CN104931554B - A kind of transducer production method and application based on screen printing carbon electrode - Google Patents
A kind of transducer production method and application based on screen printing carbon electrode Download PDFInfo
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Abstract
The invention discloses a kind of transducer production method based on screen printing carbon electrode and applications, belong to biosensor technology field.Method provided by the present invention is to prepare dicyclohexyl phthalate/multi-walled carbon nanotube nano material, drop coating is layered on screen printing carbon electrode surface, then by the screen printing work electrode surface of acetylcholinesterase drop coating to dicyclohexyl phthalate/multi-walled carbon nanotube modification, the acetylcholinesterasebiosensor biosensor for detecting Organophosphorus and carbamate pesticides class pesticide is prepared.Sensor prepared by the present invention has the characteristics that process is simple and convenient to operate, is of low cost, detection sensitivity is high, electric signal is strong and electrode pretreatment is simple, and detection limit reaches 0.05 μ g/L, suitable for detecting pesticide residue.
Description
Technical field
The present invention relates to a kind of transducer production method based on screen printing carbon electrode and applications, belong to biosensor
Technical field.
Background technology
Pesticide is important capital goods in the agricultural sector, and the prevention endangered in agricultural product production disease, worm, grass, mouse is played non-
Normal important role.But pollution by pesticides and its hazard analysis and HACCP of generation are serious, loss caused by pesticide environmental pollution
Many, including the pollution to water environment, the pollution to soil, the pollution to air, influence to environmental organism and
Harm to health etc..At present due to the use of person general lack of scientifical use pesticide knowldgde, covet yield, causes mesh
For the preceding agricultural products to circulate in the market all to some extent there are Pesticide Residue, harm also increasingly causes the pass of the public
Note.China is a large agricultural country, and as our people's living standard is continuously improved, the quality security problem of agricultural product is increasingly
It attracts attention, especially Pesticide Residues in Vegetables problem has become the focus of public attention.The insecticide that China produces and uses
Most kinds are Organophosphorus and carbamate pesticides class pesticide (accounting for 70%), wherein highly toxic organophosphor and carbamic acid
Ester pesticide is 70% or so, therefore easily causes food pesticide residual poisoning.As it can be seen that strengthen to Residual Pesticides in Farm Produce
Detection to preserving the ecological environment, especially ensure that human health has a very profound significance, and the weight of Detecting Pesticide
Point should be placed on Organophosphorus and carbamate pesticides class pesticide.
The main method of pesticide residue analysis is gas chromatograph, liquid chromatograph, gas chromatograph-mass spectrometer etc. at present, these sides
Quantitative accurate although method analysis precision is high, the complex pretreatment of its sample, time-consuming for detection, of high cost, needs are skilled in technique
Operating personnel.The fast determining method of China's pesticide residue is that enzyme inhibits test paper method and enzyme inhibits spectrophotometry (the residual quick inspection of agriculture
Survey instrument), it can realize the field quick detection of organophosphorus pesticide and carbamate chemicals for agriculture, there is preferable practical value.
Quick measuring card is the color change for observing by the naked eye card, therefore the vegetable sample progress for being generally only used for severely exceeding is qualitative
Measurement.Enzyme inhibits the application of spectrophotometry also than wide, and it is former that the existing Multiple Pesticides residual tacheometer in the country is all based on this
Reason.The principle for being divided light method is that the variation based on absorbance is detected, but substantial amounts of pigment can be to light splitting in vegetable and fruit
Photometry causes very big influence, causes the inaccuracy of testing result.And the above method there are the rate of recovery is low, false retrieval, missing inspection
Ratio is higher, poor repeatability, is difficult to the shortcomings of requirement for meeting low-residual and quantitatively detecting.
The content of the invention
It is an object of the invention to overcome above-mentioned the shortcomings of the prior art, a kind of simple in structure, operation side is provided
Just screen printing electrode biosensor preparation method, at low cost and high detection sensitivity, the technical solution of use are as follows：
A kind of transducer production method and application based on screen printing carbon electrode, it is characterised in that：It will prepare
Dicyclohexyl phthalate/multi-walled carbon nanotube is layered drop coating to the screen printing carbon electrode surface activated through over cleaning, obtains
Modified electrode, then the surface by acetylcholinesterase drop coating to modified electrode are obtained, is obtained after dry cleaning based on phthalic acid
The acetylcholinesterasebiosensor biosensor of the screen printing carbon electrode of dicyclohexyl maleate-multi-walled carbon nanotube modification.
The method step is：
1) preparation of dicyclohexyl phthalate/multi-walled carbon nanotube；
2) clean, activate screen printing carbon electrode, obtain pretreatment screen printing carbon electrode；
3) dicyclohexyl phthalate for preparing step 1)/multi-walled carbon nanotube solution is layered drop coating in step 2)
It pre-processes on screen printing carbon electrode surface, obtains modified graphite electrode；
4) on the working electrode surface of modified electrode, will be obtained obtained by acetylcholinesterase drop coating to step 3) after dry cleaning
Obtain the acetylcholinesterasebiosensor biosensor based on screen printing carbon electrode.
Step 2) the screen printing carbon electrode, the substrate including printed electrode, the external insulation being printed on substrate
At least two contact conductors, substrate printing are respectively a working electrode, one to electrode and a reference there are three electrode
Electrode, each electrode pair should be connected with a contact conductor.
Step 2) the cleaning, activation screen printing carbon electrode, process are：First, screen printing carbon electrode is placed in 1mM
Ultrasound 5min, ultrapure water, N in NaOH solution2Secondly electrode, is placed in ultrasound 5min in 1mM HCl solutions by drying, surpass
Pure water rinsing, N2Drying again, electrode, N is rinsed with absolute ethyl alcohol2Drying, finally, is scanned in pH5.0 phosphate buffers
Current versus time curve 300s, scan cycle volt-ampere curve is until performance is stablized afterwards.
Step 3) is described molten in pretreatment electrode surface layering drop coating dicyclohexyl phthalate and multi-walled carbon nanotube
Liquid is first by the dicyclohexyl phthalate drop coating of 5~10 μ L on pretreatment screen printing carbon electrode, does in air
It is dry, with ultrapure water surface, the dicyclohexyl phthalate for being not fixed to electrode surface is rinsed out, obtains O-phthalic
The screen printing carbon electrode of sour dicyclohexyl maleate modification；Afterwards, by 5~10 μ L multi-walled carbon nanotube solution drop coatings in obtained adjacent benzene
Working electrode surface in the screen printing carbon electrode of dioctyl phthalate dicyclohexyl maleate modification, is dried, in air with ultrapure water table
Face rinses out the multi-walled carbon nanotube solution for being not fixed to electrode surface, obtains dicyclohexyl phthalate-multi wall carbon and receives
The screen printing carbon electrode of mitron modification.
Step 4) the drop coating acetylcholinesterase on the working electrode (s is the 0.02U/ μ L acetyl courages of 5~10 μ L of drop coating
Alkali esterase solution, and the dry 2h at 4 DEG C, the enzyme on unadsorbed is washed off with the phosphate buffer solution of pH7.5, is obtained afterwards
To acetylcholinesterasebiosensor biosensor.
The method is as follows：
1) 0.1g chitosans are added to 50mL concentration in 1.0% acetic acid solution, to be made into the chitosan that concentration is 0.2%
Solution, more than magnetic agitation 8h are completely dissolved chitosan.2mg dicyclohexyl phthalates are scattered in 4mL concentration is
In 0.2% chitosan solution, and in 65 DEG C of water-baths ultrasonic disperse to obtain stable dispersion liquid；The high degree of dispersion obtained
Suspension is 0.5mg/mL dicyclohexyl phthalate suspension；
2) 2mg multi-walled carbon nanotubes are scattered in the chitosan solution of 4mL 0.2%, and ultrasonic disperse 6h at room temperature
To obtain stable black dispersion liquid；The high degree of dispersion black suspension obtained is gathered for 0.5mg/mL multi-walled carbon nanotubes-shell
Sugared nano-complex stores the multi-walled carbon nanotube solution prepared at 4 DEG C；
3) screen printing carbon electrode is placed in ultrasound 5min, ultrapure water, N in 1mM NaOH solutions2Drying, by electrode
It is placed in ultrasound 5min, ultrapure water, N in 1mM HCl solutions2Drying rinses electrode, N with absolute ethyl alcohol2Drying, in pH5.0
Sweep current-time graph 300s in phosphate buffer, scan cycle volt-ampere curve obtain pretreatment silk until performance stabilization
Wire mark brush carbon electrode；
4) first dicyclohexyl phthalate hanging drop prepared by the step 1) of 5~10 μ L is coated in obtained by step 3)
It pre-processes on screen printing carbon electrode, dries in air, with ultrapure water surface, the adjacent benzene of electrode surface will be not fixed to
Dioctyl phthalate dicyclohexyl maleate suspension rinses out, and obtains the screen printing carbon electrode of dicyclohexyl phthalate suspension modification；
Afterwards, multi-walled carbon nanotube solution drop coating prepared by 5~10 μ L steps 2) is suspended in obtained dicyclohexyl phthalate
Working electrode surface in the screen printing carbon electrode of liquid modification, dries, with ultrapure water surface, will be not fixed in air
The multi-walled carbon nanotube solution of electrode surface rinses out, and obtains the silk of dicyclohexyl phthalate-multi-walled carbon nanotube modification
Wire mark brush carbon electrode；
5) working electrode of modified electrode obtained by the 0.02U/ μ L acetylcholine esters enzyme solutions of 5~10 μ L of drop coating to step 4)
On surface, and it is dry at 4 DEG C, the enzyme on unadsorbed is washed off with the phosphate buffer solution of pH7.5, obtains acetylcholine
Esterase biological sensor saves backup under 4 DEG C of dry environments.
The method of the invention, which is characterized in that for detecting Organophosphorus and carbamate pesticides class pesticide.
The method, it is characterised in that：The preparation of dicyclohexyl phthalate/multi-walled carbon nanotube, silk-screen printing carbon
The cleaning of electrode, structure and the process characterization at acetylcholinesterase sensor sensing interface, acetylcholinesterase working sensor
The foundation of curve, the detection of acetylcholinesterase sensor performance, detection of the acetylcholine ester enzyme sensor to actual sample；Second
The detection of acetylcholinesterase sensor performance includes accuracy, stability and acetylcholine ester enzyme sensor to sample recovery rate
It measures.
The preparation method, it is characterised in that：Prepared current mode acetylcholine ester enzyme sensor detection chlopyrifos
Concentration range is 0.05~105μ g/L, detection are limited to 0.05 μ g/L.
The method preparation principle is：Since organophosphorus pesticide and acetylcholinesterase have higher specific binding,
Therefore molecular recognition elements of the common acetylcholinesterase as detection organophosphorus pesticide, acetylcholinesterasebiosensor biosensor are
Acetylcholinesterase is fixed on electrode surface, substrate for enzymatic activity acetylcholine hydrolyzation generation choline and acetic acid.Pesticide is in structure
Upper similar with substrate acetyl choline, it can effectively be combined with the activated centre of acetylcholine, the work of acetylcholine esterase inhibition
Property.Choline is a kind of electroactive material, oxidation reaction can occur under certain potentials, pass through thiocholine during voltammetric scan
The size of oxidation peak can measure the concentration of pesticide residue, and enzymatic reaction current signal changes when whetheing there is pesticide by comparing
To pesticide to the inhibiting rate of enzyme, the concentration of the inhibiting rate and pesticide can measure organophosphor or carbamate into correspondence
The content of class pesticide.
To achieve the above objectives, following technical scheme is taken to realize：One kind is based on dicyclohexyl phthalate-multi wall carbon
The preparation method of the sensor of nanometer tube modified screen printing carbon electrode, it is characterised in that：
(1) cleaning of screen printing electrode before prepared by current mode acetylcholine ester enzyme sensor.Screen printing electrode is put
Ultrasound 5min, ultrapure water, N in 1mM NaOH solutions2Drying.Afterwards, electrode is placed in 1mM HCl solutions ultrasonic
5min, ultrapure water, N2Drying.Finally, electrode, N are rinsed with absolute ethyl alcohol2Drying；
(2) activation of electrode before prepared by current mode acetylcholine ester enzyme sensor.It is scanned in pH5.0 phosphate buffers
Current versus time curve 300s, scan cycle volt-ampere curve is until performance is stablized afterwards；
(3) preparation of dicyclohexyl phthalate/multi-walled carbon nanotube.0.1g chitosans are added to 50mL concentration is
In 1.0% acetic acid solution, the chitosan solution that concentration is 0.2% is made into, more than magnetic agitation 8h is completely dissolved chitosan；It will
2mg dicyclohexyl phthalates are scattered in the chitosan solution that 4mL concentration is 0.2%, and the ultrasound point in 65 DEG C of water-baths
It dissipates to obtain stable dispersion liquid；The high degree of dispersion suspension obtained suspends for 0.5mg/mL dicyclohexyl phthalates
Liquid；2mg multi-walled carbon nanotubes are scattered in the chitosan solution of 4mL 0.2%, and at room temperature ultrasonic disperse 6h to obtain
Stable black dispersion liquid；The high degree of dispersion black suspension obtained is 0.5mg/mL multi-walled carbon nanotubes-chitosan nano
Compound stores the multi-walled carbon nanotube solution prepared at 4 DEG C；
(4) it is first in pretreatment electrode surface layering drop coating dicyclohexyl phthalate and multi-walled carbon nanotube solution
The dicyclohexyl phthalate hanging drop of 5~10 μ L is coated on pretreatment screen printing carbon electrode, is dried in air,
With ultrapure water surface, the dicyclohexyl phthalate suspension for being not fixed to electrode surface is rinsed out, obtains adjacent benzene
The screen printing carbon electrode of dioctyl phthalate dicyclohexyl maleate modification；Afterwards, 5~10 μ L multi-walled carbon nanotube solution drop coatings are being obtained
Working electrode surface in the screen printing carbon electrode of dicyclohexyl phthalate modification, dries, is rushed with ultra-pure water in air
Surface is washed, the multi-walled carbon nanotube solution for being not fixed to electrode surface is rinsed out, obtains dicyclohexyl phthalate-multi wall
Carbon nano tube modified screen printing carbon electrode.Then 5~10 μ L acetylcholine ester enzyme solutions of drop coating, and it is dry at 4 DEG C；With
PH is that 7.5 phosphate buffer solution washes off the enzyme on unadsorbed, thus obtains AChE/DCHP-MWCNTs/SPE biographies
Sensor saves backup under 4 DEG C of dry environments.
To achieve the above objectives, following technical scheme is taken to realize：One kind is based on dicyclohexyl phthalate-multi wall carbon
The preparation method of the sensor of nanometer tube modified screen printing carbon electrode, it is characterised in that：(1) a series of chlopyrifos marks are configured
Quasi- liquid carries out cyclic voltammetry scanning, and be inhibited rate, further obtains the current mode acetylcholinesterase biology of above-mentioned preparation
Working curve, detection range and the detection limit of sensor；
(2) accuracy and stability of acetylcholinesterasebiosensor biosensor are evaluated；(3) to actual fruits and vegetables sample
Carry out the rate of recovery and reappearance that analysis draws the sensor.
The preparation process of the acetylcholinesterasebiosensor biosensor is as follows：By the dicyclohexyl of 5~10 μ L
Ester drop coating is dried on pretreatment screen printing carbon electrode, with ultrapure water surface, will be not fixed to electrode table in air
The dicyclohexyl phthalate liquid wash in face is fallen, and obtains the screen printing carbon electrode of dicyclohexyl phthalate modification；
Afterwards, the silk-screen printing carbon 5~10 μ L multi-walled carbon nanotube solution drop coatings modified in obtained dicyclohexyl phthalate
Working electrode surface in electrode, is dried in air, and with ultrapure water surface, the multi wall carbon for being not fixed to electrode surface is received
Mitron solution rinses out, and obtains the screen printing carbon electrode of dicyclohexyl phthalate-multi-walled carbon nanotube modification.Then drip
5~10 μ L acetylcholine ester enzyme solutions are applied, and it is dry at 4 DEG C；It is washed off with the phosphate buffer solution that pH is 7.5 and not inhaled
The enzyme enclosed thus obtains AChE/DCHP-MWCNTs/SPE sensors, is saved backup under 4 DEG C of dry environments.
Advantageous effect of the present invention：The present invention only can be in its two ring of surface modification phthalic acid with simple drop coating technology
Own ester (DCHP)-multi-walled carbon nanotube (MWCNTs) nano material, the acetylcholinesterase biology overcome in previous research pass
The various unfavorable factors that sensor LBL self-assembly is brought.Screen printing carbon electrode used in the present invention can adjust it according to actual demand
Specification, have the advantages that flexible design, it is at low cost and can batch making, well solved front electrode pretreatment cumbersome journey
Sequence, and there is good homogeneity with batch electrode, it can produce or even realize commercialization in batches, application is more and more extensive.It is adjacent
Dicyclo-hexyl phthlate is excellent powder plasticizer.Multi-walled carbon nanotube is a kind of perfect carbon nanomaterial, has spy
Different hot property and mechanical performance, and with high-specific surface area and excellent electric conductivity.Chitosan have excellent functionality and
Compatibility can be passed as the material of fixed macro-molecular protein for preparing the electrochemica biological of stability and high sensitivity
Sensor.The present invention repaiies screen printing carbon electrode using dicyclohexyl phthalate-multi-walled carbon nanotube nano material
Decorations, can effectively increase the current-responsive of sensor, and higher specific surface area provides good base for the fixation of next step material
Plinth, makes the enzyme sensor of preparation there is relatively low detection to limit.
Acetylcholine ester enzyme sensor made of the method according to the present invention overcomes multiple to Detecting Pesticide operation
It is miscellaneous and the problem of spend the time long, provide that a kind of preparation section is simple, and easy to operate, of low cost, electric signal is strong, sensitivity
Height, high specificity, detection limit is low, and scope is wide, and precision is high, can qualitative and quantitative analysis pesticide residue concentration suitable for Site Detection
Disposable biosensor；It can be in vegetable using current mode acetylcholinesterasebiosensor biosensor made of the present invention
Fruit harvesting, listing before, carry out the quick measure of pesticide residue, directly to pesticide residue, whether superscale is detected, avoid because
Food causes poisoning containing the vegetables and fruits of remains of pesticide, provides the technical support of residue detection with consumption for crop production safety,
Very important meaning is respectively provided in food security and field of environment protection.
Description of the drawings
Fig. 1 SEM phenograms：A. empty screen printing electrode；b.DCHP/SPE；c.DCHP-MWCNTs/SPE
With scanning electron microscope (SEM) to empty screen printing electrode surface and modification in the O-phthalic of electrode surface
The micro-structure diagram of sour dicyclohexyl maleate, dicyclohexyl phthalate-multi-walled carbon nanotube compound is characterized, such as Fig. 1 institutes
Show.It is empty screen printing electrode surface to scheme a, and there are graininess and laminated structure in surface, and figure b is electrode face finish phthalic acid
Dicyclohexyl maleate is schemed in c it will be clear that the substance of many thin tube-likes is evenly distributed in electrode surface, and preparation multi wall carbon is received
Mitron-chitosan nano compound is successfully modified in electrode surface.
The cyclic voltammetric characterization of Fig. 2A ChE/DCHP-MWCNTs/SPE sensor assembling process：A. empty screen printing electrode；
b.AChE/DCHP-MWCNTs/SPE；c.DCHP/SPE；d.MWCNTs/SPE；e.DCHP-MWCNTs/SPE
This experiment is including 5.0mmol/L [Fe (CN)6]3-/4-With the 0.1mol/L pH7.4 phosphate of 0.1mol/L KCl
Cyclic voltammetric characterization is carried out in buffer solution；As shown in Fig. 2, empty screen printing electrode shows apparent symmetrical redox peaks
(curve a)；After dicyclohexyl phthalate is attached on electrode, peak point current significantly increases (curve c)；Curve c is more bent
The peak current of line a increases about 20 μ A, this be primarily due to dicyclohexyl phthalate frequently as adhesive use, have compared with
Good adhesiveness；After multi-walled carbon nanotube is attached on electrode, peak current increase (curve d)；Peak electricity of the curve d compared with curve a
Stream increases about 28 μ A, this is because the chemical property that multi-walled carbon nanotube is excellent；When multi-walled carbon nanotube drop coating to surface
The screen printing electrode surface of dicyclohexyl phthalate is modified, peak current reaches maximum (curve e)；Curve e is compared with curve a
About 50 μ A are increased, and current potential slightly reduces, it can be seen that, dicyclohexyl phthalate has very with multi-walled carbon nanotube
Multi-walled carbon nanotube is preferably bonded in electrode surface, increases electrode by good synergistic effect, dicyclohexyl phthalate
Surface specific surface area shows excellent electrochemical stability.Acetylcholinesterase in the drop coating of DCHP-MWCNTs/SPE surfaces
Afterwards, electric signal be substantially reduced (curve b) because acetylcholinesterase be protein molecule, shield the electroactive of electrode surface,
Electron transmission is hindered, thus there is the trend reduced in peak current.
Influences of Fig. 3 pH to curent change
The pH value of test bottom liquid is one of key factor for influencing sensor performance.The optimum pH of AChE is 7.0 to 8.0
Between.AChE/DCHP-MWCNTs/SPE electrodes are contained into 1.0mmol/L chlorinated thio acetyl courages in different pH value (6 to 8)
The test of current-responsive is carried out in the phosphate buffer of alkali (ATCl)；As shown in figure 3, response current becomes with the variation of pH
Change.Between 6.0~7.5, response current increases pH value with the increase of pH；PH value between 7.5~8.0, response current with
The increase of pH and reduce.So it for 7.5 is optimum determining pH value to select pH value.
Influence of Fig. 4 enzymes load capacity to enzyme sensor curent change
Acetylcholinesterase is in one of factor that the fixed amount of electrode surface is also influence sensor performance.As shown in figure 4,
With the increase of enzyme load capacity, response current first increases and then decreases.It is that 0.25U is to reach maximum in enzyme load capacity.Enzyme loads
When amount is less than 0.25U, response current increases with the increase of acetylcholinesterase load capacity；But when enzyme load capacity is more than
During 0.25U, with the increase of acetylcholinesterase load capacity, response current tends towards stability, and has the trend being slightly reduced, this is
Since limited electrode area causes substantial amounts of enzyme to be accumulated in electrode surface, the transmission of electronics is affected.Therefore, we select
Therefore 0.25U, is fixed in test the amount of the acetylcholinesterase of electrode surface as optimal acetylcholinesterase fixed amount
For 0.25U.
Fig. 5 acetyl chloride thiocholine concentration is to the shadow of enzyme sensor curent change
It rings：a.0.5mM；b.1mM；c.2mM；d.5mM；e.8mM；f.10mM
The AChE/DCHP-MWCNTs/SPE sensors prepared are immersed to a series of chlorinated thio acetyl of various concentrations
Cyclic voltammetry scan is carried out in choline (ATCl) solution, as shown in figure 5, with the increase of concentration of substrate, peak current accordingly increases.
Illustrate the chlorinated thio acetylcholine (ATCl) in the fixed rapid binding soln of AChE energy in interface and be catalyzed its hydrolysis.
Influence of Fig. 6 sweep speeds to enzyme sensor curent change:a.5mV；b.10mV；c.20mV；d.40mV；
e.80mV；f.100mV；g.150mV；h.200mV
We with cyclic voltammetry further study sweep speed to AChE/DCHP-MWCNTs/SPE containing
The influence of electrochemical behavior in the phosphate buffer of 1.0mmol/L pH7.5 chlorinated thios acetylcholines (ATCl), such as Fig. 6 institutes
Show：In the range of 5~200mV/s, with the increase for sweeping speed, peak current gradually increases, the variation of sweep speed and curent change
Positive correlation shows that electrode Scanning rate variation is surface-controlled process.
Influence of Fig. 7 pesticide concentrations to enzyme sensor curent change:a.1×105μg/L；b.1×103μg/L；c.5×102
μg/L；d.2×102μg/L；e.1×102μg/L；f.10μg/L；g.5μg/L；h.1μg/L；i.5μg/L；j.0.05μg/L
The chlopyrifos concentration for being fixed on electrode surface has a significant impact to the performance of enzyme sensor.
Fig. 7 shows the curent change of electrode face finish various concentration chlopyrifos.With the increase of chlopyrifos concentration, electricity
Rheology rate gradually increases.
Influence of Fig. 8 pesticide concentrations to enzyme sensor curent change
As shown in figure 8, the chlopyrifos of various concentration has acetylcholinesterase different inhibiting rates.In optimal conditions,
Chlopyrifos is 0.05~1 × 105Inhibiting rate in μ g/L concentration ranges with pesticide has preferable linear relationship, and linear equation is：y
=4.1765x+4.9171, related coefficient 0.9918.
Fig. 9 AChE/IL-MWCNTs/SPE sensor actual samples recovery of standard addition detects
Fresh vegetables is bought from local supermarket, rotten blade is removed, and cleans up.After vegetables are dried, it is cut into 2 ×
The vanelets of 2mm weigh 2g blades in small beaker, spray 10 μ g/L chlopyrifos in vegetable surface, place 3h at room temperature.Then
1mL acetone and the phosphate buffer of 9mL 0.1mol/L pH7.5 are added in, by suspension ultrasound 15min after centrifuge transfer
Speed centrifuges 10min for 1000rpm, and supernatant is taken directly to measure, need not extract and be enriched with.The concentration of pesticide is according to school in sample
Positive curve calculates；
Using the screen printing electrode second based on dicyclohexyl phthalate-multi-walled carbon nanotube nano-complex modification
Acetylcholinesterase sensor carries out actual sample (cabbage, romaine lettuce, leek, pakchoi) detection of recovery of standard addition, such as Fig. 9
Shown, for the rate of recovery between 98.9%~105.8%, relative standard deviation is 3.56%~4.12%, shows that the enzyme senses
The actual sample detection result of device is good.
Specific embodiment
With reference to specific embodiment, the present invention will be further described, but the present invention should not be limited by the examples.
Embodiment 1：A kind of transducer production method based on screen printing carbon electrode
(1) cleaning of screen printing carbon electrode before prepared by current mode acetylcholine ester enzyme sensor, by silk-screen printing carbon electricity
Pole is placed in ultrasound 5min, ultrapure water, N in 1mM NaOH solutions2Drying.Afterwards, electrode is placed in 1mM HCl solutions and surpassed
Sound 5min, ultrapure water, N2Drying.Finally, electrode, N are rinsed with absolute ethyl alcohol2Drying；
(2) activation of electrode, is scanned in pH5.0 phosphate buffers before prepared by current mode acetylcholine ester enzyme sensor
Current versus time curve 300s, scan cycle volt-ampere curve is until performance is stablized afterwards；
(3) preparation of dicyclohexyl phthalate/multi-walled carbon nanotube, 0.1g chitosans are added to 50mL concentration is
In 1.0% acetic acid solution, the chitosan solution that concentration is 0.2% is made into, more than magnetic agitation 8h is completely dissolved chitosan.It will
2mg dicyclohexyl phthalates are scattered in the chitosan solution that 4mL concentration is 0.2%, and the ultrasound point in 65 DEG C of water-baths
It dissipates to obtain stable dispersion liquid；The high degree of dispersion suspension obtained suspends for 0.5mg/mL dicyclohexyl phthalates
Liquid；2mg multi-walled carbon nanotubes are scattered in the chitosan solution of 4mL 0.2%, and at room temperature ultrasonic disperse 6h to obtain
Stable black dispersion liquid.The high degree of dispersion black suspension obtained is 0.5mg/mL multi-walled carbon nanotubes-chitosan nano
Compound stores the multi-walled carbon nanotube solution prepared at 4 DEG C；
(4) by the dicyclohexyl phthalate drop coating of 5~10 μ L on pretreatment screen printing carbon electrode, in air
It is dry, with ultrapure water surface, the dicyclohexyl phthalate for being not fixed to electrode surface is rinsed out, obtains adjacent benzene two
The screen printing carbon electrode of formic acid dicyclohexyl maleate modification；Afterwards, by 5~10 μ L multi-walled carbon nanotube solution drop coatings obtain from
Working electrode surface in the screen printing carbon electrode of sub- liquid modification, dries, with ultrapure water surface, will not consolidate in air
The multi-walled carbon nanotube solution for being scheduled on electrode surface rinses out, and obtains dicyclohexyl phthalate-multi-walled carbon nanotube modification
Screen printing carbon electrode；Then 5~10 μ L acetylcholine ester enzyme solutions of drop coating, and it is dry at 4 DEG C.With the phosphorus that pH is 7.5
Hydrochlorate buffer solution washes off the enzyme on unadsorbed, thus obtains AChE/DCHP-MWCNTs/SPE sensors, dry at 4 DEG C
It is saved backup under dry environment.
Embodiment 2：Vegetable sample is detected using prepared current mode acetylcholine ester enzyme sensor
(1) by the acetylcholine ester enzyme sensor prepared according to embodiment 1 in the chlorinated thio acetyl courage containing 1.0mM
Cyclic voltammetry is carried out with 50mV/s sweep speeds in the phosphate buffer solution of the pH7.5 of alkali (ATCl), potential window is
0V~1.0V；
(2) chlopyrifos standard solution is configured, when pesticide measures, above-mentioned acetylcholine ester enzyme sensor is immersed in different dense
Then 10min in the pesticide standard solution of degree adds in 15~20mL in reaction tank and contains 1.0mM chlorinated thio acetylcholines
(ATCl) phosphate buffer solution, carries out cyclic voltammetry scan, and inhibiting rate I can be acquired by following formula：
I (%)=(IP,control-IP,exp)/IP,control× 100%
Wherein IP,controlAnd IP,expRespectively modified electrode inhibits without pesticide and after pesticide inhibits, in chlorination
Peak current in acetylthiocholine solution, pesticide concentration, in certain linear relationship, are made working curve diagram, are obtained with inhibiting rate
To the linear relationship between pesticide concentration and inhibiting rate and detection limit；
(3) accuracy of acetylcholinesterasebiosensor biosensor is studied by deviation experiment in group, same branch electrode
The coefficient of variation in six replication acquisition groups is carried out, is 5.0%；It repeats to prepare different electrode pair normal concentration pesticides six times
It measures and obtains between-group variation coefficient, be 7.3%.When electrode is stored in when not used in 4 DEG C of refrigerators, current-responsive after 7 days and 30 days
Variation obtain the stability of this sensor, after 7 days, electric current does not have a significant change, and current-responsive value is reduced to former electricity after 30 days
The 80% of flow valuve；
(4) fresh vegetables is bought from local supermarket, removes rotten blade, and clean up.After vegetables are dried, it is cut into
The vanelets of 2 × 2mm weigh 2g blades in small beaker, spray 10 μ g/L chlopyrifos in vegetable surface, place 3h at room temperature.
Then 1mL acetone and the phosphate buffer of 9mL 0.1mol/L pH7.5 are added in, by suspension ultrasound 15min after centrifuge
Middle rotating speed centrifuges 10min for 1000rpm, and supernatant is taken directly to measure, need not extract and be enriched with.The concentration root of pesticide in sample
It is calculated according to calibration curve, the rate of recovery can reach 98.9%~105.8%.
The present invention carries out screen printing carbon electrode using dicyclohexyl phthalate-multi-walled carbon nanotube nano material
Modification, can promote the transmission of electronics in electrochemical reaction, increase effectively the current-responsive of sensor.Two ring of phthalic acid
Own ester is excellent powder plasticizer.Multi-walled carbon nanotube can improve the microenvironment of electrode surface, have larger specific surface
Product, good basis is provided for the fixation of next step material, makes the enzyme sensor of preparation there is relatively low detection to limit.Chitosan has
There are excellent functionality and compatibility, it can be as the material of fixed macro-molecular protein, for preparing stability and sensitivity
High electrochemica biological sensor.Simultaneously screen printing carbon electrode it is at low cost, can batch making, the acetylcholine for preparing of the present invention
Esterase sensor improves sensitivity and selectivity, simple with preparation section, of low cost, and detection limit is low, and scope is wide, precision
Height, the advantages that suitable for Site Detection.
It can be harvested, before listing in vegetables and fruits using current mode acetylcholinesterasebiosensor biosensor made of the present invention, into
The quick measure of row pesticide residue, directly to pesticide residue, whether superscale is detected, and is avoided because eating containing remains of pesticide
Vegetables and fruits and cause poisoning, provide the technical support of residue detection for crop production safety and consumption.Meet China's acetylcholine
Esterase transducer production method develops and international requirement.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this
The people of technology without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection of the present invention
Scope should be subject to what claims were defined.
Claims (6)
1. a kind of transducer production method based on screen printing carbon electrode, it is characterised in that：It is the O-phthalic that will be prepared
Sour dicyclohexyl maleate/multi-walled carbon nanotube is layered drop coating to the screen printing carbon electrode surface activated through over cleaning, obtains modification electricity
Pole, then the surface by acetylcholinesterase drop coating to modified electrode, obtain after dry cleaning based on dicyclohexyl phthalate-
The acetylcholinesterasebiosensor biosensor of the screen printing carbon electrode of multi-walled carbon nanotube modification；Step is as follows：
The preparation of dicyclohexyl phthalate/multi-walled carbon nanotube；
Cleaning, activation screen printing carbon electrode, obtain pretreatment screen printing carbon electrode；
Dicyclohexyl phthalate prepared by step 1)/multi-walled carbon nanotube solution layering drop coating is in the pretreatment of step 2)
On screen printing carbon electrode surface, modified graphite electrode is obtained；
It on the working electrode surface of modified electrode, will be based on obtained by acetylcholinesterase drop coating to step 3) after dry cleaning
The acetylcholinesterasebiosensor biosensor of screen printing carbon electrode；
It is as follows：
0.1g chitosans are added to 50mL concentration in 1.0% acetic acid solution, to be made into the chitosan solution that concentration is 0.2%,
More than magnetic agitation 8h is completely dissolved chitosan；It is 0.2% that 2mg dicyclohexyl phthalates are scattered in 4mL concentration
In chitosan solution, and in 65 DEG C of water-baths ultrasonic disperse to obtain stable dispersion liquid；The high degree of dispersion suspension obtained
For 0.5mg/mL dicyclohexyl phthalate suspension；The chitosan that 2mg multi-walled carbon nanotubes are scattered in 4mL 0.2% is molten
In liquid, and at room temperature ultrasonic disperse 6h to obtain stable black dispersion liquid；The high degree of dispersion black suspension obtained is
0.5mg/mL multi-walled carbon nanotubes-chitosan nano compound store the multi-walled carbon nanotube solution prepared at 4 DEG C；
Screen printing carbon electrode is placed in ultrasound 5min, ultrapure water, N in 1mM NaOH solutions2Drying, 1mM is placed in by electrode
Ultrasound 5min, ultrapure water, N in HCl solution2Drying rinses electrode, N with absolute ethyl alcohol2Drying, delays in pH5.0 phosphate
Sweep current-time graph 300s in fliud flushing, scan cycle volt-ampere curve obtain pretreatment silk-screen printing carbon until performance stabilization
Electrode；Dicyclohexyl phthalate hanging drop prepared by the step 1) of 5~10 μ L is first coated in the pre- place obtained by step 3)
It manages on screen printing carbon electrode, dries in air, with ultrapure water surface, the O-phthalic of electrode surface will be not fixed to
Sour dicyclohexyl maleate suspension rinses out, and obtains the screen printing carbon electrode of dicyclohexyl phthalate suspension modification；Afterwards,
By 5~10 μ L steps 2) prepare multi-walled carbon nanotube solution drop coating obtained dicyclohexyl phthalate suspension silk
Working electrode surface in wire mark brush carbon electrode, is dried in air, with ultrapure water surface, will be not fixed to electrode surface
Multi-walled carbon nanotube solution rinses out, and obtains the silk-screen printing carbon electricity of dicyclohexyl phthalate-multi-walled carbon nanotube modification
Pole；Obtained by the 0.02U/ μ L acetylcholine esters enzyme solutions of 5~10 μ L of drop coating to step 4) on the working electrode surface of modified electrode,
And it is dry at 4 DEG C, the enzyme on unadsorbed is washed off with the phosphate buffer solution of pH7.5, obtains acetylcholinesterase life
Object sensor saves backup under 4 DEG C of dry environments.
2. method according to claim 1, which is characterized in that the step 2) screen printing carbon electrode, including printed electrode
Substrate, the external insulation being printed on substrate and at least two contact conductors, substrate printing is respectively one there are three electrode
A working electrode, one a contact conductor should be connected with to electrode and a reference electrode, each electrode pair.
3. method according to claim 1, which is characterized in that the step 2) cleaning, activation screen printing carbon electrode, process
It is：First, screen printing carbon electrode is placed in ultrasound 5min, ultrapure water, N in 1mM NaOH solutions2Drying, secondly, will
Electrode is placed in ultrasound 5min, ultrapure water, N in 1mM HCl solutions2Drying again, electrode, N is rinsed with absolute ethyl alcohol2It blows
Dry, finally, sweep current-time graph 300s in pH5.0 phosphate buffers, scan cycle volt-ampere curve is until property afterwards
It can stablize.
4. method according to claim 1, which is characterized in that in step 3) pretreatment electrode surface layering drop coating neighbour's benzene
Dioctyl phthalate dicyclohexyl maleate and multi-walled carbon nanotube solution are first to locate the dicyclohexyl phthalate drop coating of 5~10 μ L in advance
It manages on screen printing carbon electrode, dries in air, with ultrapure water surface, the O-phthalic of electrode surface will be not fixed to
Sour dicyclohexyl maleate rinses out, and obtains the screen printing carbon electrode of dicyclohexyl phthalate modification；Afterwards, it is 5~10 μ L are more
Wall carbon nano tube solution drop coating working electrode table in the screen printing carbon electrode of obtained dicyclohexyl phthalate modification
Face is dried in air, and with ultrapure water surface, the multi-walled carbon nanotube solution for being not fixed to electrode surface is rinsed out,
Obtain the screen printing carbon electrode of dicyclohexyl phthalate-multi-walled carbon nanotube modification.
5. method according to claim 1, which is characterized in that step 4) the drop coating acetylcholine ester on the working electrode (s
Enzyme is the 0.02U/ μ L acetylcholine ester enzyme solutions of 5~10 μ L of drop coating, and the dry 2h at 4 DEG C, afterwards with the phosphoric acid of pH7.5
Salt buffer solution washes off the enzyme on unadsorbed, obtains acetylcholinesterasebiosensor biosensor.
6. the application of sensor prepared by the method described in claim 1-5, which is characterized in that for detecting organophosphor and amino
Formate ester pesticide.
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| CN105223251A (en) * | 2015-10-19 | 2016-01-06 | 山东理工大学 | A kind of preparation method and application detecting the sensor of organophosphorus pesticide based on screen printing electrode |
| CN106442676A (en) * | 2016-12-22 | 2017-02-22 | 山东理工大学 | Preparation and application of nano cerium oxide/mesoporous carbon sensor for detecting pesticide |
| 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 |
| CN114166727B (en) * | 2021-11-01 | 2024-02-20 | 湖北科技学院 | Cell sensor for detecting nitric oxide and application thereof |
| CN114965634A (en) * | 2022-04-14 | 2022-08-30 | 深圳可孚生物科技有限公司 | Preparation method of silk-screen bioelectrochemical sensor |
| WO2023245284A1 (en) * | 2022-06-20 | 2023-12-28 | Glc Medical (Glcm) Inc. | Electrochemical biosensors and method of preparing electrochemical biosensors |
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