CN101178375B - Manufacturing method of biology enzymic electrode used for detecting pesticide residue - Google Patents
Manufacturing method of biology enzymic electrode used for detecting pesticide residue Download PDFInfo
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Abstract
The invention relates to a production method of a biological enzyme electrode used for the detection of pesticide residues, which pertains to the biochemistry electrodes, the production method adopts a combination method of nanoparticles and layer-by-layer self-assembly technology, the method has the steps that: a rod-shaped electrode carrier is made by using organic insulation materials, the substrate conductive materials are coated on a cross section of the lower end of the electrode carrier to form an electrode or the similar electrode is bought directly, then the outer surface of the conductive materials of the electrode uses the nanoparticles to decorate the electrode to form a nanoparticle layer, an anti-interference layer is prepared by the layer-by-layer self-assembly, an electrostatic adsorptive enzyme layer is prepared by the layer-by-layer self-assembly, so as to obtain a reaction layer which comprises the nanoparticle layer, the anti-interference layer and the electrostatic adsorptive enzyme layer, and the biological enzyme electrode used for detection of the pesticide residues is produced further. The production method of the invention is simple; a biosensor which is assembled by the biological enzyme electrode which is produced by the method has high sensitivity, stable performance and strong anti-interference capacity, so the invention is applicable to the actual application of the industrialization of the biological sensors for the detection of the pesticide residues.
Description
Technical field
Technical scheme of the present invention relates to biochemical electrode, specifically, is the method for making that is used to detect the biologic enzyme electrode of residues of pesticides.
Background technology
In recent years, the residues of pesticides problem that exceeds standard has caused people's extensive concern in the agricultural product.The biology sensor that is used to detect residues of pesticides is is also constantly researched and developed and is utilized.
(Flow injection amperometric enzyme biosensor for directdetermination of organphosphate nerve agents such as Mulchandani, Environ.Sci.Technol.2001, (35)) the flow injection ampere-type organophosphor hydrolytic enzyme biology sensor of developing in conjunction with the flow injection technology (FIAB), by detecting the hydrolysate p-nitrophenol of organophosphorus, judge the concentration of organophosphorus.(Detection of paraoxonby continuous flow system based enzyme sensor such as Jeanty, Biosensors and Bioelectronics.1998, (13)) detect organophosphorus pesticide paraoxon and derivant thereof with flow injection acetylcholinesterase biology sensor.Wei Fuxiang etc. (acetylcholinesterase biology sensor method is measured organophosphorus pesticide in the vegetable and fruit, Food Science, 2007 (28)) have introduced a kind of acetylcholinesterase biosensor technology that utilizes and have measured organophosphorus pesticide in the vegetable and fruit.Said method is the performance of attempting to improve by the change of enzyme fixing means and material biology sensor, but have or the range of linearity narrow, shortcoming such as perhaps not high the or antijamming capability of detection sensitivity is not strong, and the neither use that can control the enzyme amount.(Electroanalysis such as Haibin Shi, 2005,17, (14): 1285-1290) reported utilize molecule one enzyme layer by layer self assembly built up film technology on electrode, prepare conducting polymer film, utilizing high molecular polymer PDDA and choline oxidase to prepare the choline sensor is Pt/ (PDDA/ChOx) n.(Sensor ﹠amp such as Zhao Song; Actuator B, 2006,115:626-633) reported that modifying platinum electrode with CNT is basic electrode, adopt fixedly choline oxidase (ChOx) of sol-gel process, made up current mode choline detection of biological sensor.(sensor technology, 2005,24 (7): 16-18) reported with the screen printing electrode to be carrier, be that main method has prepared the amperometric biosensor enzyme electrode based on acetylcholinesterase and choline oxidase with the gelatin embedding to people such as CN2723998 and Song Zhao.CN01253887.6 has disclosed a kind of calorimetric biosensor that residues of pesticides are used of surveying, form by test solution processing unit and detecting unit, the test solution processing unit comprises front processor, filtrator, sample liquid container, buffer container, conduit, peristaltic pump, pulse damper, micro-injection pump, detecting unit comprises thermostat, semiconductor cooler, the threeway that inside and outside courage is formed, detect enzyme post, reference enzyme post, detector unit places enzyme post both sides, detect the temperature difference of enzyme column outlet liquid, output is connected with processing, display unit and microprocessor.The composition complex structure of this calorimetric biosensor, cost costliness, particularly because during actual detected used, the activity of repressed enzyme was difficult to reverse, enzyme reaction layer once more fixedly activation can become matter of utmost importance.CN1699985 has disclosed the method for making of Pesticides Testing with nano-modified miniature electrochemica biological sensor, form by nano composite enzyme electrode, miniature silver/silver chloride (Ag/AgCl) contrast electrode with to electrode, concrete structure is: nano material and enzyme are compound, combine with the working electrode of film like, electrode is formed film like formula three electrode type sensors jointly with miniature silver/silver chloride (Ag/AgCl) contrast electrode of film like and film like.It directly mixes dropping on working electrode with enzyme and CNT, then substrate is detected, have following shortcoming unavoidably: (1) can not be controlled the enzyme amount well and use (2) testing process to be subject to the influence of chaff interference, be that antijamming capability is not strong, (3) enzyme is fixing firmly causes electrode stability bad inadequately, poor repeatability.
In a word, in the existing method that adopts the biosensor test residues of pesticides, the sensitivity of biology sensor, stability and antijamming capability still remain further to be improved.Therefore, research and develop highly sensitive, good stability, antijamming capability is strong, and to be used for the biologic enzyme electrode that remains of pesticide detects very necessary.
Summary of the invention
Technical matters to be solved by this invention is: the method for making that is provided for detecting the biologic enzyme electrode of residues of pesticides, wherein adopt the method that nano particle and layer-by-layer are combined, further improved the sensitivity, stability and the antijamming capability that are used for the biologic enzyme electrode that remains of pesticide detects.
The present invention solves this technical problem the technical scheme that is adopted: be used to detect the method for making of the biologic enzyme electrode of residues of pesticides, adopt the method that nano particle and layer-by-layer are combined, concrete steps are as follows:
Make the stick electrode carrier with organic insulation, on the xsect of its lower end, coat the matrix conductive material, form electrode, perhaps directly buy such electrode, make the responding layer that constitutes by nano-particle layer, anti-interference layer and Electrostatic Absorption enzyme layer at the outside surface of this conductive material of electrodes with operation as described below again, be made into the biologic enzyme electrode that is used to detect residues of pesticides thus.
The first step, the processing of electrode lower end conductive material outside surface
Polish above-mentioned electrode lower end conductive material outer surface part to cleaning with aluminium powder, move into then and clean 2～3 minutes in the ultrasound bath, repeat 3 times, make this electrode lower end conductive material outside surface clean with the thorough supersound washing of ethanol, watery hydrochloric acid and distilled water again as minute surface, standby after drying;
In second step, form nano-particle layer with nano-particle modified electrode
It is the concentrated sulphuric acid that nano particle is placed volume ratio: the mixed acid solution of red fuming nitric acid (RFNA)=3: 1, ultrasonic concussion 4 hours, be dispersed into even light/dark balance solution, centrifugal with 10000 rev/mins in hydro-extractor, outwell the supernatant acid solution, add the ultrasonic dispersion washing of water 5 minutes again, the repeated multiple times washing is closely neutral to this dark solution, make the nanoparticles solution that concentration is 1g/L, place 4 ℃ of refrigerators to deposit stand-by, to immerse in this nanoparticles solution 25 minutes by the electrode lower end that the first step is handled well, at its conductive material outside surface absorption nano particle, form nano-particle layer, then this electrode is placed damping fluid to clean 5 minutes, flush away adsorbs not firm nano particle, finishes the operation of nano-particle modified electrode thus;
In the 3rd step, self assembly layer by layer prepares anti-interference layer
Second step was immersed in the cation high molecular polymer solution at least a in polypropylene amine that concentration is 2mg/mL, polyethyleneimine, the diallyl dimethyl ammoniumchloride 25 minutes with the lower end of nano-particle modified electrode, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the anionic polymer polymer solution at least a in poly-sulfonated ethylene that concentration is 2mg/mL, the poly-sulfonated phenylethylene 25 minutes again, repeat this process until alternately plating 1～3 floor height Molecularly Imprinted Polymer built up film, finish the preparation of anti-interference layer;
In the 4th step, self assembly layer by layer prepares Electrostatic Absorption enzyme layer
A. self assembly layer by layer prepares choline oxidase enzyme layer
It is the polypropylene amine of 2mg/mL that the 3rd lower end that goes on foot the electrode of finishing anti-interference layer preparation is immersed in the concentration that is mixed with 5% nano particle, polyethyleneimine, in at least a cation high molecular polymer solution in the diallyl dimethyl ammoniumchloride 25 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the choline oxidase solution that concentration is 2mg/mL 25 minutes again, repeat this process until the composite membrane that alternately plates 2～15 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of choline oxidase enzyme layer, make the choline biologic enzyme electrode that detects choline thus;
B. self assembly layer by layer prepares acetylcholinesterase enzyme layer
It is the polypropylene amine of 2mg/mL that the lower end that the 4th step a is finished the electrode of choline oxidase enzyme layer preparation is immersed in the concentration that is mixed with 5% nano particle, polyethyleneimine, in at least a cation high molecular polymer solution in the diallyl dimethyl ammoniumchloride 25 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the acetylcholinesterase solution that concentration is 2mg/mL 25 minutes again, repeat this process until the composite membrane that alternately plates 1～5 floor height Molecularly Imprinted Polymer and enzyme, finish the preparation of acetylcholinesterase enzyme layer, also finish the making of responding layer, and finally made the biologic enzyme electrode that is used to detect acetylcholine and residues of pesticides;
The above-mentioned method for making that is used for detecting the biologic enzyme electrode of residues of pesticides, the concentration of used solution is weight/volume percent concentration, and the compound method of used damping fluid is with NaOH 1.874 grams, KH
2PO
46.805 the gram, constant volume in the volumetric flask of 1000mL, pH8.0.
The above-mentioned method for making that is used for detecting the biologic enzyme electrode of residues of pesticides, described matrix conductive material as electrode is gold, carbon, platinum or silver.
The above-mentioned method for making that is used for detecting the biologic enzyme electrode of residues of pesticides, the described stick electrode carrier made from organic insulation, its bar-shaped xsect are square, rectangle, circle, ellipse or other figures.
The above-mentioned method for making that is used for detecting the biologic enzyme electrode of residues of pesticides, described nano particle is CNT or nm of gold, its grain size scope is at 8～100nm.CNT or nm of gold are the known raw material article that buy.
The above-mentioned method for making that is used for detecting the biologic enzyme electrode of residues of pesticides, the selected organic insulation of biologic enzyme electrode is well known to those skilled in the art.
The above-mentioned method for making that is used for detecting the biologic enzyme electrode of residues of pesticides, the method that coats conductive material on the organic insulation carrier is well known to those skilled in the art.
The invention has the beneficial effects as follows:
(1) the inventive method is made is used to detect the detection range of linearity of the biologic enzyme electrode of residues of pesticides to acetylcholine: 5 * 10
-6～8 * 10
-4Mol/L detects and is limited to 5 * 10
-6Mol/L; The detection range of linearity to metrifonate is: 0.01～0.6 μ g/mL, detection limit are 1 * 10
-10Mol/L.
(2) in the above-mentioned method for making that is used for detecting the biologic enzyme electrode of residues of pesticides, the 4th step a finishes the choline biologic enzyme electrode of making, to the detection range of linearity of choline: 5 * 10
-7～1 * 10
-4Mol/L; Sensitivity: 12.53 μ A/mmol; Can be controlled in detection time 7.6 seconds, and detected and be limited to 2 * 10
-7Mol/L.Its overall performance is better than the choline biologic enzyme electrode of previous report.
(3) biologic enzyme electrode that is used to detect residues of pesticides of the inventive method making is modified with nano particle.Because the nano particle synergy, the response sensitivity of this sensor obviously improves, than exceeding about 20% of unmodified nano particle.
(4) biologic enzyme electrode that the inventive method is made is used to detect residues of pesticides has used the high molecular polymer multilayer film as anti-interference layer, make the whole biology sensor of assembling with this biologic enzyme electrode have the strong characteristics of antijamming capability, the influence of chaff interference almost can be ignored.
(5) because the present invention is used for detecting the method for making of the biologic enzyme electrode of residues of pesticides, adopted nano particle and accumulated the method that self-assembling technique combines layer by layer, both improved sensitivity, use that again can better controlled enzyme amount with the biology sensor of this biologic enzyme electrode assembling.Accumulate self assembly layer by layer and be meant molecule and molecule under certain condition, utilize molecular recognition, rely on the spontaneous process that connects into constitutionally stable molecule aggregate of intermolecular force.
Beneficial effect of the present invention specifically is reflected in embodiment 11～12 and Fig. 3～12.
In a word, the present invention is used to detect the method for making of the biologic enzyme electrode of residues of pesticides, and method is simple, highly sensitive with the biology sensor that this biologic enzyme electrode constitutes, stable performance, antijamming capability is strong, is applicable to the practical application of the biology sensor industrialization that detects residues of pesticides.
Description of drawings
The present invention is further described below in conjunction with drawings and Examples.
The longitudinal profile synoptic diagram of the biologic enzyme electrode of the bar-shaped detection residues of pesticides of Fig. 1 the present invention.
Fig. 2 the present invention detects the synoptic diagram that the responding layer of the lower end conductive material of biologic enzyme electrode of residues of pesticides and outside surface thereof constitutes.
Fig. 3 CNT modified electrode (a) and the current-responsive of naked platinum electrode (b) to the 1mmol/L hydrogen peroxide.
The different number of plies choline oxidase of Fig. 4 laminated film is modified the cyclic voltammetry curve of choline biology sensor.
The number of plies of Fig. 5 choline oxidase laminated film is to the influence of sensor response current.
Fig. 6 choline oxidase laminated film is modified the working curve of sensor to the Choline Chloride response.
Fig. 7 choline oxidase laminated film is modified the electric current-time curve of sensor to the Choline Chloride response.
Fig. 8 chaff interference is to Pt/MWCNT/ (PAA/PVS)
3(PDDA/ChOx)
8The influence of choline sensor response current,
Among this figure, a. choline, b. ascorbic acid, c. acetaminophen, d. uric acid, e. choline.
Fig. 9 Pt/MWCNT/ (PAA/PVS)
3(PDDA/ChOx)
8The stability study of choline sensor.
The number of plies of Figure 10 acetylcholinesterase laminated film is to the influence of sensor response current.
Figure 11 Pt/MWCNT/ (PAA/PVS)
3(PDDA/ChOx)
8+ (PDDA/AChE)
2The laminated film modified electrode is to the electric current-time curve of acetylcholine response.
Figure 12 residues of pesticides sensor Pt/MWCNT/ (PAA/PVS)
3(PDDA/ChOx)
8+ (PDDA/AChE)
2Response to metrifonate.
Among the figure, 1. the stick electrode carrier made from organic insulation, 2. make the conductive material gold, carbon, platinum or the silver that coat on the xsect of stick electrode carrier lower end and the responding layer of conductive material outside surface at organic insulation, 3. conductive material gold, carbon, platinum or silver, 4. nano-particle layer, 5. anti-interference layer, 6. choline oxidase enzyme layer, 7. acetylcholinesterase enzyme layer.
Embodiment
Making xsect with organic insulation is square stick electrode carrier (1), coats gold (3) on the xsect of its lower end, is made into bar-shaped working electrode as shown in Figure 1.
Remove with carbon and replace gold, xsect is outside the rectangle, and other are with embodiment 1.
Remove with platinum and replace gold, xsect is outside the circle, and other are with embodiment 1.
Remove with silver and replace gold, xsect is outside the ellipse, and other are with embodiment 1.
The first step, the processing of electrode lower end conductive material outside surface
The outer surface part of the gold (3) that the electrode lower end that is made by embodiment 1 with the aluminium powder polishing coats is to cleaning, move into then and clean 2～3 minutes in the ultrasound bath, repeat 3 times, make the lower end conductive material outside surface of working electrode clean with the thorough supersound washing of ethanol, diluted acid and distilled water again as minute surface, standby after drying;
In second step, modify working electrode with CNT and form nano-particle layer
It is the concentrated sulphuric acid that the grain size scope is placed volume ratio at the CNT of 8～100nm: the mixed acid solution of red fuming nitric acid (RFNA)=3: 1, ultrasonic concussion was dispersed into even light/dark balance solution in 4 hours, centrifugal with 10000 rev/mins in hydro-extractor, outwell the supernatant acid solution, add the ultrasonic dispersion washing of water 5 minutes again, the repeated multiple times washing is closely neutral to this dark solution, making concentration is the CNT solution of 1g/L, place 4 ℃ of refrigerators to deposit stand-by, the lower end of the electrode that will be handled well by the first step was immersed in this CNT solution 25 minutes, at its conductive material outside surface absorption CNT, then this electrode is placed damping fluid to clean 5 minutes, the CNT that flush away absorption is not firm forms nano-particle layer (4), finishes the operation that CNT is modified working electrode thus;
In the 3rd step, self assembly layer by layer prepares anti-interference layer
The lower end of the electrode that second step was modified with CNT was immersed in the polypropylene amine cation high molecular polymer solution that concentration is 2mg/mL 25 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the poly-sulfonated ethylene anionic polymer polymer solution that concentration is 2mg/mL 25 minutes again, repeat this process until alternately plating 1 floor height Molecularly Imprinted Polymer built up film, finish the preparation of anti-interference layer (5);
In the 4th step, self assembly layer by layer prepares Electrostatic Absorption enzyme layer
A. self assembly layer by layer prepares choline oxidase enzyme layer
The lower end that the 3rd step was finished the electrode of anti-interference layer preparation was immersed in the polypropylene amine cation high molecular polymer solution that the concentration that is mixed with 5% CNT is 2mg/mL 20 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the choline oxidase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 2 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of choline oxidase enzyme layer (6), make the choline biologic enzyme electrode that detects choline thus;
B. self assembly layer by layer prepares acetylcholinesterase enzyme layer
It is the polypropylene amine of 2mg/mL that the lower end of electrode that the 4th step a is finished the preparation of choline oxidase enzyme layer is immersed in the concentration that is mixed with 5% CNT, polyethyleneimine, in at least a cation high molecular polymer solution in the diallyl dimethyl ammoniumchloride 20 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the acetylcholinesterase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 1 floor height Molecularly Imprinted Polymer and enzyme, finish the preparation of acetylcholinesterase enzyme layer (7), also finish the making of responding layer (2) as shown in Figure 2, and finally made the biologic enzyme electrode that detects acetylcholine and residues of pesticides.
The first step, the processing of electrode lower end conductive material outside surface
The outer surface part of the carbon (3) that the electrode lower end that is made by embodiment 2 with the aluminium powder polishing coats is to cleaning, move into then and clean 2～3 minutes in the ultrasound bath, repeat 3 times, make the lower end conductive material outside surface of electrode clean with the thorough supersound washing of ethanol, diluted acid and distilled water again as minute surface, standby after drying;
In second step, form nano-particle layer with the decorated by nano-gold electrode
It is the concentrated sulphuric acid that the grain size scope is placed volume ratio in the nm of gold of 8～100nm: the mixed acid solution of red fuming nitric acid (RFNA)=3: 1, ultrasonic concussion was dispersed into even light/dark balance solution in 4 hours, 10000 rev/mins centrifugal, outwell the supernatant acid solution, add the ultrasonic dispersion washing of water 5 minutes again, the repeated multiple times washing is closely neutral to this dark solution, make the nano-Au solution that concentration is 1g/L, place 4 ℃ of refrigerators to deposit stand-by, the lower end of the electrode that will be handled well by the first step was immersed in this nano-Au solution 25 minutes, in its conductive material outside surface absorption nm of gold, then this electrode is placed damping fluid to clean 5 minutes, the nm of gold that flush away absorption is not firm forms nano-particle layer (4), finishes the operation of decorated by nano-gold electrode thus;
In the 3rd step, self assembly layer by layer prepares anti-interference layer
Second step was immersed in the polyethyleneimine cation high molecular polymer solution that concentration is 2mg/mL 20 minutes with the lower end of the electrode of decorated by nano-gold, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the poly-sulfonated phenylethylene anionic polymer polymer solution that concentration is 2mg/mL 20 minutes again, repeat this process until alternately plating 2 floor height Molecularly Imprinted Polymer built up films, finish the preparation of anti-interference layer (5);
In the 4th step, self assembly layer by layer prepares Electrostatic Absorption enzyme layer
A. self assembly layer by layer prepares choline oxidase enzyme layer
The lower end that the 3rd step was finished the electrode of anti-interference layer preparation was immersed in the polyethyleneimine cation high molecular polymer solution that the concentration that is mixed with 5% nm of gold is 2mg/mL 20 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the choline oxidase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 6 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of choline oxidase enzyme layer (6), make the choline biologic enzyme electrode that detects choline thus;
B. self assembly layer by layer prepares acetylcholinesterase enzyme layer
The lower end of electrode that the 4th step a is finished the preparation of choline oxidase enzyme layer was immersed in the polyethyleneimine cation high molecular polymer solution that the concentration that is mixed with 5% nm of gold is 2mg/mL 20 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the acetylcholinesterase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 2 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of acetylcholinesterase enzyme layer (7), also finish the making of responding layer (2) as shown in Figure 2, and finally made the biologic enzyme electrode that detects acetylcholine and residues of pesticides.
The first step, the processing of electrode lower end conductive material outside surface
The outer surface part of the platinum (3) that the electrode lower end that is made by embodiment 3 with the aluminium powder polishing coats is to cleaning, move into then and clean 2～3 minutes in the ultrasound bath, repeat 3 times, make the lower end conductive material outside surface of electrode clean with the thorough supersound washing of ethanol, diluted acid and distilled water again as minute surface, standby after drying;
In second step, form nano-particle layer with the CNT modified electrode
It is the concentrated sulphuric acid that the grain size scope is placed volume ratio at the CNT of 8～100nm: the mixed acid solution of red fuming nitric acid (RFNA)=3: 1, ultrasonic concussion was dispersed into even light/dark balance solution in 4 hours, 10000 rev/mins centrifugal, outwell the supernatant acid solution, add the ultrasonic dispersion washing of water 5 minutes again, the repeated multiple times washing is closely neutral to this dark solution, making concentration is the CNT solution of 1g/L, place 4 ℃ of refrigerators to deposit stand-by, the lower end of the electrode that will be handled well by the first step was immersed in this CNT solution 25 minutes, at its conductive material outside surface absorption CNT, then this electrode is placed damping fluid to clean 5 minutes, the CNT that flush away absorption is not firm forms nano-particle layer (4), finishes the operation of CNT modified electrode thus;
In the 3rd step, self assembly layer by layer prepares anti-interference layer
The lower end of the electrode that second step was modified with CNT was immersed in the diallyl dimethyl ammoniumchloride cation high molecular polymer solution that concentration is 2mg/mL 20 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again, to be immersed in concentration again be in the anionic polymer polymer solution that mixes with arbitrary proportion of poly-sulfonated ethylene and the poly-sulfonated phenylethylene of 2mg/mL 20 minutes, repeat this process until alternately plating 3 floor height Molecularly Imprinted Polymer built up films, finish the preparation of anti-interference layer (5);
In the 4th step, self assembly layer by layer prepares Electrostatic Absorption enzyme layer
A. self assembly layer by layer prepares choline oxidase enzyme layer
The lower end that the 3rd step was finished the electrode of anti-interference layer preparation was immersed in the diallyl dimethyl ammoniumchloride macromolecule polymer solution that the concentration that is mixed with 5% CNT is 2mg/mL 20 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the choline oxidase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 8 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of choline oxidase enzyme layer (6), make the choline biologic enzyme electrode that detects choline thus;
B. self assembly layer by layer prepares acetylcholinesterase enzyme layer
The lower end of electrode that the 4th step a is finished the preparation of choline oxidase enzyme layer was immersed in the diallyl dimethyl ammoniumchloride cation high molecular polymer solution that the concentration that is mixed with 5% CNT is 2mg/mL 20 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the acetylcholinesterase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 3 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of acetylcholinesterase enzyme layer (7), also finish the making of responding layer (2) as shown in Figure 2, and finally made the biologic enzyme electrode that detects acetylcholine and residues of pesticides.
The first step, the processing of electrode lower end conductive material outside surface
The outer surface part of the silver (3) that the electrode lower end that is made by embodiment 4 with the aluminium powder polishing coats is to cleaning, move into then and clean 2～3 minutes in the ultrasound bath, repeat 3 times, make the lower end conductive material outside surface of electrode clean with the thorough supersound washing of ethanol, diluted acid and distilled water again as minute surface, standby after drying;
In second step, form nano-particle layer with the CNT modified electrode
It is the concentrated sulphuric acid that the grain size scope is placed volume ratio at the CNT of 8～100nm: the mixed acid solution of red fuming nitric acid (RFNA)=3: 1, ultrasonic concussion was dispersed into even light/dark balance solution in 4 hours, 10000 rev/mins centrifugal, outwell the supernatant acid solution, add the ultrasonic dispersion washing of water 5 minutes again, the repeated multiple times washing is closely neutral to this dark solution, making concentration is the CNT solution of 1g/L, place 4 ℃ of refrigerators to deposit stand-by, the lower end of the electrode that will be handled well by the first step was immersed in this CNT solution 25 minutes, at its conductive material outside surface absorption CNT, then this electrode is placed damping fluid to clean 5 minutes, the CNT that flush away absorption is not firm forms nano-particle layer (4), finishes the operation of CNT modified electrode thus;
In the 3rd step, self assembly layer by layer prepares anti-interference layer
With embodiment 7;
In the 4th step, self assembly layer by layer prepares Electrostatic Absorption enzyme layer
A. self assembly layer by layer prepares choline oxidase enzyme layer
It is in the cation high molecular polymer solution that mixes with arbitrary proportion of polypropylene amine and the polyethyleneimine of 2mg/mL 20 minutes that the lower end that the 3rd step was finished the electrode of anti-interference layer preparation is immersed in the concentration that is mixed with 5% CNT, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the choline oxidase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 10 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of choline oxidase enzyme layer (6), make the choline biologic enzyme electrode that detects choline thus;
B. self assembly layer by layer prepares acetylcholinesterase enzyme layer
It is in the cation high molecular polymer solution that mixes with arbitrary proportion of polypropylene amine and the diallyl dimethyl ammoniumchloride of 2mg/mL 20 minutes that the lower end of electrode that the 4th step a is finished the preparation of choline oxidase enzyme layer is immersed in the concentration that is mixed with 5% CNT, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the acetylcholinesterase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 4 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of acetylcholinesterase enzyme layer (7), also finish the making of responding layer (2) as shown in Figure 2, and finally made the biologic enzyme electrode that detects acetylcholine and residues of pesticides.
The first step, the processing of electrode lower end conductive material outside surface
With embodiment 7;
In second step, form nano-particle layer with the CNT modified electrode
With embodiment 7;
In the 3rd step, self assembly layer by layer prepares anti-interference layer
With embodiment 7;
In the 4th step, self assembly layer by layer prepares Electrostatic Absorption enzyme layer
A. self assembly layer by layer prepares choline oxidase enzyme layer
It is the polypropylene amine of 2mg/mL that the 3rd lower end that goes on foot the electrode of finishing anti-interference layer preparation is immersed in the concentration that is mixed with 5% CNT, in the cation high molecular polymer solution that polyethyleneimine and diallyl dimethyl ammoniumchloride are mixed with arbitrary proportion 20 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the choline oxidase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 12 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of choline oxidase enzyme layer (6), make the choline biologic enzyme electrode that detects choline thus;
B. self assembly layer by layer prepares acetylcholinesterase enzyme layer
It is the polypropylene amine of 2mg/mL that the lower end of electrode that the 4th step a is finished the preparation of choline oxidase enzyme layer is immersed in the concentration that is mixed with 5% CNT, in the cation high molecular polymer solution that polyethyleneimine and diallyl dimethyl ammoniumchloride are mixed with arbitrary proportion 20 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the acetylcholinesterase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 5 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of acetylcholinesterase enzyme layer (7), also finish the making of responding layer (2) as shown in Figure 2, and finally made the biologic enzyme electrode that detects acetylcholine and residues of pesticides.
The first step, the processing of electrode lower end conductive material outside surface
With embodiment 7;
In second step, use the CNT modified electrode
With embodiment 7;
In the 3rd step, self assembly layer by layer prepares anti-interference layer
With embodiment 7;
In the 4th step, self assembly layer by layer prepares Electrostatic Absorption enzyme layer
A. self assembly layer by layer prepares choline oxidase enzyme layer
It is in the cation high molecular polymer solution that mixes with arbitrary proportion of polypropylene amine and the diallyl dimethyl ammoniumchloride of 2mg/mL 20 minutes that the lower end that the 3rd step was finished the electrode of anti-interference layer preparation is immersed in the concentration that is mixed with 5% CNT, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the choline oxidase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 15 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of choline oxidase enzyme layer (6), make the choline biologic enzyme electrode that detects choline thus;
B. self assembly layer by layer prepares acetylcholinesterase enzyme layer
It is in the cation high molecular polymer solution that mixes with arbitrary proportion of polypropylene amine and the diallyl dimethyl ammoniumchloride of 2mg/mL 20 minutes that the lower end of electrode that the 4th step a is finished the preparation of choline oxidase enzyme layer is immersed in the concentration that is mixed with 5% CNT, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the acetylcholinesterase solution that concentration is 2mg/mL 20 minutes again, repeat this process until the composite membrane that alternately plates 1 floor height Molecularly Imprinted Polymer and enzyme, finish the preparation of acetylcholinesterase enzyme layer (7), also finish the making of responding layer (2) as shown in Figure 2, and finally made the biologic enzyme electrode that detects acetylcholine and residues of pesticides.
In the foregoing description 5～10, the concentration of used solution is weight/volume percent concentration, and the prescription of used damping fluid is with NaOH 1.874 grams, KH
2PO
46.805 the gram, constant volume in the volumetric flask of 1000mL, pH8.0.
Embodiment 11
The choline biologic enzyme electrode that makes among the 4th step of the foregoing description 7 a is done as working electrode, with the biology sensor that electrode (platinum filament) and contrast electrode (Ag/AgCl) is constituted the three-electrode system that detects choline, its assemble method is well-known to those skilled in the art.Phosphate buffer with 20mL 0.10mol/L is an electrolyte, measures the response current of this biology sensor to choline under+0.6V, and this assay method and process are well-known to those skilled in the art, and measurement result is seen Fig. 3～9.This shows that the choline biologic enzyme electrode that makes with method for making of the present invention is to the detection range of linearity of choline: 5 * 10
-7～1 * 10
-4Mol/L; Sensitivity: 12.53 μ A/mmol; Can be controlled in detection time 7.6 seconds, and detected and be limited to 2 * 10
-7Mol/L.
Embodiment 12
Being used to of making in the foregoing description 7 detected the biologic enzyme electrode of residues of pesticides as working electrode, with the biology sensor that electrode (platinum filament) and contrast electrode (Ag/AgCl) is constituted the three-electrode system that detects residues of pesticides, its assemble method and assay method are with embodiment 11, and measurement result is seen Figure 10～12.This biologic enzyme electrode is to the detection range of linearity of acetylcholine: 5 * 10
-6～8 * 10
-4Mol/L, detection limit 5 * 10
-6Mol/L; The detection range of linearity to metrifonate is: 0.01～0.6 μ g/mL detects and is limited to 1 * 10
-10Mol/L, the biologic enzyme electrode of the detection residues of pesticides that make with method for making of the present invention can be realized the quick online detection to metrifonate, can be controlled in detection time 8 minutes.
Claims (4)
1. be used to detect the method for making of the biologic enzyme electrode of residues of pesticides, it is characterized in that: adopt the method that nano particle and layer-by-layer are combined, concrete steps are as follows:
Make the stick electrode carrier with organic insulation, on the xsect of its lower end, coat the matrix conductive material, form electrode, perhaps directly buy such electrode, make the responding layer that constitutes by nano-particle layer, anti-interference layer and Electrostatic Absorption enzyme layer of operation as described below at the outside surface of this conductive material of electrodes again, be made into the biologic enzyme electrode that is used to detect residues of pesticides thus
The first step, the processing of electrode lower end conductive material outside surface
Polish above-mentioned electrode lower end conductive material outer surface part to cleaning with aluminium powder, move into then and clean 2～3 minutes in the ultrasound bath, repeat 3 times, make this electrode lower end conductive material outside surface clean with the thorough supersound washing of ethanol, watery hydrochloric acid and distilled water again as minute surface, standby after drying;
In second step, form nano-particle layer with nano-particle modified electrode
It is the concentrated sulphuric acid that nano particle is placed volume ratio: the mixed acid solution of red fuming nitric acid (RFNA)=3: 1, ultrasonic concussion 4 hours, be dispersed into even light/dark balance solution, centrifugal with 10000 rev/mins in hydro-extractor, outwell the supernatant acid solution, add the ultrasonic dispersion washing of water 5 minutes again, the repeated multiple times washing is closely neutral to this dark solution, make the nanoparticles solution that concentration is 1g/L, place 4 ℃ of refrigerators to deposit stand-by, to immerse in this nanoparticles solution 25 minutes by the electrode lower end that the first step is handled well, at its conductive material outside surface absorption nano particle, form nano-particle layer, then this electrode is placed damping fluid to clean 5 minutes, flush away adsorbs not firm nano particle, finishes the operation of nano-particle modified electrode thus;
In the 3rd step, self assembly layer by layer prepares anti-interference layer
Second step was immersed in the cation high molecular polymer solution at least a in polypropylene amine that concentration is 2mg/mL, polyethyleneimine, the diallyl dimethyl ammoniumchloride 25 minutes with the lower end of nano-particle modified electrode, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the anionic polymer polymer solution at least a in poly-sulfonated ethylene that concentration is 2mg/mL, the poly-sulfonated phenylethylene 25 minutes again, repeat this process until alternately plating 1～3 floor height Molecularly Imprinted Polymer built up film, finish the preparation of anti-interference layer;
In the 4th step, self assembly layer by layer prepares Electrostatic Absorption enzyme layer
A. self assembly layer by layer prepares choline oxidase enzyme layer
It is the polypropylene amine of 2mg/mL that the 3rd lower end that goes on foot the electrode of finishing anti-interference layer preparation is immersed in the concentration that is mixed with 5% nano particle, polyethyleneimine, in at least a cation high molecular polymer solution in the diallyl dimethyl ammoniumchloride 25 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the choline oxidase solution that concentration is 2mg/mL 25 minutes again, repeat this process until the composite membrane that alternately plates 2～15 floor height Molecularly Imprinted Polymers and enzyme, finish the preparation of choline oxidase enzyme layer, make the choline biologic enzyme electrode that detects choline thus;
B. self assembly layer by layer prepares acetylcholinesterase enzyme layer
It is the polypropylene amine of 2mg/mL that the lower end that the 4th step a is finished the electrode of choline oxidase enzyme layer preparation is immersed in the concentration that is mixed with 5% nano particle, polyethyleneimine, in at least a cation high molecular polymer solution in the diallyl dimethyl ammoniumchloride 25 minutes, taking out this electrode immersed in the damping fluid 5 minutes again, taking out this electrode again was immersed in the acetylcholinesterase solution that concentration is 2mg/mL 25 minutes again, repeat this process until the composite membrane that alternately plates 1～5 floor height Molecularly Imprinted Polymer and enzyme, finish the preparation of acetylcholinesterase enzyme layer, also finish the making of responding layer, and finally made the biologic enzyme electrode that is used to detect acetylcholine and residues of pesticides;
The above-mentioned method for making that is used for detecting the biologic enzyme electrode of residues of pesticides, the concentration of used solution is weight/volume percent concentration, and the compound method of used damping fluid is with NaOH 1.874 grams, KH
2PO
46.805 the gram, constant volume in the volumetric flask of 1000mL, pH8.0.
2. according to the method for making of the biologic enzyme electrode of the described detection residues of pesticides of claim 1, it is characterized in that: described matrix conductive material as electrode is gold, carbon, platinum or silver.
3. according to the method for making of the biologic enzyme electrode of the described detection residues of pesticides of claim 1, it is characterized in that: the described stick electrode carrier made from organic insulation, its bar-shaped xsect are square, rectangle, circle or oval.
4. according to the method for making of the biologic enzyme electrode of the described detection residues of pesticides of claim 1, it is characterized in that: described nano particle is CNT or nm of gold, and its grain size scope is at 8～100nm.
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| CN101339156B (en) * | 2008-08-19 | 2011-12-07 | 西北农林科技大学 | Pesticide residue biosensor thin layer electrode and its making process |
| CN102221529A (en) * | 2011-03-31 | 2011-10-19 | 吉林大学 | Method for rapidly detecting residuals of organophosphorus pesticides in vegetables by utilizing Au nano-particle colorimetric method |
| CN103175872A (en) * | 2011-12-23 | 2013-06-26 | 长沙中生众捷生物技术有限公司 | Portable electrochemical detection test strip and preparation method thereof |
| CN102672197B (en) * | 2012-05-29 | 2013-09-18 | 江南大学 | Method for preparing multipurpose isoionic gold nanoparticle film |
| CN103018305A (en) * | 2012-12-11 | 2013-04-03 | 江苏大学 | Portable enzyme sensor based on carbon nanometer tube |
| CN103592337B (en) * | 2013-11-19 | 2015-09-09 | 山东理工大学 | A kind of pesticide multi-residues detection method based on array-type sensor |
| CN107505373A (en) * | 2017-10-11 | 2017-12-22 | 浙江海洋大学 | A kind of biological sensor electrode for being used to detect a variety of residues of veterinary drug |
| CN107764880A (en) * | 2017-10-11 | 2018-03-06 | 浙江海洋大学 | A kind of biosensor thin layer electrode of quick detection miocardial infarction protein marker |
| CN111089889A (en) * | 2019-12-25 | 2020-05-01 | 北京农业质量标准与检测技术研究中心 | Silk-screen printing carbon nanotube sensor, preparation method thereof and pesticide detection application |
| CN111413381B (en) * | 2020-03-26 | 2022-03-11 | 上海大学 | Biosensor for detecting organophosphorus pesticide and preparation method thereof |
| CN114113247B (en) * | 2020-08-31 | 2024-05-14 | 浙江纳智汇生物科技有限公司 | Electrochemical detection method, detection chip, detection kit and detection system based on enzyme inhibition method |
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| CN2723998Y (en) * | 2004-08-23 | 2005-09-07 | 南开大学 | Enzyme electrode for residual pesticide test |
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