immunity combination, after flushing, the parathion-methyl two dripping 10 μ L horseradish peroxidase HRP labellings again resists, and after hatching 30min, the parathion-methyl two of HRP labelling is anti-carries out immunity combination with parathion-methyl, after flushing, prepare W2 to be measured；
(2) utilizing electrochemical workstation, adopt the method for step voltage that W1 applies step voltage on W1, initial voltage is 0v, and step voltage is 0.7v, and snap time is 10s；Meanwhile, when adopting on W2 to be measured, W2 to be measured is applied constant voltage by m-current methods, and voltage is 0v；Electric current on W2 to be measured can increase accordingly along with the increase of parathion-methyl concentration, according to the relation between gained electric current increase value and parathion-methyl concentration, drawing curve；
(3) parathion-methyl solution to be measured is replaced the standard solution of parathion-methyl, parathion-methyl detection method described in (1) and (2) detects, and draws the concentration of parathion-methyl solution to be measured according to obtained electric current increase value and the working curve drawn；
The concentration that the parathion-methyl two of described HRP labelling is anti-is 300 μ g/mL.
Embodiment 5 one kinds is based on the application of the parathion-methyl immunosensor of two kinds of electrochemical method couplings
A kind of parathion-methyl immunosensor based on two kinds of electrochemical method couplings of embodiment 2 preparation is applied to the detection of hydrogen peroxide, and its detecting step is except following steps, and all the other steps are with embodiment 4:
Step (2) utilizes electrochemical workstation, adopts the method for step voltage that W1 applies step voltage on W1, and initial voltage is 0v, and step voltage is 0.8v, and snap time is 20s；Meanwhile, when adopting on W2 to be measured, W2 to be measured is applied constant voltage by m-current methods, and voltage is 0.3v；Electric current on W2 to be measured can increase accordingly along with the increase of parathion-methyl concentration, according to the relation between gained electric current increase value and parathion-methyl concentration, drawing curve.
The application of the parathion-methyl immunosensor of 6 one kinds of two kinds of electrochemical method couplings of embodiment
A kind of parathion-methyl immunosensor based on two kinds of electrochemical method couplings of embodiment 3 preparation is applied to the detection of hydrogen peroxide, and its detecting step is except following steps, and all the other steps are with embodiment 4:
Step (2) utilizes electrochemical workstation, adopts the method for step voltage that W1 applies step voltage on W1, and initial voltage is 0v, and step voltage is 0.9v, and snap time is 30s；Meanwhile, when adopting on W2 to be measured, W2 to be measured is applied constant voltage by m-current methods, and voltage is 0.6v；Electric current on W2 to be measured can increase accordingly along with the increase of parathion-methyl concentration, according to the relation between gained electric current increase value and parathion-methyl concentration, drawing curve.
Embodiment 7Parathion-methyl immunosensor prepared by embodiment 1-3, is applied to the detection of parathion-methyl according to the detecting step of embodiment 4-6, has excellent Detection results, and detection is limited to 14pmol/L.

Claims (2)

1. the preparation method based on the parathion-methyl immunosensor of two kinds of electrochemical method couplings, it is characterised in that preparation process is:
(1) preparation of electrogenerated chemiluminescence working electrode W1:
1) with ITO electro-conductive glass for working electrode, at the molybdenum bisuphide two-dimension nano materials Cd-MoS of electrode surface drop coating cadmium doping2Solution, area coverage is 1cm × 1cm, dries under room temperature；
2) by 1) working electrode that obtains, immerse in electrolyte, immersion area is Cd-MoS2The area covered, utilizes three-electrode system that working electrode is carried out electrochemical deposition, takes out working electrode after deposition, uses ultra-pure water to clean, and at 4 DEG C, lucifuge dries, and prepares electrogenerated chemiluminescence working electrode W1；
The molybdenum bisuphide two-dimension nano materials Cd-MoS of described cadmium doping2Solution is Cd-MoS2Aqueous solution, described Cd-MoS2For the molybdenum bisuphide two-dimension nano materials doped with cadmium ion, described two-dimension nano materials is scraps of paper shape, and thickness is about 10 ~ 20nm；
Described electrolyte is the sulfuric acid solution containing luminol, and in described electrolyte, the concentration of luminol is 1 ~ 10mmol/L, and sulfuric acid concentration is 0.1 ~ 1.0mol/L；
Described three-electrode system, including working electrode, reference electrode and to electrode, described reference electrode is saturated calomel electrode, and described is platinum electrode to electrode；
Described electrochemical deposition process, the electrochemical method of employing is cyclic voltammetry, and starting voltage is-0.2V, and final voltage is 1.5V, sweeps speed for 100mv/s, circulation 20 ~ 30 circle；
(2) preparation of Optical Electro-Chemistry working electrode W2:
1) with ITO electro-conductive glass for working electrode, at electrode surface drop coating 8 ~ 12 μ L titanium dioxide nano-particle colloidal sol TiO2NPs, dries under room temperature；
2) by 1) in the working electrode that obtains put in Muffle furnace, be annealed processing at 450 DEG C, after process, be cooled to room temperature；
3) by 2) in working electrode surface drop coating 8 ~ 12 μ L parathion-methyl antibody-solutions that obtains, dry at 4 DEG C, clean with ultra-pure water after drying, dry at 4 DEG C, prepare Optical Electro-Chemistry working electrode W2；
Described TiO2NPs is the titanium dioxide nano-particle aqueous solution of 1mg/mL；
The concentration of described parathion-methyl antibody-solutions is 300 μ g/mL；
(3) based on the preparation method of the parathion-methyl immunosensor of two kinds of electrochemical method couplings:
1) being inserted face to face in electrolyzer by W1 and W2, W1 and W2 spacing is 0.5cm ~ 1.5cm；
2) with Ag/AgCl be reference electrode RE, platinum electrode be to electrode CE, insert in electrolyzer, collectively constitute four electrode systems with W1 and W2；
3) in electrolyzer add 10mLpH value be 11 ~ 13 NaOH solution and 0.2mL concentration be the hydrogenperoxide steam generator of 1mmol/L；
4) by 1) ~ 3) obtained by four electrode systems and electrolyzer be placed in magazine, namely prepare the parathion-methyl immunosensor based on two kinds of electrochemical method couplings.
2. the parathion-methyl immunosensor that prepared by preparation method according to claim 1, it is characterised in that described parathion-methyl immunosensor is applied to the detection of parathion-methyl, and detecting step is:
(1) on W2, parathion-methyl solution to be measured is dripped, after the parathion-methyl in parathion-methyl solution to be measured parathion-methyl antibody mediated immunity on W2 is combined, rinse out all the other materials, drip that the parathion-methyl two of horseradish peroxidase HRP labelling is anti-carries out immunity combination again, the parathion-methyl two washing unnecessary HRP labelling in conjunction with backlash off resists, and prepares W2 to be measured；
(2) utilizing electrochemical workstation, in the electrolyte containing fixed concentration hydrogen peroxide, adopt the method for step voltage that W1 applies step voltage on W1, initial voltage is 0v, and step voltage is 0.7 ~ 0.9v, and snap time is 10 ~ 30s；Meanwhile, when adopting on W2 to be measured, W2 to be measured is applied constant voltage by m-current methods, and voltage is 0 ~ 0.6v；Electric current on W2 to be measured can along with in parathion-methyl solution to be measured the concentration of parathion-methyl increase and increase accordingly, increasing according to gained electric current is worth in parathion-methyl solution to be measured the concentration of parathion-methyl.
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