CN103558269A - Preparation method of enzyme sensor for detecting chlorpyrifos pesticide residues - Google Patents
Preparation method of enzyme sensor for detecting chlorpyrifos pesticide residues Download PDFInfo
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Abstract
The invention relates to a preparation method of an enzyme sensor for detecting chlorpyrifos pesticide residues, belonging to the field of preparation of biosensors. The preparation method comprises the following steps: (1) preparing a multi-walled carbon-stannic oxide-chitosan nano composite material and a 0.5 percent of Nafion solution, specifically, modifying a working electrode to promote electron transfer by taking multi-walled carbon, stannic oxide and chitosan as raw materials by utilizing high electrochemical redox activity, high biocompatibility and high chemical stability of the multi-walled carbon-stannic oxide-chitosan nano composite material; and (2) preparing the enzyme sensor with a common modified electrode of acetylcholin esterase and a multi-walled carbon-stannic oxide-chitosan nano composite film. The acetylcholin esterase biosensor is short in detection time, high in sensitivity, high in selectivity, high in stability and high in regeneration capability, and the recovery rate meets the requirement.
Description
Technical field
The invention provides a kind of enzyme sensor preparation method who detects chlorpyrifos pesticide residue, belong to biology sensor preparation field.
Background technology
In recent years, the poisoning that residues of pesticides cause occurs repeatedly, and the residue problem harmful, highly toxic pesticide in fruits and vegetables is the focus that people pay close attention to always, and after being also China's entry into the WTO now, the export of farm produce meets with one of the major influence factors of " green barrier ".Along with China's living standards of the people improve constantly, the quality security problem of agricultural product more and more receives publicity, and especially fruit, Pesticide Residues in Vegetables problem have become the focus of public attention.The whole nation has the edible acute poisoning event being caused by the agricultural product of pollution by pesticides of causes up to a hundred every year, have a strong impact on the healthy of consumers in general, therefore the food-safety problem that is exceeded standard and caused by residues of pesticides, is more and more subject to national governments and the public's attention.Organophosphorus pesticide is the class agricultural chemicals that China is most widely used, and its Chlorpyrifos is one of insecticide variety of current produced worldwide and sales volume maximum.Chlopyrifos is the efficient poisoning broad spectrum pesticide herbicide of holding concurrently, and has certain interior absorption, can be transferred to human body by the inrichment of food chain, and human body is had to potential carcinogenesis.Visible, reinforcement to preserving the ecological environment, especially ensures that human health has very profound significance to the detection of Residual Pesticides in Farm Produce, and its Chlorpyrifos is the big event that environment and Pesticide Residues detect always.
Traditional Detecting Pesticide method mainly contains: gas chromatography (GC), high performance liquid chromatography (HPLC), Chromatography/Mass Spectrometry coupling technique (GC/LC-MS), capillary electrophoresis (CE), fluorescence analysis, euzymelinked immunosorbent assay (ELISA) (ELISA).Although these method selectivity are good, highly sensitive, accuracy is high, detection limit is low, can detect multiple element or compound, but it needs expensive instrument and equipment, sample pretreatment process is loaded down with trivial details, time-consuming simultaneously, and the technical merit requirement to analyst is very high, is unsuitable for field quick detection.Conventional Fast Determination of Pesticide Residue method has enzyme to suppress method (enzyme suppresses test paper method and enzyme inhibition-spectrophotometry), can realize the field quick detection of organophosphorus pesticide, has good practical value.But quick measuring card is by the change color of visual inspection card, therefore generally can only be for the observational measurement of the vegetable sample of severe overweight.The principle of enzyme inhibition-spectrophotometry is that the variation based on absorbance detects, but in vegetable and fruit, a large amount of pigments can affect the accuracy of testing result.And said method exists, and the recovery is low, false retrieval, undetected ratio are higher, poor repeatability, be difficult to the shortcomings such as requirement that meet low-residual and quantitatively detect.Enzyme sensor is the sensor that utilizes the toxicity principle of organophosphorus to develop, and compares with traditional analytical approach, and it has the advantages such as high specificity, analysis speed be fast, simple in structure, with low cost.The key of enzyme sensor is the technique for fixing of enzyme, and the technique for fixing of enzyme comprises the fixing means of enzyme and the carrier material of immobilized enzyme.It directly affects the main performances such as the immobilization of biomolecule, the stability of enzyme sensor and sensitivity.
The object of invention is to provide a kind of enzyme sensor preparation method that can overcome above-mentioned defect and detection chlorpyrifos pesticide residue simple to operate, highly sensitive, to detect limit for height.
Its technical scheme is: a kind of enzyme sensor preparation method who detects chlorpyrifos pesticide residue; it is characterized in that: the responsive interface of mediated enzyme biosensors forms and comprises nano composite membrane and the 0.5%Nafion diaphragm of being prepared by many walls carbon, tin ash, shitosan, and then the technology of immobilized enzyme.
Described a kind of enzyme sensor preparation method who detects chlorpyrifos pesticide residue, it is characterized in that: the cleaning of gold electrode (d=1mm), (preparing many walls carbon-stannic oxide nanometer compound substance the shitosan of usining disperses as spreading agent for the structure at the responsive interface of enzyme sensor and process sign, utilize the common modified electrode of synergy of many walls carbon-tin ash), the foundation of enzyme sensor typical curve, the detection of enzyme sensor performance, the detection of enzyme sensor to actual sample.
Described a kind of enzyme sensor preparation method who detects chlorpyrifos pesticide residue, is characterized in that: the optimization of experiment condition, mainly comprises that pH and the agricultural chemicals of enzyme dripping quantity, test end liquid suppresses the time; Enzyme sensor Performance Detection comprises reappearance, stability, reproducibility and the enzyme sensor mensuration to the vegetable sample recovery.
Its preparation principle is: take acetylcholinesterase catalytic as basic enzyme electrochemical sensor be that acetylcholinesterase is fixed on to gold electrode surfaces, catalytic substrate acetyl thio choline (ATCl) hydrolysis, produce the electroactive sulfydryl choline of tool, sulfydryl choline can be in gold electrode surfaces generation oxidation reaction, the electric signal producing can be converted into electric current, under certain condition the activity of the large I reflection acetylcholinesterase of this electric current.Due to acetylcholinesterase be a kind of take three serine residues conjuncted be active enzyme, when any one amino acid residue is wherein destroyed, will make enzyme loss of catalytic activity.Organophosphorus pesticide and acetylcholinesterase interaction energy form stable phosphorylated cholinesterase and destroy its amino acid residue structure, thereby acetylcholinesterase is lost activity, and reduce the cholinolytic ability of catalysis acetyl thio.Therefore,, in the substrate solution adding containing organophosphorus pesticide, because part enzymatic activity is suppressed, cause its corresponding electric current to reduce.By comparing the size of the current signal of the suppressed front and back of enzyme, can carry out qualitative detection to organophosphorus pesticide.The present invention adopts usings many walls carbon-tin ash compound and the 0.5%Nafion solution that shitosan prepared as spreading agent gold electrode is modified.Multi-walled carbon nano-tubes (MWCNTs) has unique electrochemical properties, higher chemical stability and physical strength, is widely used in the making at the responsive interface of enzyme sensor.Shitosan (CHIT) belongs to polysaccharide, it has excellent film forming, adsorbability, gas penetration potential and perviousness, after film forming, have good adsorbability, stability and good biocompatibility, its abundant amino, cellular structure make it be widely used in the preparation of the fixing and modified electrode of biomolecule.The aminoterminal of shitosan connects tin ash, for enzyme fixedly providing of electrode surface a good bioelectric interface, the biologically active that has kept enzyme is to be used for and agricultural chemicals carries out inhibitory reaction.In addition, tin ash has transmissibility and the biocompatibility that higher specific surface area, stability and electric conductivity can increase electrode surface electronics, and then increases the response current of enzyme sensor.Before the mediated enzyme biosensors that adopts the present invention to make can be gathered at vegetables and fruits, be gone on the market, carry out the Fast Measurement of residues of pesticides, directly whether persticide residue is exceeded standard and detected, avoid because of the edible vegetables and fruits that contain remains of pesticide cause poisoning, for crop production safety and consumption provide the technical support of residue detection.
For reaching above object, take following technical scheme to realize: a kind of enzyme sensor preparation method who detects chlorpyrifos pesticide residue, it is characterized in that: cleaning, activation and the performance test of naked gold electrode before the preparation of (1) enzyme sensor, if the spike potential in test loop volt-ampere curve is poor below 120mV, oxidation peak and reduction peak are symmetrical, described gold electrode can be used, otherwise will return in cleaning step, until meet the requirements.(2) cleaned naked gold electrode surfaces is dripped and is coated with finely dispersed many walls carbon-tin ash-shitosan complex liquid, then modifies 0.5%Nafion solution, then fixing acetylcholinesterase.After enzyme sensor preparation finishes, put in refrigerator 4 ℃ and save backup.
For reaching above object, take following technical scheme to realize: a kind of enzyme sensor preparation method who detects chlorpyrifos pesticide residue, it is characterized in that: (1) does cyclic voltammetric method scanning with the different speed of sweeping by the above-mentioned enzyme sensor preparing in work end liquid, can obtain peak value and sweep fast relation, obtaining being subject to diffusion control.(2) configure a series of chlopyrifos titer, carry out cyclic voltammetry scan, the rate that is inhibited, further draws typical curve, sensing range and the detectability of the enzyme sensor of above-mentioned preparation; (3) configure the pesticide solution that a series of frequent mixing are used, to detect the selectivity of prepared enzyme sensor; (4) by cyclic voltammetry scan multistage, verify the stability of above-mentioned enzyme sensor; (5) actual fruits and vegetables sample is analyzed to the recovery that draws this enzyme sensor.
The present invention is usingd the shitosan with good biocompatibility and film forming and is disperseed the prepared many walls carbon-tin ash-chitosan nano complexes membrane becoming of many walls carbon and tin ash can promote the transmission of electronics in electrochemical reaction as spreading agent, improve the response current on electrode, improve the microenvironment of electrode surface, thereby can be used as carrier material, in order to prepare the strong and highly sensitive enzyme sensor of response signal; 0.5%Nafion diaphragm has higher stability and high-sequential, has improved the effectively fixing of sensor surface enzyme, thereby has increased accuracy of detection.
The preparation technology of described enzyme sensor is as follows: (1) is got many walls carbon-tin ash-chitosan nano composite membrane that 2.5 μ L prepare and dripped and be coated in the gold electrode surfaces that pre-service is good, and standing 1h under normal temperature, rinses electrode surface with the PBS damping fluid of PH=7.5; (2) after electrode surface dries, get 2.5 μ L acetylcholinesterases and drip and be coated in electrode surface, standing 2h at 4 ℃, then rinses surface with the phosphate buffer of PH=7.5, and nitrogen dries up; (3) get 2.5 μ L0.5%Nafion diaphragms and drip and be coated in electrode surface, standing 1.5h under normal temperature, then rinses surface with the PBS damping fluid of PH=7.5, and nitrogen dries up; (4) by above-mentioned, through nanometer-material-modified good electrode, be immersed in chlopyrifos solution, the lower immersion of normal temperature 14min, rinses surface with PBS damping fluid after taking out, and dries stand-by.
accompanying drawing explanation
The cyclic voltammetric assembly drawing of accompanying drawing 1 biology sensor modification.
Accompanying drawing 2 biosensor test liquid pH values are optimized.
Accompanying drawing 3 biosensor tests suppress time-optimized.
Embodiment
Embodiment: the preparation of (1) many walls carbon-tin ash-chitosan nano composite membrane: add 0.1g shitosan to be prepared into 0.2% chitosan solution in 50mL acetic acid solution.In 4mL 0.2% chitosan solution, add 1mg tin ash, 3mg multi-walled carbon nano-tubes, in 25 ℃ of water, ultrasonic concussion is 30 minutes, and many walls carbon-tin ash-chitosan nano composite membrane is successfully prepared; (3) cleaning of gold electrode: before gold electrode is modified, first immerse the middle 15min of immersion of " piranha " solution (H2SO4:30% H2O2=3:1) of heat, water cleans up, next with the Al2O3 slurry of 0.3 μ m, 30nm, on chamois leather, be polished to minute surface, after polishing, with distilled water, wash away except surface contaminants, then move in ultrasonic water bath and clean, each 5min, repeat secondary, then use successively HNO3, absolute ethyl alcohol and the deionized water ultrasonic cleaning of 6mol/L, dry under nitrogen environment.(4) activation of gold electrode: after thoroughly cleaning, electrode is with cyclic voltammetry activation in 0.5mol/L H2SO4 solution, and sweep limit 1.0V～-1.0V, scans repeatedly until reach stable cyclic voltammogram.(5) test of the gold electrode that pre-service is good: run cyclic voltammetry curve in containing the 0.20 mol/L KNO3 solution of 1 * 10-3 mol/L K3Fe (CN) 6, to test the performance of described gold electrode, sweep velocity 50mV/S, sweep limit is-0.1V～0.6V; Spike potential in described cyclic voltammetry curve is poor below 80mV, and approaches as far as possible 64mV, and described gold electrode can be used, otherwise will return in step (3), processes described gold electrode, until meet the requirements.(6) get the compound drop of the finely dispersed many walls carbon-tin ash-chitosan nano of 2.5 μ L and be coated in electrode surface, 1h under normal temperature, then rinses surface with the phosphate buffer of pH7.5, and nitrogen dries up.(7) after electrode surface dries, get 2.5 μ L acetylcholinesterases and drip and be coated in electrode surface, standing 2h at 4 ℃, then rinses surface with the phosphate buffer of PH=7.5, and nitrogen dries up.(8) get 2.5 μ L0.5%Nafion diaphragms and drip and be coated in electrode surface, standing 1.5h under normal temperature, then rinses surface with the PBS damping fluid of PH=7.5, and nitrogen dries up.(9) by above-mentioned, through nanometer-material-modified electrode, be immersed in chlopyrifos solution, under room temperature, soak 14min, after taking out, with PBS damping fluid, rinse surface, dry stand-by.(10) from chlopyrifos solution concentration, test end liquid pH and incubation time three aspects:, the experiment condition of prepared enzyme sensor is optimized, the scope of chlopyrifos solution concentration is 0.1-20 μ g/mL, the scope of pH is 6.0-8.0, and the scope of incubation time is 6-14min.(11) configure the chlopyrifos standard solution of 0. 05-100000 ng/mL, the above-mentioned enzyme sensor preparing is immersed respectively to the chlopyrifos standard solution of variable concentrations, hatch at normal temperatures 14min, before and after detection reaction, curent change obtains its working curve.(12) select 5 enzyme sensors that prepare under the same terms to detect its reappearance; The continuous chlopyrifos solution that detects successively same concentrations for 7 days is to detect its stability.(13) choose representational vegetable sample, with moist rag by the earth wiped clean of vegetable surface, be chopped into the square fragment of 2cm left and right, remove sample 2g, put into extraction flask, add 10ml phosphate buffer, put into ultrasonic extraction apparatus, ultrasonic extraction 3 minutes, pours out the standing 2min of extract, standby.
The detection method operating procedure of this kind of enzyme sensor detection chlorpyrifos pesticide residue is simple, detection time is shorter, detect pesticide concentration scope wide, highly sensitive, good stability, power of regeneration is high and analysis has the good recovery and reappearance to actual sample, meets China's Fast Determination of Pesticide Residue technical development and internationalization requirement.
Claims (2)
1. an enzyme sensor preparation method who detects chlorpyrifos pesticide residue, is characterized in that: a kind of preparation of novel many walls carbon-tin ash-chitosan nano composite material, and preparation process is:
In 50mL acetic acid solution, add 0.1g shitosan to be prepared into 0.2% chitosan solution, in 4mL 0.2% chitosan solution, add 1mg tin ash, 3mg multi-walled carbon nano-tubes, in 25 ℃ of water, ultrasonic concussion is 30 minutes, obtains uniform many walls carbon-tin ash-chitosan nano composite membrane dispersion liquid.
2. a kind of enzyme sensor preparation method who detects chlorpyrifos pesticide residue as claimed in claim 1, is characterized in that: utilize the act synergistically enzyme sensor preparation process of common modified electrode of many walls carbon-tin ash-chitosan nano composite membrane to be:
Get many walls carbon-tin ash-chitosan nano composite membrane that 2.5 μ L prepare and drip the gold electrode surfaces that is coated in Φ=1mm that pre-service is good, standing 1h under normal temperature, rinses electrode surface with the phosphate buffer of pH7.5; After electrode surface dries, get 2.5 μ L acetylcholinesterases and drip and be coated in electrode surface, standing 2h at 4 ℃, then rinses surface with the phosphate buffer of pH7.5, and nitrogen dries up; Get 2.5 μ L 0.5% Nafion diaphragms and drip and be coated in electrode surface, standing 1.5h under normal temperature, then rinses surface with the phosphate buffer of pH7.5, and nitrogen dries up; By above-mentioned, through nanometer-material-modified good electrode, be immersed in chlopyrifos solution, under normal temperature, soak 14min, after taking-up, with phosphate buffer, rinse surface, dry stand-byly, enzyme sensor completes, and is kept under 4 ° of C conditions standby.
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| CN104634843A (en) * | 2015-03-02 | 2015-05-20 | 淄博瑞谷自动化控制设备有限公司 | Preparation method and application of sensor based on screen-printed carbon electrode |
| 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 |
| CN105301077A (en) * | 2015-10-19 | 2016-02-03 | 山东理工大学 | Preparation method of aptamer sensor for detecting chlorpyrifos |
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| CN102788827A (en) * | 2012-09-10 | 2012-11-21 | 山东理工大学 | One-step preparation method for electro-deposition acetylcholinesterase biosensor |
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| CHEN ZHAI ET AL.: "Acetylcholinesterasebiosensorbasedonchitosan/prussianblue/multiwall carbon nanotubes/hollow gold nanospheres nanocomposite film by one-step electrodeposition", 《BIOSENSORS AND BIOELECTRONICS》, 29 October 2012 (2012-10-29) * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104634843A (en) * | 2015-03-02 | 2015-05-20 | 淄博瑞谷自动化控制设备有限公司 | Preparation method and application of sensor based on screen-printed carbon electrode |
| 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 |
| CN105301077A (en) * | 2015-10-19 | 2016-02-03 | 山东理工大学 | Preparation method of aptamer sensor for detecting chlorpyrifos |
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