CN103558269B - A kind of enzyme sensor preparation method detecting chlorpyrifos pesticide residue - Google Patents
A kind of enzyme sensor preparation method detecting chlorpyrifos pesticide residue Download PDFInfo
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Abstract
The present invention relates to a kind of enzyme sensor preparation method detecting chlorpyrifos pesticide residue, belong to biology sensor preparation field.The first step of the present invention is the preparation of many walls carbon-tin ash-chitosan nano composite material and 0.5%Nafion solution: with many walls carbon, tin ash, shitosan for raw material, utilize the chemical stability of the good electrical chemistry redox activity of many walls carbon-tin ash-Chitosan Composites, good Bc and height, modify working electrode to promote electro transfer; Second step is the enzyme sensor preparation of acetylcholinesterase and many walls carbon-tin ash-common modified electrode of chitosan nano composite membrane.Acetylcholinesterasebiosensor biosensor of the present invention, detection time is shorter, highly sensitive, selectivity, good stability, and power of regeneration is good, and the recovery meets the requirements.
Description
Technical field
The invention provides a kind of enzyme sensor preparation method detecting 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 that is harmful, highly toxic pesticide in fruits and vegetables is the focus that people pay close attention to always, and be also after 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 our people's living standard improves 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 cause up to a hundred to eat the acute poisoning event caused by the agricultural product of pollution by pesticides every year, have a strong impact on the healthy of consumers in general, therefore to be exceeded standard the food-safety problem caused by residues of pesticides, be more and more subject to the attention of national governments and the public.Organophosphorus pesticide is the class agricultural chemicals that China is most widely used, and its Chlorpyrifos is one of maximum insecticide variety of current produced worldwide and sales volume.Chlopyrifos is that efficiently poisoning broad spectrum pesticide is held concurrently herbicide, has certain interior absorption, can be transferred to human body, have potential carcinogenesis to human body by the inrichment of food chain.Visible, strengthening to the detection of Residual Pesticides in Farm Produce to preserving the ecological environment, especially ensureing that human health has very profound significance, its Chlorpyrifos is the big event that environment and Pesticide Residues detect always.
Traditional pesticide residue detection 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 require very high to the technical merit of analyst, be unsuitable for field quick detection.Conventional Fast Determination of Pesticide Residue method has inhibiting AChE (enzyme level test paper method and enzyme level spectrophotometric method), can realize the field quick detection of organophosphorus pesticide, have good practical value.But quick measuring card is changed by the color of visual inspection card, therefore generally can only be used for the observational measurement of the vegetable sample of severe overweight.Enzyme level spectrophotometric ratio juris carries out detecting based on the change of absorbance, but pigments a large amount of in vegetable and fruit 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 meet the shortcomings such as low-residual and the requirement that quantitatively detects.Enzyme sensor is the sensor utilizing the toxicity principle of organophosphorus to develop, compared with traditional analytical approach, and the advantages such as it has high specificity, analysis speed is fast, structure is simple, 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 detecting chlorpyrifos pesticide residue; it is characterized in that: the sensitive interface composition of mediated enzyme biosensors comprises the nano composite membrane and 0.5%Nafion diaphragm prepared by many walls carbon, tin ash, shitosan, and then the technology of immobilized enzyme.
Described a kind of enzyme sensor preparation method detecting chlorpyrifos pesticide residue, it is characterized in that: the cleaning of gold electrode (d=1mm), the structure of enzyme sensor sensitive interface and process characterize (to be prepared many walls carbon-stannic oxide nanometer compound substance and disperses as spreading agent using shitosan, 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, enzyme sensor is to the detection of actual sample.
Described a kind of enzyme sensor preparation method detecting chlorpyrifos pesticide residue, is characterized in that: the optimization of experiment condition, mainly comprises enzyme dripping quantity, the pH testing end liquid and agricultural chemicals and 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: the enzyme electrochemical sensor based on the catalytic of acetylcholinesterase is that acetylcholinesterase is fixed on gold electrode surfaces, catalytic substrate acetylthiocholine (ATCl) is hydrolyzed, produce the electroactive sulfydryl choline of tool, sulfydryl choline can in gold electrode surfaces generation oxidation reaction, the electric signal produced can be converted into electric current, under certain condition the activity of the large I reflection acetylcholinesterase of this electric current.Because acetylcholinesterase is a kind of conjuncted for active enzyme with three serine residues, when any one amino acid residue is wherein destroyed, enzyme loss of catalytic activity will be made.Organophosphorus pesticide and acetylcholinesterase effect can form stable phosphorylated cholinesterase and destroy its amino acid residue structure, thus acetylcholinesterase is lost activity, and reduce the ability of catalysis acetylthiocholine hydrolysis.Therefore, adding in the substrate solution containing organophosphorus pesticide, causing because part enzymatic activity is suppressed its phase induced current to reduce.By comparing the size of the current signal of the suppressed front and back of enzyme, qualitative detection can be carried out to organophosphorus pesticide.The present invention adopts many walls carbon-tin ash compound of preparing using shitosan as spreading agent and 0.5%Nafion solution to modify gold electrode.Multi-walled carbon nano-tubes (MWCNTs) has unique electrochemical properties, higher chemical stability and physical strength, is widely used in the making of enzyme sensor sensitive interface.Shitosan (CHIT) belongs to polysaccharide, it has excellent film forming, adsorbability, gas penetration potential and perviousness, have good adsorbability, stability and good biocompatibility after film forming, its abundant amino, cellular structure make it be widely used in the preparation of the fixing of biomolecule and modified electrode.The aminoterminal of shitosan connects tin ash, and for enzyme fixedly provides a good bioelectric interface at electrode surface, the biologically active maintaining enzyme is to be used for and agricultural chemicals carries out suppressions and reacts.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 adopting the mediated enzyme biosensors 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 whether persticide residue is exceeded standard and detect, avoid the vegetables and fruits because eating containing remains of pesticide and cause poisoning, for crop production safety and consumption provide the technical support of residue detection.
For reaching above object, following technical scheme is taked to realize: a kind of enzyme sensor preparation method detecting chlorpyrifos pesticide residue, it is characterized in that: the cleaning of naked gold electrode before the preparation of (1) enzyme sensor, activation and performance test, if the spike potential difference in test loop volt-ampere curve is at below 120mV, oxidation peak and reduction peak symmetry, then described gold electrode can use, 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 fixes acetylcholinesterase.After enzyme sensor preparation terminates, to put in refrigerator 4 DEG C and save backup.
For reaching above object, following technical scheme is taked to realize: a kind of enzyme sensor preparation method detecting chlorpyrifos pesticide residue, it is characterized in that: the above-mentioned enzyme sensor prepared is done the scanning of cyclic voltammetric method with different speed of sweeping by (1) in work end liquid, peak value can be obtained and sweep fast relation, obtain being control by diffusion.(2) configure a series of chlopyrifos titer, carry out cyclic voltammetry scan, be inhibited rate, draws the typical curve of the enzyme sensor of above-mentioned preparation, sensing range and detectability further; (3) a series of the pesticide solution often used in combination is configured, to detect the selectivity of prepared enzyme sensor; (4) the stability of above-mentioned enzyme sensor is verified by cyclic voltammetry scan multistage; (5) recovery drawing this enzyme sensor is analyzed to actual fruits and vegetables sample.
The present invention using the shitosan with good biocompatibility and film forming as dispersant many walls carbon with tin ash prepared by many walls carbon-tin ash-chitosan nano complexes membrane of becoming can promote the transmission of electronics in electrochemical reaction, improve the response current on electrode, improve the microenvironment of electrode surface, thus can 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, improves the effectively fixing of sensor surface enzyme, thus increases 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 good gold electrode surfaces of pre-service, 1h is left standstill, with the PBS wash buffer electrode surface of PH=7.5 under normal temperature; (2) after electrode surface dries, get 2.5 μ L acetylcholinesterases and drip and be coated in electrode surface, leave standstill 2h at 4 DEG C, then rinse surface with the phosphate buffer of PH=7.5, nitrogen dries up; (3) get 2.5 μ L0.5%Nafion diaphragms to drip and be coated in electrode surface, leave standstill 1.5h under normal temperature, then use the PBS wash buffer surface of PH=7.5, nitrogen dries up; (4) be immersed in chlopyrifos solution by above-mentioned through nanometer-material-modified good electrode, under under normal temperature, soak 14min, with PBS wash buffer surface after taking out, dry stand-by.
Accompanying drawing explanation
The cyclic voltammetric assembly drawing of accompanying drawing 1 biology sensor modification.
Accompanying drawing 2 biosensor test liquid pH value is optimized.
Accompanying drawing 3 biosensor test suppresses time-optimized.
Embodiment
Embodiment: the preparation of (1) many walls carbon-tin ash-chitosan nano composite membrane: add 0.1g shitosan and be prepared into 0.2% chitosan solution in 50mL acetic acid solution.In 4mL0.2% chitosan solution, add 1mg tin ash, 3mg multi-walled carbon nano-tubes, ultrasonic vibration 30 minutes in 25 DEG C of water, then many walls carbon-tin ash-chitosan nano composite membrane is successfully prepared.
(3) cleaning of gold electrode: before gold electrode is modified, first immerse in " piranha " solution (H2SO4:30%H2O2=3:1) of heat and soak 15min, clean up with water, the Al2O3 slurry of following use 0.3 μm, 30nm is polished to minute surface on chamois leather, washes away except surface contaminants, then move in ultrasonic water bath and clean after polishing with distilled water, each 5min, repeat secondary, then use the HNO3 of 6mol/L, absolute ethyl alcohol and deionized water ultrasonic cleaning successively, dry under nitrogen environment.(4) activation of gold electrode: thoroughly after cleaning, electrode is with cyclic voltammetry activation in 0.5mol/LH2SO4 solution, and sweep limit 1.0V ~-1.0V, scanning is till reaching stable cyclic voltammogram repeatedly.(5) test of the gold electrode that pre-service is good: run cyclic voltammetry curve in containing the 0.20mol/LKNO3 solution of 1 × 10-3mol/LK3Fe (CN) 6, to test the performance of described gold electrode, sweep velocity 50mV/S, sweep limit is-0.1V ~ 0.6V; When the spike potential difference in described cyclic voltammetry curve is at below 80mV, and as far as possible close to 64mV, described gold electrode can use, otherwise will return in step (3), processes described gold electrode, until meet the requirements.(6) get the finely dispersed many walls carbon-tin ash-chitosan nano compound drop of 2.5 μ L and be coated in electrode surface, 1h under normal temperature, then rinse surface with the phosphate buffer of pH7.5, nitrogen dries up.(7) after electrode surface dries, get 2.5 μ L acetylcholinesterases and drip and be coated in electrode surface, leave standstill 2h at 4 DEG C, then rinse surface with the phosphate buffer of PH=7.5, nitrogen dries up.(8) get 2.5 μ L0.5%Nafion diaphragms to drip and be coated in electrode surface, leave standstill 1.5h under normal temperature, then use the PBS wash buffer surface of PH=7.5, nitrogen dries up.(9) be immersed in chlopyrifos solution by above-mentioned through nanometer-material-modified electrode, under room temperature, soak 14min, with PBS wash buffer surface after taking out, dry stand-by.(10) be optimized from chlopyrifos solution concentration, test end liquid pH and incubation time tripartite in the face of the experiment condition of prepared enzyme sensor, 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 0.05-100000ng/mL, the above-mentioned enzyme sensor prepared is immersed respectively the chlopyrifos standard solution of variable concentrations, hatch 14min at normal temperatures, before and after detection reaction, curent change obtains its working curve.(12) 5 enzyme sensors prepared under the same terms are selected to detect its reappearance; The continuous chlopyrifos solution detecting same concentrations for 7 days is successively to detect its stability.(13) representational vegetable sample is chosen, with the rag of humidity by the earth wiped clean of vegetable surface, be chopped into the square fragment of about 2cm, remove sample 2g, put into extraction flask, add 10ml phosphate buffer, put into ultrasonic extraction apparatus, ultrasonic extraction 3 minutes, pours out extract and leaves standstill 2min, for subsequent use.
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 have the good recovery and reappearance to actual sample analysis, meets China's Fast Determination of Pesticide Residue technical development and internationalization requirement.
Claims (1)
1. one kind is detected the enzyme sensor preparation method of chlorpyrifos pesticide residue, it is characterized in that: a kind of preparation of novel many walls carbon-tin ash-chitosan nano composite material, preparation process is: in 50mL acetic acid solution, add 0.1g shitosan be prepared into 0.2% chitosan solution, 1mg tin ash, 3mg multi-walled carbon nano-tubes is added in 4mL0.2% chitosan solution, ultrasonic vibration 30 minutes in 25 DEG C of water, obtains uniform many walls carbon-tin ash-chitosan nano composite membrane dispersion liquid; Get many walls carbon-tin ash-chitosan nano composite membrane that 2.5 μ L prepare and drip the gold electrode surfaces being coated in the good Φ=1mm of pre-service, under normal temperature, leave standstill 1h, rinse 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, leave standstill 2h at 4 DEG C, then rinse surface with the phosphate buffer of pH7.5, nitrogen dries up; Get 2.5 μ L0.5%Nafion diaphragms to drip and be coated in electrode surface, leave standstill 1.5h under normal temperature, then rinse surface with the phosphate buffer of pH7.5, nitrogen dries up; Be immersed in chlopyrifos solution by above-mentioned through nanometer-material-modified good electrode, soak 14min under normal temperature, rinse surface with phosphate buffer after taking-up, dry stand-by, enzyme sensor completes, for subsequent use under being kept at 4 DEG C of conditions.
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| WO2020065042A1 (en) * | 2018-09-28 | 2020-04-02 | Klearia | Method for cleaning and/or regenerating a glass microfluidic sensor for the analysis of metals |
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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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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020065042A1 (en) * | 2018-09-28 | 2020-04-02 | Klearia | Method for cleaning and/or regenerating a glass microfluidic sensor for the analysis of metals |
| FR3086554A1 (en) * | 2018-09-28 | 2020-04-03 | Klearia | METHOD FOR CLEANING AND / OR REGENERATION OF A GLASS MICROFLUIDIC SENSOR FOR METAL ANALYSIS |
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