CN105021679A - Silk-screen printing electrode sensor preparation method based on ionic liquid-multi-wall carbon nano-tube modification - Google Patents
Silk-screen printing electrode sensor preparation method based on ionic liquid-multi-wall carbon nano-tube modification Download PDFInfo
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Abstract
The present invention discloses a silk-screen printing electrode sensor preparation method based on ionic liquid-multi-wall carbon nano-tube modification, and belongs to the technical field of biological sensors. According to the method, an ionic liquid-multi-wall carbon nano-tube nanometer material is prepared and then coats the surface of a silk-screen printing carbon electrode in a dropwise manner, and then acetylcholine esterase coats the surface of the ionic liquid-multi-wall carbon nano-tube nanometer material modified silk-screen printing work electrode in a dropwise manner so as to prepare the acetylcholine esterase biological sensor for detecting organic phosphorus and carbamate pesticides. According to the present invention, the prepared sensor has characteristics of simple process, easy operation, low cost, high detection sensitivity, strong electrical signal and simple electrode pretreatment, and is suitable for detection of pesticide residues, wherein the detection limit is 0.05 [mu]g/L.
Description
Technical field
The present invention relates to a kind of transducer production method of the screen printing electrode based on ionic liquid-multi-walled carbon nano-tubes modification, belong to biosensor technology field.
Background technology
Agricultural chemicals is important capital goods in the agricultural sector, in agricultural product are produced, play very important effect to the control of disease, worm, grass, mouse harm.But the hazard analysis and HACCP of pollution by pesticides and generation thereof is serious, and the loss that agricultural chemicals environmental pollution causes is many-sided, comprise the pollution to water environment, to the pollution of soil, to the pollution of air, the impact on environmental organism and the harm etc. to health.At present because user generally lacks scientifical use pesticide knowldgde, covet output, causes the agricultural products circulated in the market to there is Pesticide Residue all to some extent, and its harm also causes the concern of the public day by day.China is a large agricultural country, and along with our people's living standard improves constantly, the quality security problem of agricultural product more and more receives publicity, and especially Pesticide Residues in Vegetables problem has become the focus of public attention.China produces and the most kind of pesticide of use is Organophosphorus and carbamate pesticides class agricultural chemicals (accounting for 70%), and wherein highly toxic Organophosphorus and carbamate pesticides insecticides is about 70%, therefore easily causes food pesticide to remain poisoning.Visible, strengthening to the detection of Residual Pesticides in Farm Produce to preserving the ecological environment, especially ensure that human health has very profound significance, and the emphasis of Detecting Pesticide should be placed on Organophosphorus and carbamate pesticides class agricultural chemicals.
The main method of current pesticide residue analysis is gas chromatograph, liquid chromatograph, gas chromatograph-mass spectrometer etc., although these method analysis precisions are high, quantitatively accurately, but the complex pretreatment of its sample, detect length consuming time, cost high, need the operating personnel that are skilled in technique.The fast determining method of China's residues of pesticides is enzyme level test paper method and enzyme level spectrophotometric method (the residual fast detector of agriculture), can realize the field quick detection of organophosphorus pesticide and carbamate chemicals for agriculture, have good practical value.Quick measuring card is changed by the color of visual inspection card, and the vegetable sample that therefore generally can only be used for severe overweight carries out observational measurement.The application of enzyme level spectrophotometric method is also relatively more extensive, and it is all based on this principle that domestic existing Multiple Pesticides remains tacheometer.Light splitting light ratio juris carries out detecting based on the change of absorbance, but pigments a large amount of in vegetable and fruit can cause very large impact to spectrophotometric method, causes the inaccurate of testing result.And said method exists, and the recovery is low, false retrieval, undetected ratio are higher, poor repeatability, be difficult to meet the shortcomings such as low-residual and the requirement that quantitatively detects.
Summary of the invention
The object of the invention is to overcome the deficiency that above-mentioned prior art exists, provide that a kind of structure is simple, easy to operate, cost is low and the screen printing electrode biology sensor preparation method that detection sensitivity is high, the technical scheme of employing is as follows:
A kind of transducer production method of the screen printing electrode based on ionic liquid-multi-walled carbon nano-tubes modification, it is characterized in that: be the ionic liquid prepared/multi-walled carbon nano-tubes layering is dripped the screen printing carbon electrode surface be coated onto through cleaning activation, obtain modified electrode, again acetylcholinesterase is dripped the surface being coated onto modified electrode, after dry cleaning, obtain the acetylcholinesterasebiosensor biosensor of the screen printing carbon electrode modified based on ionic liquid-multi-walled carbon nano-tubes.
Described method step is:
1) preparation of ionic liquid/multi-walled carbon nano-tubes;
2) clean, activate screen printing carbon electrode, obtain pre-service screen printing carbon electrode;
3) ionic liquid step 1) prepared/multi-walled carbon nano-tubes solution layering is dripped and is coated in step 2) pre-service screen printing carbon electrode on the surface, obtain modified graphite electrode;
4) acetylcholinesterase is dripped on the working electrode surface being coated onto step 3) gained modified electrode, after dry cleaning, obtain the acetylcholinesterasebiosensor biosensor based on screen printing carbon electrode.
Step 2) described screen printing carbon electrode, comprise the substrate of printed electrode, be printed on on-chip external insulation and at least two contact conductors, substrate is printed with three electrodes, be respectively a working electrode, one to electrode and a contrast electrode, each electrode pair should be connected with a contact conductor.
Step 2) described cleaning, activation screen printing carbon electrode, process is: first, screen printing carbon electrode is placed in the ultrasonic 5min of 1mM NaOH solution, ultrapure water, N
2dry up, secondly, electrode is placed in the ultrasonic 5min of 1mM HCl solution, ultrapure water, N
2dry up, again, rinse electrode with absolute ethyl alcohol, N
2dry up, finally, sweep current-time curve 300s in pH5.0 phosphate buffer, scan cycle volt-ampere curve is until stable performance afterwards.
Drip in the layering of pre-service electrode surface described in step 3) and be coated with ionic liquid and multi-walled carbon nano-tubes solution, first the ionic liquid of 5 ~ 10 μ L is dripped to be coated on pre-service screen printing carbon electrode, dry in atmosphere, with ultrapure water surface, the ionic liquid not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode of Ionic Liquid Modified; Afterwards, 5 ~ 10 μ L multi-walled carbon nano-tubes solution are dripped working electrode surface in the screen printing carbon electrode being coated in the Ionic Liquid Modified obtained, dry in atmosphere, with ultrapure water surface, the multi-walled carbon nano-tubes solution not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode that ionic liquid-multi-walled carbon nano-tubes is modified.
Painting acetylcholinesterase is dripped on the working electrode (s described in step 4), it is the 0.02U/ μ L acetylcholine ester enzyme solutions dripping painting 5 ~ 10 μ L, and at 4 DEG C dry 2h, rinse the enzyme removed and do not adsorb afterwards by the phosphate buffered solution of pH7.5, obtain acetylcholinesterasebiosensor biosensor.
Described method concrete steps are as follows:
1) 4mg N-octylpyridinium hexafluorophosphate (ionic liquid) is scattered in 4mL absolute ethyl alcohol, ultrasonic disperse 6h is to obtain stable dispersion liquid, the high degree of dispersion suspending liquid obtained is 1mg/mL ionic liquid solution, is stored by the ionic liquid solution prepared at 4 DEG C;
2) joined in 50mL 1.0% acetic acid solution by 0.1g shitosan, be made into the chitosan solution that concentration is 0.2%, magnetic agitation makes more than 8h that shitosan is dissolved completely.2mg multi-walled carbon nano-tubes is 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 nano-tubes-chitosan nano compound, is stored by the multi-walled carbon nano-tubes solution prepared at 4 DEG C;
3) screen printing carbon electrode is placed in the ultrasonic 5min of 1mM NaOH solution, ultrapure water, N
2dry up, electrode is placed in the ultrasonic 5min of 1mM HCl solution, ultrapure water, N
2dry up, rinse electrode with absolute ethyl alcohol, N
2dry up, sweep current-time curve 300s in pH5.0 phosphate buffer, scan cycle volt-ampere curve, until stable performance, obtains pre-service screen printing carbon electrode;
4) first ionic liquid prepared by the step 1) of 5 ~ 10 μ L is dripped and be coated on the pre-service screen printing carbon electrode of step 3) gained, dry in atmosphere, with ultrapure water surface, the ionic liquid not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode of Ionic Liquid Modified; Afterwards, by 5 ~ 10 μ L steps 2) the multi-walled carbon nano-tubes solution prepared drips working electrode surface in the screen printing carbon electrode being coated in the Ionic Liquid Modified obtained, dry in atmosphere, with ultrapure water surface, the multi-walled carbon nano-tubes solution not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode that ionic liquid-multi-walled carbon nano-tubes is modified;
5) the 0.02U/ μ L acetylcholine ester enzyme solutions of painting 5 ~ 10 μ L is dripped on the working electrode surface of step 4) gained modified electrode, and it is dry at 4 DEG C, the enzyme removed and do not adsorb is rinsed by the phosphate buffered solution of pH7.5, obtain acetylcholinesterasebiosensor biosensor, save backup under 4 DEG C of dry environments.
The method of the invention, is characterized in that, for detecting Organophosphorus and carbamate pesticides class agricultural chemicals.
Described method, it is characterized in that: the preparation of ionic liquid/multi-walled carbon nano-tubes, the cleaning of screen printing carbon electrode, the structure at acetylcholinesterase sensor sensing interface and process characterize, the foundation of acetylcholinesterase working sensor curve, the detection of acetylcholinesterase sensor performance, acetylcholine ester enzyme sensor is to the detection of actual sample.Acetylcholinesterase sensor performance detects and comprises accuracy, stability and acetylcholine ester enzyme sensor to the mensuration of sample recovery rate.
Described preparation method, is characterized in that: the concentration range that prepared current mode acetylcholine ester enzyme sensor detects chlopyrifos is 0.05 ~ 10
5μ g/L, detects and is limited to 0.05 μ g/L.
Described method preparation principle is: because organophosphorus pesticide and acetylcholinesterase have higher specific binding, and therefore conventional acetylcholinesterase is as the molecular recognition elements detecting organophosphorus pesticide.Acetylcholinesterasebiosensor biosensor is that acetylcholinesterase is fixed on electrode surface, and substrate for enzymatic activity acetylcholine hydrolyzation generates choline and acetic acid.Agricultural chemicals structurally with substrate acetyl choline seemingly, it effectively can combine with the activated centre of acetylcholine, the activity of acetylcholine esterase inhibition.Choline is a kind of electroactive material, oxidation reaction can be there is under certain potentials, the concentration of residues of pesticides can be measured by the size of thiocholine oxidation peak in voltammetric scan process, the inhibiting rate of agricultural chemicals to enzyme is obtained with or without the change of enzymatic reaction current signal during agricultural chemicals by comparing, this inhibiting rate becomes corresponding relation with the concentration of agricultural chemicals, just can record the content of organophosphorus or carbamate chemicals for agriculture.
For reaching above object, following technical scheme is taked to realize: a kind of preparation method of sensor of the screen printing carbon electrode based on ionic liquid-multi-walled carbon nano-tubes modification, is characterized in that:
(1) cleaning of the front screen printing electrode of current mode acetylcholine ester enzyme sensor preparation.Screen printing electrode is placed in the ultrasonic 5min of 1mM NaOH solution, ultrapure water, N
2dry up.Afterwards, electrode is placed in the ultrasonic 5min of 1mM HCl solution, ultrapure water, N
2dry up.Finally, rinse electrode with absolute ethyl alcohol, N
2dry up; (2) activation of the front electrode of current mode acetylcholine ester enzyme sensor preparation.Sweep current-time curve 300s in pH5.0 phosphate buffer, scan cycle volt-ampere curve is until stable performance afterwards; (3) preparation of ionic liquid/multi-walled carbon nano-tubes.4mg N-octylpyridinium hexafluorophosphate (ionic liquid) is scattered in 4mL absolute ethyl alcohol, ultrasonic disperse 6h is to obtain stable dispersion liquid, the high degree of dispersion suspending liquid obtained is 1mg/mL ionic liquid solution, is stored by the ionic liquid solution prepared at 4 DEG C; Joined by 0.1g shitosan in 50mL 1.0% acetic acid solution, be made into the chitosan solution that concentration is 0.2%, magnetic agitation makes more than 8h that shitosan is dissolved completely.2mg multi-walled carbon nano-tubes is 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 nano-tubes-chitosan nano compound, is stored by the multi-walled carbon nano-tubes solution prepared at 4 DEG C;
(4) painting ionic liquid and multi-walled carbon nano-tubes solution is dripped in the layering of pre-service electrode surface, first the ionic liquid of 5 ~ 10 μ L is dripped to be coated on pre-service screen printing carbon electrode, dry in atmosphere, with ultrapure water surface, the ionic liquid not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode of Ionic Liquid Modified; Afterwards, 5 ~ 10 μ L multi-walled carbon nano-tubes solution are dripped working electrode surface in the screen printing carbon electrode being coated in the Ionic Liquid Modified obtained, dry in atmosphere, with ultrapure water surface, the multi-walled carbon nano-tubes solution not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode that ionic liquid-multi-walled carbon nano-tubes is modified.Then painting 5 ~ 10 μ L acetylcholine ester enzyme solutions is dripped, and dry at 4 DEG C.Rinse by the phosphate buffered solution that pH is 7.5 enzyme removed and do not adsorb, so just obtain AChE/IL-MWCNTs/SPE sensor, save backup under 4 DEG C of dry environments.
For reaching above object, following technical scheme is taked to realize: a kind of transducer production method of the screen printing electrode based on ionic liquid-multi-walled carbon nano-tubes modification, it is characterized in that: (1) configures a series of chlopyrifos titer, carry out cyclic voltammetry scanning, be inhibited rate, obtains the working curve of the current mode acetylcholinesterasebiosensor biosensor of above-mentioned preparation, sensing range and detectability further; (2) degree of accuracy of acetylcholinesterasebiosensor biosensor and stability are evaluated; (3) actual fruits and vegetables sample is analyzed to the recovery and reappearance that draw this sensor.
The preparation technology of described acetylcholinesterasebiosensor biosensor is as follows: dripped by the ionic liquid of 5 ~ 10 μ L and be coated on pre-service screen printing carbon electrode, dry in atmosphere, with ultrapure water surface, the ionic liquid not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode of Ionic Liquid Modified; Afterwards, 5 ~ 10 μ L multi-walled carbon nano-tubes solution are dripped working electrode surface in the screen printing carbon electrode being coated in the Ionic Liquid Modified obtained, dry in atmosphere, with ultrapure water surface, the multi-walled carbon nano-tubes solution not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode that ionic liquid-multi-walled carbon nano-tubes is modified.Then painting 5 ~ 10 μ L acetylcholine ester enzyme solutions is dripped, and dry at 4 DEG C.Rinse by the phosphate buffered solution that pH is 7.5 enzyme removed and do not adsorb, so just obtain AChE/IL-MWCNTs/SPE sensor, save backup under 4 DEG C of dry environments.
Beneficial effect of the present invention: the present invention only just in its finishing ionic liquid (IL)-multi-walled carbon nano-tubes (MWCNTs) nano material, can overcome the various unfavorable factors that the acetylcholinesterasebiosensor biosensor LBL self-assembly in research in the past brings by simple painting technology of dripping.The present invention's screen printing carbon electrode used can adjust its specification according to the actual requirements, there is flexible design, cost low and can the advantage of batch making, solve the pretreated complicated procedures of front electrode well, and with a batch electrode, there is good homogeneity, can produce in batches, even realize commercialization, apply more and more extensive.It is good that ionic liquid has dissolubility, and conductance is high, and electrochemical window is wide, and viscosity is high, chemical stability and the high feature of thermal stability.Multi-walled carbon nano-tubes is a kind of perfectly carbon nanomaterial, has special thermal behavior and mechanical property, and has the electric conductivity of high-ratio surface sum excellence.Shitosan has excellent functionality and compatibility, can, as the material of fixing macro-molecular protein, be used for preparing stability and highly sensitive electrochemica biological sensor.The present invention adopts ionic liquid-multi-walled carbon nano-tubes nano material to modify screen printing carbon electrode, effectively can increase the current-responsive of sensor, what higher specific surface area was next step material fixedly provides good basis, makes the enzyme sensor of preparation have lower detectability.
According to the acetylcholine ester enzyme sensor that the method for the invention is made, overcome Detecting Pesticide complicated operation and the problem of spended time length, provide a kind of preparation section simple, easy to operate, with low cost, electric signal is strong, highly sensitive, high specificity, detectability is low, and scope is wide, and precision is high, be applicable to Site Detection, can the disposable biology sensor of qualitative and quantitative analysis residues of pesticides concentration.Before adopting the current mode acetylcholinesterasebiosensor biosensor made of the present invention can gather at vegetables and fruits, going on the market; carry out the Fast Measurement of residues of pesticides; directly to residues of pesticides, whether superscale detects; avoid because food contains the vegetables and fruits of remains of pesticide and cause poisoning; for crop production safety and consumption provide the technical support of residue detection, all have very important significance in food security and field of environment protection.
Accompanying drawing explanation
The empty screen printing electrode of Fig. 1 SEM phenogram: a.; B. IL/SPE; C. IL-MWCNTs/SPE
Scanning electron microscope (SEM) is used to characterize empty screen printing electrode surface and the micro-structure diagram modified in the ionic liquid of electrode surface, ionic liquid-multi-walled carbon nano-tubes compound, as shown in Figure 1.Figure a is empty screen printing electrode surface, surface is graininess and schistose texture, figure b is electrode face finish ionic liquid, can be clear that the material of many thin tube-like is evenly distributed in electrode surface in figure c, prepare multi-walled carbon nano-tubes-chitosan nano compound and be successfully modified at electrode surface.
The cyclic voltammetric of Fig. 2 AChE/IL-MWCNTs/SPE sensor assembling process characterizes: the empty screen printing electrode of a.; B. AChE/IL-MWCNTs/SPE; C. IL/SPE; D. MWCNTs/SPE; E. IL-MWCNTs/SPE
This test is comprising 5.0mmol/L [Fe (CN)
6]
3-/4-cyclic voltammetry curve sign is carried out with in the PBS damping fluid of the 0.1mol/L pH7.4 of 0.1mol/L KCl.As shown in Figure 2, empty screen printing electrode presents obviously symmetrical redox peak (curve a); When ionic liquid is attached to after on electrode, peak current obviously increases (curve c); Curve c increases about 17 μ A compared with the peak current of curve a, and this is mainly because ionic liquid has good conductance; When multi-walled carbon nano-tubes is attached to after on electrode, peak current increases (curve d); Curve d increases about 27 μ A compared with the peak current of curve a, this is because the chemical property of multi-walled carbon nano-tubes excellence; When multi-walled carbon nano-tubes drips the screen printing electrode surface being coated onto finishing ionic liquid, peak current reaches maximal value (curve e); Curve e comparatively curve a increases about 47 μ A, and current potential obviously reduces, and as can be seen here, ionic liquid and multi-walled carbon nano-tubes have extraordinary synergy, shows the advantages such as large, the outstanding conductance of specific surface area.After IL-MWCNTs/SPE drips on surface and coats acetylcholinesterase, electric signal obviously reduces (curve b), because acetylcholinesterase is protein molecule, shields the electroactive of electrode surface, hinders electron transmission, and thus the trend reduced has appearred in peak current.
Fig. 3 pH is on the impact of curent change
The pH value of test end liquid is one of key factor affecting sensor performance.The optimum pH of AChE is between 7.0 to 8.0.AChE/IL-MWCNTs/SPE electrode is carried out in 1.0mmol/L chlorinated thio acetylcholine (ATCl) phosphate buffer of different pH value (6 to 8) test of current-responsive.As shown in Figure 3, the current-responsive of modified electrode presents the trend of first increases and then decreases with the increase of pH.PH value is between 6.0 ~ 7.5, and response current increases with the increase of pH; PH value is between 7.5 ~ 8.0, and response current reduces with the increase of pH.Can find out, when pH value is 7.5, electrode response is the strongest, and therefore pH7.5 is optimum determining pH value.
Fig. 4 enzyme charge capacity is on the impact of enzyme sensor curent change
Acetylcholinesterase is also one of factor affecting enzyme sensor performance in the charge capacity of electrode surface.As shown in Figure 4, along with the increase of electrode surface acetylcholinesterase charge capacity, response current presents the trend of first increases and then decreases, and response current reaches maximal value when enzyme charge capacity is 0.25U.When enzyme charge capacity is lower than 0.25U, along with the increase of enzyme charge capacity, response current increases gradually; When enzyme charge capacity is higher than 0.25U, response current tends towards stability and has the trend of reduction, this is because electrode area is limited, and a large amount of enzyme that makes of excessive enzyme charge capacity forms multilayer film in electrode surface enrichment, accumulation, thus have impact on the transmittance process of electronics at electrode surface.Therefore, the acetylcholinesterase fixed amount being fixed on electrode surface in this test is 0.25U.
Fig. 5 acetyl chloride thiocholine concentration is on the impact of enzyme sensor curent change: a. 0.5mM; B. 1mM; C. 2mM; D. 5mM; E. 8mM; F. 10mM
The AChE/IL-MWCNTs/SPE sensor prepared is immersed in chlorinated thio acetylcholine (ATCl) solution of a series of variable concentrations and carry out cyclic voltammetry scan, as shown in Figure 5, along with the increase of concentration of substrate, the corresponding increase of peak current, shows the acecoline in the rapid binding soln of acetylcholinesterase energy fixed at interface and its hydrolysis of catalysis.
Fig. 6 sweep speed is on the impact of enzyme sensor curent change: a.5mV; B.10mV; C.20mV; D.40mV; E.80mV; F.100mV; G.150mV; H.200mV
We further study by cyclic voltammetry and sweep the impact of speed on AChE/IL-MWCNTs/SPE sensor electrochemical behavior in chlorinated thio acetylcholine (ATCl) phosphate buffer containing 1.0mmol/L pH7.5, as shown in Figure 6: within the scope of sweep speed 5 ~ 200mV/s, along with the increase of sweeping speed, peak current increases gradually, sweep speed and curent change are proportionate, and show that electrode process is surface-controlled process.
Fig. 7 pesticide concentration is on the impact of enzyme sensor curent change: a. 1 × 10
5μ g/L; B. 1 × 10
3μ g/L; C. 5 × 10
2μ g/L; D. 2 × 10
2μ g/L; E. 1 × 10
2μ g/L; F. 10 μ g/L; G. 5 μ g/L; H. 1 μ g/L; I. 5 μ g/L; J. 0.05 μ g/L
The performance of chlopyrifos concentration to enzyme sensor being fixed on electrode surface has a significant impact.Fig. 7 shows the current changing rate of electrode face finish variable concentrations chlopyrifos.Along with the increase of chlopyrifos concentration, current changing rate increases gradually.
Fig. 8 pesticide concentration is on the impact of enzyme sensor curent change
As shown in Figure 8, the chlopyrifos of variable concentrations has different inhibiting rates to acetylcholinesterase.In optimal conditions, chlopyrifos is 0.05 ~ 1 × 10
5have good linear relationship with the inhibiting rate of agricultural chemicals in μ g/L concentration range, linear equation is: y=3.7794x+8.3039, and related coefficient is 0.9926.
Fig. 9 AChE/IL-MWCNTs/SPE sensor actual sample recovery of standard addition detects
Buy fresh vegetables from local supermarket, remove the blade rotted, and clean up.After vegetables are dried, be cut into the vanelets of 2 × 2mm, take 2g blade in small beaker, spray 10 μ g/L chlopyrifos at vegetable surface, ambient temperatare puts 3h.Then add the phosphate buffer of 1mL acetone and 9mL 0.1mol/L pH7.5, by being the centrifugal 10min of 1000rpm in hydro-extractor medium speed after ultrasonic for suspending liquid 15min, getting supernatant and directly measuring, do not need to extract and enrichment.The concentration of sample Pesticides calculates according to calibration curve.
The screen printing electrode acetylcholine ester enzyme sensor modified based on ionic liquid-multi-walled carbon nano-tubes nano-complex is adopted actual sample (cabbage, romaine lettuce, leek, pakchoi) to be carried out to the detection of recovery of standard addition, as shown in Figure 9, the recovery is positioned between 98.7% ~ 110.4%, relative standard deviation is 3.88% ~ 4.63%, shows that the actual sample Detection results of this enzyme sensor is good.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not by the restriction of embodiment.
Embodiment 1: a kind of preparation method of sensor of the screen printing carbon electrode based on ionic liquid-multi-walled carbon nano-tubes modification
(1) cleaning of the front screen printing carbon electrode of current mode acetylcholine ester enzyme sensor preparation.Screen printing carbon electrode is placed in the ultrasonic 5min of 1mM NaOH solution, ultrapure water, N
2dry up.Afterwards, electrode is placed in the ultrasonic 5min of 1mM HCl solution, ultrapure water, N
2dry up.Finally, rinse electrode with absolute ethyl alcohol, N
2dry up; (2) activation of the front electrode of current mode acetylcholine ester enzyme sensor preparation.Sweep current-time curve 300s in pH5.0 phosphate buffer, scan cycle volt-ampere curve is until stable performance afterwards; (3) preparation of ionic liquid/multi-walled carbon nano-tubes.4mg N-octylpyridinium hexafluorophosphate (ionic liquid) is scattered in 4mL absolute ethyl alcohol, ultrasonic disperse 6h is to obtain stable dispersion liquid, the high degree of dispersion suspending liquid obtained is 1mg/mL ionic liquid solution, is stored by the ionic liquid solution prepared at 4 DEG C; Joined by 0.1g shitosan in 50mL 1.0% acetic acid solution, be made into the chitosan solution that concentration is 0.2%, magnetic agitation makes more than 8h that shitosan is dissolved completely.2mg multi-walled carbon nano-tubes is 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 nano-tubes-chitosan nano compound, is stored by the multi-walled carbon nano-tubes solution prepared at 4 DEG C; (4) ionic liquid of 5 ~ 10 μ L is dripped be coated on pre-service screen printing carbon electrode, dry in atmosphere, with ultrapure water surface, the ionic liquid not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode of Ionic Liquid Modified; Afterwards, 5 ~ 10 μ L multi-walled carbon nano-tubes solution are dripped working electrode surface in the screen printing carbon electrode being coated in the Ionic Liquid Modified obtained, dry in atmosphere, with ultrapure water surface, the multi-walled carbon nano-tubes solution not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode that ionic liquid-multi-walled carbon nano-tubes is modified.Then painting 5 ~ 10 μ L acetylcholine ester enzyme solutions is dripped, and dry at 4 DEG C.Rinse by the phosphate buffered solution that pH is 7.5 enzyme removed and do not adsorb, so just obtain AChE/IL-MWCNTs/SPE sensor, save backup under 4 DEG C of dry environments.
Embodiment 2: the current mode acetylcholine ester enzyme sensor prepared by utilization detects vegetable sample
(1) the acetylcholine ester enzyme sensor prepared according to embodiment 1 is carried out cyclic voltammetry with 50mV/s sweep velocity in the phosphate buffered solution of the pH7.5 of the chlorinated thio acetylcholine (ATCl) containing 1.0mM, potential window is 0V ~ 1.0V; (2) chlopyrifos standard solution is configured, when agricultural chemicals is measured, above-mentioned acetylcholine ester enzyme sensor is immersed in 10min in the pesticide standard solution of variable concentrations, then in reaction tank, add the phosphate buffered solution that 15 ~ 20mL contains 1.0mM chlorinated thio acetylcholine (ATCl), carry out cyclic voltammetry scan, inhibiting rate I can be tried to achieve by following formula:
I(%)=(I
P, control?I
P, exp)/I
P, control×100%
Wherein I
p, controland I
p, expbe respectively modified electrode without agricultural chemicals suppression with after agricultural chemicals suppresses, peak current in chlorinated thio acetylcholine solution, pesticide concentration and inhibiting rate are certain linear relationship, make working curve diagram, obtain the linear relationship between pesticide concentration and inhibiting rate, and detectability; (3) accuracy of acetylcholinesterasebiosensor biosensor is studied by deviation experiment in group, and same electrode carries out the coefficient of variation in six replication acquisition groups, is 5.3%; The electrode pair normal concentration evaluation of pesticides repeating six preparations different obtains between-group variation coefficient, is 7.6%.When electrode, the used time is not kept in 4 DEG C of refrigerators, and the change of 7 days and the response of 30 days after-currents obtains the stability of this sensor, and after 7 days, electric current does not have significant change, and after 30 days, current-responsive value reduces to 79% of primary current value; (4) buy fresh vegetables from local supermarket, remove the blade rotted, and clean up.After vegetables are dried, be cut into the vanelets of 2 × 2mm, take 2g blade in small beaker, spray 10 μ g/L chlopyrifos at vegetable surface, ambient temperatare puts 3h.Then add the phosphate buffer of 1mL acetone and 9mL 0.1mol/L pH7.5, by being the centrifugal 10min of 1000rpm in hydro-extractor medium speed after ultrasonic for suspending liquid 15min, getting supernatant and directly measuring, do not need to extract and enrichment.The concentration of sample Pesticides calculates according to calibration curve.Its recovery can reach 98.7% ~ 110.4%.
The present invention adopts ionic liquid-multi-walled carbon nano-tubes nano material to modify screen printing carbon electrode, the transmission of electronics in electrochemical reaction can be promoted, increase effectively the current-responsive of sensor, it is good that ionic liquid has dissolubility, conductance is high, electrochemical window is wide, and viscosity is high, chemical stability and the high feature of thermal stability.Multi-walled carbon nano-tubes can improve the microenvironment of electrode surface, has larger specific surface area, fixedly provides good basis for next step material, makes the enzyme sensor of preparation have lower detectability.Shitosan has excellent functionality and compatibility, can, as the material of fixing macro-molecular protein, be used for preparing stability and highly sensitive electrochemica biological sensor.Simultaneously screen printing carbon electrode cost low, can batch making, acetylcholine ester enzyme sensor prepared by the present invention improves sensitivity and selectivity, and have preparation section simple, with low cost, detectability is low, and scope is wide, and precision is high, is applicable to the advantages such as Site Detection.
Before adopting the current mode acetylcholinesterasebiosensor biosensor made of the present invention can gather at vegetables and fruits, going on the market, carry out the Fast Measurement of residues of pesticides, directly to residues of pesticides, whether superscale detects, avoid because food contains the vegetables and fruits of remains of pesticide and cause poisoning, for crop production safety and consumption provide the technical support of residue detection.Meet China's acetylcholinesterase transducer production method development and international requirement.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; can do various change and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (8)
1. the transducer production method of the screen printing electrode modified based on ionic liquid-multi-walled carbon nano-tubes, it is characterized in that: be the ionic liquid prepared/multi-walled carbon nano-tubes layering is dripped the screen printing carbon electrode surface be coated onto through cleaning activation, obtain modified electrode, again acetylcholinesterase is dripped the surface being coated onto modified electrode, after dry cleaning, obtain the acetylcholinesterasebiosensor biosensor of the screen printing carbon electrode modified based on ionic liquid-multi-walled carbon nano-tubes.
2. method according to claim 1, it is characterized in that, step is as follows:
The preparation of ionic liquid/multi-walled carbon nano-tubes;
Cleaning, activation screen printing carbon electrode, obtain pre-service screen printing carbon electrode;
Ionic liquid step 1) prepared/multi-walled carbon nano-tubes solution layering is dripped and is coated in step 2) pre-service screen printing carbon electrode on the surface, obtain modified graphite electrode;
Acetylcholinesterase is dripped on the working electrode surface being coated onto step 3) gained modified electrode, after dry cleaning, obtain the acetylcholinesterasebiosensor biosensor based on screen printing carbon electrode.
3. method according to claim 2, it is characterized in that, step 2) described screen printing carbon electrode, comprise the substrate of printed electrode, be printed on on-chip external insulation and at least two contact conductors, substrate is printed with three electrodes, be respectively a working electrode, one to electrode and a contrast electrode, each electrode pair should be connected with a contact conductor.
4. method according to claim 2, is characterized in that, step 2) described cleaning, activation screen printing carbon electrode, process is: first, screen printing carbon electrode is placed in the ultrasonic 5min of 1mM NaOH solution, ultrapure water, N
2dry up, secondly, electrode is placed in the ultrasonic 5min of 1mM HCl solution, ultrapure water, N
2dry up, again, rinse electrode with absolute ethyl alcohol, N
2dry up, finally, sweep current-time curve 300s in pH5.0 phosphate buffer, scan cycle volt-ampere curve is until stable performance afterwards.
5. method according to claim 2, it is characterized in that, drip in the layering of pre-service electrode surface described in step 3) and be coated with ionic liquid and multi-walled carbon nano-tubes solution, first the ionic liquid of 5 ~ 10 μ L is dripped to be coated on pre-service screen printing carbon electrode, dry in atmosphere, with ultrapure water surface, the ionic liquid not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode of Ionic Liquid Modified; Afterwards, 5 ~ 10 μ L multi-walled carbon nano-tubes solution are dripped working electrode surface in the screen printing carbon electrode being coated in the Ionic Liquid Modified obtained, dry in atmosphere, with ultrapure water surface, the multi-walled carbon nano-tubes solution not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode that ionic liquid-multi-walled carbon nano-tubes is modified.
6. method according to claim 2, it is characterized in that, painting acetylcholinesterase is dripped on the working electrode (s described in step 4), it is the 0.02U/ μ L acetylcholine ester enzyme solutions dripping painting 5 ~ 10 μ L, and at 4 DEG C dry 2h, rinse by the phosphate buffered solution of pH7.5 the enzyme removed and do not adsorb afterwards, obtain acetylcholinesterasebiosensor biosensor.
7. method according to claim 2, it is characterized in that, concrete steps are as follows:
4mg N-octylpyridinium hexafluorophosphate (ionic liquid) is scattered in 4mL absolute ethyl alcohol, ultrasonic disperse 6h is to obtain stable dispersion liquid, the high degree of dispersion suspending liquid obtained is 1mg/mL ionic liquid solution, is stored by the ionic liquid solution prepared at 4 DEG C;
Joined by 0.1g shitosan in 50mL 1.0% acetic acid solution, be made into the chitosan solution that concentration is 0.2%, magnetic agitation makes more than 8h that shitosan is dissolved completely; 2mg multi-walled carbon nano-tubes is 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 nano-tubes-chitosan nano compound, is stored by the multi-walled carbon nano-tubes solution prepared at 4 DEG C; Screen printing carbon electrode is placed in the ultrasonic 5min of 1mM NaOH solution, ultrapure water, N
2dry up, electrode is placed in the ultrasonic 5min of 1mM HCl solution, ultrapure water, N
2dry up, rinse electrode with absolute ethyl alcohol, N
2dry up, sweep current-time curve 300s in pH 5.0 phosphate buffer, scan cycle volt-ampere curve, until stable performance, obtains pre-service screen printing carbon electrode; First being dripped by ionic liquid prepared by the step 1) of 5 ~ 10 μ L is coated on the pre-service screen printing carbon electrode of step 3) gained, dry in atmosphere, with ultrapure water surface, the ionic liquid not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode of Ionic Liquid Modified; Afterwards, by 5 ~ 10 μ L steps 2) the multi-walled carbon nano-tubes solution prepared drips working electrode surface in the screen printing carbon electrode being coated in the Ionic Liquid Modified obtained, dry in atmosphere, with ultrapure water surface, the multi-walled carbon nano-tubes solution not being fixed on electrode surface is rinsed out, obtains the screen printing carbon electrode that ionic liquid-multi-walled carbon nano-tubes is modified; Drip the 0.02U/ μ L acetylcholine ester enzyme solutions of painting 5 ~ 10 μ L on the working electrode surface of step 4) gained modified electrode, and it is dry at 4 DEG C, the enzyme removed and do not adsorb is rinsed by the phosphate buffered solution of pH7.5, obtain acetylcholinesterasebiosensor biosensor, save backup under 4 DEG C of dry environments.
8. the method described in claim 1-7, is characterized in that, for detecting Organophosphorus and carbamate pesticides class agricultural chemicals.
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