CN101726521B - Biosensor for rapidly detecting sterigmatocystin and assembling method thereof - Google Patents
Biosensor for rapidly detecting sterigmatocystin and assembling method thereof Download PDFInfo
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- CN101726521B CN101726521B CN2009101942606A CN200910194260A CN101726521B CN 101726521 B CN101726521 B CN 101726521B CN 2009101942606 A CN2009101942606 A CN 2009101942606A CN 200910194260 A CN200910194260 A CN 200910194260A CN 101726521 B CN101726521 B CN 101726521B
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Abstract
The invention discloses a biosensor for rapidly detecting sterigmatocystin and an assembling method thereof; the biosensor comprises a substrate electrode and a reaction layer assembled on the substrate electrode, the reaction layer comprises a polymer film, an electronic carrier, chitosan gel and 6-methoxy group furocoumarin coumarin oxidase, the 6-methoxy group furocoumarin coumarin oxidase is fixed in the electronic carrier-chitosan mixing membrane, and then is assembled on the substrate electrode decorated by the polymer film, thereby obtaining the biosensor for detecting the sterigmatocystin; the biosensor has high sensor sensitivity, rapid response time and strong anti-jamming capability, the detection lower limit to the sterigmatocystin can reach 3ng/ml, the biosensor can be used for practical sample detection, and the average recovery rate is 95.8 percent; the biosensor has simple manufacturing, convenient usage, good stability and long service life and provides a new detection method for rapidly detecting the sterigmatocystin content in practical samples.
Description
Technical field
The invention belongs to field of biosensors, relate to a kind of biology sensor and assemble method thereof that is used for the fast detecting sterigmatocystin.
Background technology
(Sterigmatocystin is a kind of mycotoxin of carcinogenic teratogenesis ST) to sterigmatocystin, is that (the two structural similarity all is to be connected to form by bifuran and oxa-anthraquinone to aflatoxin for Aflatoxin, synthetic precursor AFT).ST is the 2B class carcinogen that international cancer research institution divides, and is closely related with lung cancer, liver cancer, cancer of the stomach, is the main fungal contamination source in the cereal such as grain, forage grass, corn, wheat, peanut.In China, malignant tumour district occurred frequently major part is the pollution owing to ST in grain and the cereal, yet people are fewer to the acute toxicity and chronic toxicity understanding of ST, and the content of therefore measuring ST has great importance.At present; The method of measuring ST mainly contains TLC (TLC), high performance liquid chromatography (HPLC), LC-MS method (LC-MS), gas chromatography mass spectrometry method (GC-MS) and coupling mass spectroscopy (Turner NW such as (Tandem MS); Subrahmanyam S, Piletsky SA.Analytical methods fordetermination of mycotoxins:A review.Anal Chim Acta 2009; 632:168-180.).These methods respectively have its characteristics, but the needs that have use expensive instrument, the complex operation that has, the sample pre-treatments that has trouble; In addition, because the fluorescigenic intensity of ST can not show a candle to aflatoxin, use the method for derivatization then to introduce bigger error easily, sensitive and accurate detection need be used mass detector usually, thereby is difficult to realize the fast detecting of ST content in the actual sample.
Enzyme biologic sensor is a kind of alternative method preferably; Characteristics such as it is fast to have detection speed, simple to operate, the enzyme biologic sensor method of having reported; As in inventor's Chinese patent ZL 200410028018.9; Provide and utilized the Aflatoxin-detofizyme modified electrode to prepare biology sensor detection sterigmatocystin, its detection line is 10ng/mL, and the response time is 2min.But such biology sensor still remains to be improved, and improves its sensitivity, stability and reappearance etc.
Summary of the invention
The objective of the invention is to deficiency, a kind of biology sensor that can be used for aspergillus versicolor cellulose content fast detecting in the actual sample is provided to prior art.This biology sensor sensitivity is higher, and stability and reappearance are better, and the response time is faster, and can be used for biology sensor and assemble method thereof that actual sample ST detects fast.
A kind of biology sensor that is used for the fast detecting sterigmatocystin of the present invention comprises basal electrode and is assembled in the responding layer on the basal electrode, and described responding layer comprises: polymer film is made up of the poly-o-phenylenediamine of electropolymerization on basal electrode; This polymer film is modified on the said basal electrode; Electron transit mediator is organic molecule or macromolecule electron transit mediator; Chitosan gel rubber is processed by chitosan solution; And the two furocoumarin oxidase of 6-methoxyl; Wherein, be dispersed in by the SWCN after the activation and form electron transit mediator-chitosan gel rubber hybrid films in the chitosan solution; The two furocoumarin oxidase embeddings of said 6-methoxyl are fixed on electron transit mediator-chitosan gel rubber hybrid films, are assembled on the basal electrode of polymer film modification again, are formed for detecting the biology sensor of sterigmatocystin.
The preferred polymer film of the present invention is a poly-o-phenylenediamine; O-phenylenediamine can pass through electropolymerization; Methods such as organic synthesis make polymer thin film on a lot of conducting bases; It promptly is suitable in organic bath, using, and also can be used for aqueous electrolyte solution, has higher enclosed pasture efficient and stability.
Electron transit mediator of the present invention can be selected organic molecule or macromolecule electron transit mediator for use.The organic molecule electron transit mediator mainly contains ferrocene and derivant, organic dyestuff, quinone and derivant, ionic liquid etc.; Redox polymerses such as the macromolecule electron transit mediator mainly contains CNT, metal nanoparticle, the transition metal ion type that appraises at the current rate and organic oxidation reduced form etc.The preferred electron transit mediator of the present invention is the SWCN after the activation, can adopt strong acid or strong oxidizer to carry out activation processing.Among the present invention SWCN is dispersed in the chitosan solution, also can selects for use collosol and gel (sol-gel) and compound thereof, alum and some other compounds to replace shitosan with film forming ability.
A kind of assemble method that is used for the biology sensor of fast detecting sterigmatocystin of the present invention may further comprise the steps:
A, the o-phenylenediamine monomer is aggregated in the basal electrode surface through electrochemical method, obtains the basal electrode that the poly-o-phenylenediamine polymer film is modified;
B, chitosan solution is processed chitosan gel rubber;
C, with after the SWCN activation processing, join mixing in the above-mentioned chitosan solution, promptly get SWCN-shitosan mixed liquor, subsequent use;
D, the two furocoumarin oxidase solution of preparation 6-methoxyl, subsequent use;
E, with the two furocoumarin oxidase solution of 6-methoxyl and SWCN-shitosan mixed liquor mixing, it is surperficial to drip the basal electrode of modifying at the poly-o-phenylenediamine polymer film, 4 ℃ of assemblings are spent the night, and promptly get described biology sensor.
Preferred manufacturing procedure may further comprise the steps:
The preparation that a, poly-o-phenylenediamine are modified basal electrode:
The basal electrode that cleans up is before use earlier at H
2SO
4In carry out electrochemical pre-treatment, scan till electrode signal is stable with cyclic voltammetry; The basal electrode of handling well is inserted into the saturated 0.1-0.5mol/L H that contains the 0.01-0.05mol/L o-phenylenediamine of nitrogen
2SO
4In the electropolymerization liquid,, can form one deck poly-o-phenylenediamine film on the basal electrode surface with the fast cyclic voltammetric electropolymerization of sweeping of 0.05-0.1V/s 30-60 circle;
The preparation of b, chitosan solution:
With acetate dissolving shitosan powder, make its concentration between 0.1%-1.5%, fully dissolve the insoluble impurity of back elimination, the pH value of regulating chitosan solution is 5.5, places 4 ℃ of preservations subsequent use;
The preparation of c, SWCN-shitosan mixed liquor:
With strong acid or strong oxidizer activation SWCN, be dissolved in the prepared chitosan solution of step b after being washed till neutral oven dry, ultrasonic dispersion makes SWCN-chitosan solution of finely dispersed 0.1mg/mL-2.5mg/mL;
D, the two oxidasic preparations of furocoumarin of 6-methoxyl:
The clone is had the two oxidasic gene engineering colibacillus abduction deliverings of furocoumarin of methoxyl, collect thalline, carry out purifying with affinity column behind the ultrasonication cell, enzyme is cut fusion and is obtained the two furocoumarin oxidase of single 6-methoxyl; The chromatographic purifying enzyme is cut product, collects the enzymatic activity peak component in the chromatographic process, gets the two furocoumarin oxidase of 6-methoxyl after concentrating; Be mixed with protein concentration and be the enzyme liquid of 2mg/mL, subsequent use;
The preparation of the biology sensor that e, the two furocoumarin oxidase of 6-methoxyl are modified:
Get SWCN prepared among the step c-shitosan mixed liquor and drip on the basal electrode that poly-o-phenylenediamine is modified in step a, dry film forming under the room temperature; And then prepared enzyme liquid in the dropping steps d, 4 ℃ of assemblings are spent the night, and promptly get the biology sensor that the two furocoumarin oxidase of 6-methoxyl are modified.
Biology sensor of the present invention mainly is to utilize the two furocoumarin oxidase of 6-methoxyl to detect sterigmatocystin (ST) as the molecular recognition original paper; Its principle is: redox reaction takes place in ST under the two oxidasic catalytic action of furocoumarin of 6-methoxyl; Along with the transfer of electronics, but produce detected electric signal.Biology sensor of the present invention is a high specific for the detection of sterigmatocystin, high selectivity and high sensitivity, can satisfy the detection of aspergillus versicolor cellulose content in the actual sample fully.
Compared with prior art, biology sensor of the present invention has following beneficial effect:
(1) utilize biology sensor of the present invention can realize fast detecting, only need carry out simple pre-service actual sample to actual sample, just can real time and on line monitoring, its response time is fast, less than 10s, to ST response sensitivity more;
(2) biology sensor sensitivity of the present invention is higher, the following 3ng/mL that is limited to that in the PBS of 0.1mol/L pH7.0 detection architecture, ST is detected, and the range of linearity is wide, is 10ng/mL-310ng/mL;
(3) biology sensor of the present invention has good stability and reappearance; Antijamming capability is strong, can Rapid Realization the single or detection of sample in batches; The recovery that detects actual sample is 87.6-105.5%, and detection speed is fast, stablizes long service life;
(4) biology sensor of the present invention use save time, laborsaving, detection speed is fast and simple to operation, need not train and can promote the use of.
Description of drawings
Fig. 1 is the cyclic voltammetry curve of MDCO/CS-SWCNTs/POPD/Au modified electrode in the PBS that contains variable concentrations ST; Wherein, the concentration of the corresponding ST of the variation of curve from a to c is respectively 0ng/mL among the figure, 10ng/mL, 20ng/mL.
Fig. 2 is the response of chronoamperometry detecting sensor to ST; Wherein, illustration is the relation of response current and ST concentration among the figure.
Embodiment
Embodiment one: the biology sensor that is used for the fast detecting sterigmatocystin
The biology sensor that is used for fast detecting actual sample sterigmatocystin of the present invention; Comprise that gold electrode (Au) is basal electrode and is assembled in the responding layer on the gold electrode; Described responding layer comprises polymer film; Electron transit mediator, the two furocoumarin oxidase of chitosan gel rubber and 6-methoxyl.At first on gold electrode, utilize electrochemical method assembling last layer polymer film; Then electron transit mediator is dispersed in the shitosan; Again the two furocoumarin oxidase of 6-methoxyl are embedded in the potpourri of electron transit mediator-shitosan; The two furocoumarin oxidase of formed electron transit mediator-6-methoxyl-shitosan heterocomplex is assembled on the gold electrode of polymer film modification, has formed the biology sensor that the two furocoumarin oxidase of 6-methoxyl are modified.
In the present embodiment, select poly-o-phenylenediamine to be assembled on the gold electrode as polymer film, the electron transit mediator that is adopted is the SWCN after the activation.SWCN is dispersed in and forms SWCN-shitosan suspension in the shitosan, through investment fixing two furocoumarin oxidase of 6-methoxyl in hybrid films, is assembled into the enzyme biologic sensor sensitive to sterigmatocystin.
Embodiment two: be used to detect the preparation of the biology sensor of sterigmatocystin
(1), the preparation of material
(1) working electrode: select gold electrode for use, interior diameter is 2mm, available from Shanghai occasion China instrument company.
(2) o-phenylenediamine (OPD): chemical pure, available from Shanghai chemical reagent company limited.
(3) SWCN (SWCNTs): diameter 2nm, length 5～15 μ m, purity 95% is available from nanometer port, Shenzhen.
(4) shitosan (CS): the deacetylation of 85-90%, mean molecular weight are 1 * 10
6G/mol is available from ZHEJIANG AOXING BIOTECHNOLOGY CO., LTD.
(2), the preparation of poly-o-phenylenediamine modified gold electrode
With gold electrode at H by 7 volumes 98%
2SO
4With 3 volume 30%H
2O
2Soaked 30 minutes in the solution of forming, rinse well with distilled water, using particle diameter more successively is that the alumina powder of 1.0 μ m, 0.3 μ m and 0.05 μ m is polished to minute surface, dries subsequent use after cleaning up.Gold electrode is before use earlier at H
2SO
4In carry out electrochemical pre-treatment, cyclic voltammetry scans till electrode signal is stable.The gold electrode of handling well is inserted in the saturated 0.1-0.5mol/L H2SO4 electropolymerization liquid that contains the 0.01-0.05mol/L o-phenylenediamine of nitrogen; Enclose with the fast cyclic voltammetric electropolymerization of sweeping of 0.05-0.1V/s 30-60; Can form one deck poly-o-phenylenediamine film in gold electrode surfaces, be designated as POPD/Au.
(3) preparation of chitosan solution
An amount of shitosan powder is dissolved in the acetic acid solution, is configured to the chitosan solution of 0.1%-1.5%, fully after the dissolving, the pH value of regulating chitosan solution is 5.5, places 4 ℃ of preservations subsequent use.
(4) preparation of SWCN-shitosan mixed liquor
SWCN carries out purification process before use earlier; Make its activation with strong acid or strong oxidizer; Be dissolved in the prepared chitosan solution of step 3 after being washed till neutral oven dry, ultrasonic dispersion makes SWCN-chitosan solution of finely dispersed 0.1mg/mL-2.5mg/mL.
(5) the two oxidasic preparations of furocoumarin of 6-methoxyl
Two furocoumarin oxidase (MDCO) gene ORF (ORFs) of 6-methoxyl are cloned on the pMAL-C2X carrier (available from U.S. NEB) that contains fusion tag MBP (maltose-binding protein); Make up protokaryon recombinant expression plasmid pMAL-C2X-MDCO; It is imported among the Escherichia coli Rosetta (DE3) (available from Tianjin Bo Meike bio tech ltd); Promptly get the two furocoumarin oxidase gene engineering colon bacillus (Rosetta (DE3)-C2X-MDCO) of the methoxyl of recombinating; Induce with IPTG (isopropylthio β-D-galactoside), realized efficiently expressing of MBP_MDCO fusion.Collect thalline, carry out purifying with the affinity column (amylose is available from Beijing NEB) that is filled with the amylose medium behind the ultrasonication cell, collect eluting peak.Cut fusion with Factor Xa enzyme and obtain single MDCO (the two furocoumarin oxidase of 6-methoxyl).Enzyme is cut product and is further purified with PhenylSepharose 6 Fast Flow hydrophobic chromatographies that (Phenyl Sepharose 6 Fast Flow hydrophobic chromatographies are Sweden Pharmacia Company products; Phenyl sepharose hydrophobic chromatoghaphy medium 6Fast Flow model); The continuous linear gradient elution that adopts salinity to successively decrease, wash-out A liquid is 0.05mol/L PBS, pH 6.5; Wash-out B liquid is 0.05mol/L PBS, 2mol/L (NH
4) 2SO4, pH 6.5, collect the enzymatic activity peak component in the chromatographic process, get the two furocoumarin oxidase (MDCO) of 6-methoxyl after concentrating.Be mixed with protein concentration and be the enzyme liquid of 2mg/mL, subsequent use.
(6) preparation of the biology sensor of the two furocoumarin oxidase modifications of 6-methoxyl
Get SWCN prepared in an amount of step 4-shitosan mixed liquor and drip on the gold electrode that poly-o-phenylenediamine is modified in step 2, dry film forming; And then prepared enzyme liquid in an amount of dropping step 5; 4 ℃ of assemblings are spent the night; Promptly get the biology sensor that the two furocoumarin oxidase of 6-methoxyl are modified; Be designated as MDCO/CS-SWCNTs/POPD/Au, in the PBS of 0.1M pH7.0, soak 2min earlier before electrode uses, the time spent does not place 4 ℃ of kept dry.
In the present embodiment, select for use sterigmatocystin (ST) as the test substrate.
Embodiment three: the MDCO/CS-SWCNTs/POPD/Au modified electrode is to the electrochemical response of sterigmatocystin
The MDCO/CS-SWCNTs/POPD/Au modified electrode that embodiment two is prepared is formed a three-electrode system with general to electrode and optional contrast electrode as working electrode.
In the present embodiment, selecting Ag/AgCl electrode (saturated KCl) for use is contrast electrode, and Pt silk electrode is that electrode is formed three-electrode system, and the PBS of 0.1mol/L pH7.0 is that liquid is supported in electrolysis.
(1), cyclic voltammetry detects ST
Adopt CHI660C electrochemical workstation (Shanghai occasion China instrument company); Utilize three-electrode system (contrast electrode: the Ag/AgCl electrode, to electrode: Pt silk electrode, working electrode: the MDCO/CS-SWCNTs/POPD/Au modified electrode); Select the cyclic voltammetry scan pattern, it is following that electrochemical parameter is set:
Operating potential :-0.8V-+0.2V
Sweep speed: 100mV/s
The tranquillization time: 2s
After setting parameter, in the PBS of 0.1mol/L pH7.0, drip the ST of variable concentrations, adopt cyclic voltammetry to detect behind the mixing.As shown in Figure 1, in not having the electrolyte solution of ST, MDCO/CS-SWCNTs/POPD/Au shows a pair of redox peak that will definitely be contrary, and (curve a) to have realized the Direct Electrochemistry reaction of MDCO on modified electrode.Along with the increase of ST concentration (like curve b=10ng/mL ST; C=20ng/mL ST); The reduction peak current of MDCO modified electrode increases; And oxidation peak current reduces, and oxidation peak and reduction peak current potential remain unchanged basically, explains that MDCO/CS-SWCNTs/POPD/Au has good electrical catalyze reduction effect to ST.
(2), detect electric current-time curve of ST
The CHI660C electrochemical workstation is selected electric current-time detecting pattern, adopt three-electrode system (contrast electrode: the Ag/AgCl electrode, to electrode: Pt silk electrode, working electrode: the MDCO/CS-SWCNTs/POPD/Au modified electrode), it is following that electrochemical parameter is set:
Detect current potential :-0.4V
The tranquillization time: 2s
Stirring rate: 50r/min
After treating that background current is stable, equal time dropping ST in the PBS of 10mL 0.1mol/L pH7.0 can be the ST of variable concentrations, also can be isocyatic ST, the preferably every dropping of the present invention once, the ST change in concentration is 10ng/mL.Visible by Fig. 2, along with the ST concentration gradient changes, response current continues to increase, and this enzyme biologic sensor response is rapid and sensitive, and average response time is less than 10s.The response current of enzyme electrode and ST concentration linear in the 10-310ng/mL scope (Fig. 2 illustration), equation of linear regression is: I (μ A)=0.0389c (ng/ml)+2.5825 (r=0.997) detects and is limited to 3ng/mL.
Embodiment four: the stability of enzyme electrode and reappearance
A MDCO/CS-SWCNTs/POPD/Au modified electrode is placed the pH7.0PBS solution that contains 20ng/mL ST, and cyclic voltammetry scan 100 encloses continuously, and peak shape almost remains unchanged, and explains that enzyme biologic sensor of the present invention has good stability.To 20ng/mL ST replication 11 times, the average relative standard deviation (RSD) of its current-responsive is 3.9% with this biology sensor, and it is repeated to show that this electrode has good detections.In addition, prepare 7 enzyme modified electrodes simultaneously, the RSD to the response current of 20ng/mL ST under the same terms is 4.4%.Show that this modified electrode has good making reappearance.Electrode not the time spent place 4 ℃ pH 7.0 PBS solution top to preserve, during intermittent the use, response signal is still can keep 91% behind 95.5%, 30 day of initial value after 4 days, biology sensor of the present invention can use for a long time.
Embodiment five: the antijamming capability of MDCO/CS-SWCNTs/POPD/Au modified electrode
Adopt chronoamperometry that the antijamming capability of MDCO/CS-SWCNTs/POPD/Au modified electrode is studied; Test condition parameters is provided with shown in embodiment 3 (two); Contain among the PBS of 0.1mol/L pH7.0 of 50ng/mL ST at 10mL; Drip the possible chaff interference of following variable concentrations respectively, analyze its antijamming capability.
The kind of the chaff interference that drips respectively:
1000 times to the PO of ST concentration
4 3-, NO
3-, citrate, fructose, glucose;
500 times of EDTA, NH to ST concentration
4+, oxalic acid, alpha-lactose, ascorbic acid;
100 times of glycocoll, L-serine, uric acid, methyl alcohol, oleic acid to ST concentration;
10 times to the Fe of ST concentration
3+, p-nitrophenol, acetate.
The test effect: be no more than when the control relative error current-responsive value that detects 50ng/mL ST ± 5% situation under, 1000 times PO
4 3-, NO
3-, citrate, fructose, glucose; 500 times EDTA, NH
4+, oxalic acid, alpha-lactose, ascorbic acid; 100 times glycocoll, L-serine, uric acid, methyl alcohol, oleic acid; 10 times Fe
3+, p-nitrophenol, acetate etc. do not produce interference to measuring.
Embodiment six: actual sample is measured and recovery experiment
Enzyme biologic sensor of the present invention is used for actual sample measures and detect its recovery; Described actual sample can be a series of agricultural byproducts and cereal crops such as corn, peanut, rice, wheat, also can be feed, beverage, soy sauce, olive oil, a series of products that possibly contain ST of peanut wet goods.
Select for use corn sample to detect in the present embodiment.
The processing of corn sample: get 1g high-grade maize powder, add the ST of variable concentrations, oscillation extraction 45min in the methanol solution of 5ml 80%, it is centrifugal that (5000r/min, 10min), supernatant detects by 1: 5 (V/V) dilution back with the PBS of 0.1mol/L pH7.0.Other samples can detect through after the simple pre-service according to similar method.
Cyclic voltammetric detects: the test condition parameters setting is shown in embodiment three (one), and to containing 10ng/nL respectively, 50ng/nL, 100ng/nL, the corn sample of 150ng/nL ST carry out cyclic voltammetric and detect, and the corn sample of each concentration is all measured 5 times.Test result is shown in table one.
Table one: ST Determination on content result in the corn sample
As above shown in the table, the relative standard deviation of measured sample is between 2.8%～4.1%, and recovery of standard addition is between 87.6%～105.5%, and average recovery rate is 95.8%.
Claims (1)
1. a biology sensor that is used for the fast detecting sterigmatocystin comprises basal electrode and is assembled in the responding layer on the basal electrode, and it is characterized in that, described responding layer comprises:
Polymer film is made up of the poly-o-phenylenediamine of electropolymerization on basal electrode; This polymer film is modified on the said basal electrode;
Electron transit mediator is the macromolecule electron transit mediator;
Chitosan gel rubber is processed by chitosan solution; And
The two furocoumarin oxidase of 6-methoxyl;
Wherein, be dispersed in by the SWCN after the activation and form electron transit mediator-chitosan gel rubber hybrid films in the chitosan solution; The two furocoumarin oxidase embeddings of said 6-methoxyl are fixed on electron transit mediator-chitosan gel rubber hybrid films, are assembled on the basal electrode of polymer film modification again, are formed for detecting the biology sensor of sterigmatocystin.
2
.Biology sensor according to claim 1 is characterized in that: described electron transit mediator is the SWCN through strong acid or strong oxidizer activation processing.
3
.The assemble method of biology sensor according to claim 1 is characterized in that, may further comprise the steps:
A, the o-phenylenediamine monomer is aggregated in the basal electrode surface through electrochemical method, obtains the basal electrode that the poly-o-phenylenediamine polymer film is modified;
B, chitosan solution is processed chitosan gel rubber;
C, with after the SWCN activation processing, join mixing in the above-mentioned chitosan solution, promptly get SWCN-shitosan mixed liquor, subsequent use;
D, the two furocoumarin oxidase solution of preparation 6-methoxyl, subsequent use;
E, with the two furocoumarin oxidase solution of 6-methoxyl and SWCN-shitosan mixed liquor mixing, it is surperficial to drip the basal electrode of modifying at the poly-o-phenylenediamine polymer film, 4 ℃ of assemblings are spent the night, and promptly get described biology sensor.
4
.The assemble method of biology sensor according to claim 3 is characterized in that, may further comprise the steps:
The preparation that a, poly-o-phenylenediamine are modified basal electrode:
The basal electrode that cleans up is before use earlier at H
2SO
4In carry out electrochemical pre-treatment, scan till electrode signal is stable with cyclic voltammetry; The basal electrode of handling well is inserted into the saturated 0.1-0.5mol/L H that contains the 0.01-0.05mol/L o-phenylenediamine of nitrogen
2SO
4In the electropolymerization liquid,, can form one deck poly-o-phenylenediamine film on the basal electrode surface with the fast cyclic voltammetric electropolymerization of sweeping of 0.05-0.1V/s 30-60 circle;
The preparation of b, chitosan solution:
With acetate dissolving shitosan powder, make its concentration between 0.1%-1.5%, fully dissolve the insoluble impurity of back elimination, the pH value of regulating chitosan solution is 5.5, places 4 ℃ of preservations subsequent use;
The preparation of c, SWCN-shitosan mixed liquor:
With strong acid or strong oxidizer activation SWCN, be dissolved in the prepared chitosan solution of step b after being washed till neutral oven dry, ultrasonic dispersion makes SWCN-chitosan solution of finely dispersed 0.1mg/mL-2.5mg/mL;
D, the two oxidasic preparations of furocoumarin of 6-methoxyl:
The clone is had the two oxidasic gene engineering colibacillus abduction deliverings of furocoumarin of methoxyl, collect thalline, carry out purifying with affinity column behind the ultrasonication cell, enzyme is cut fusion and is obtained the two furocoumarin oxidase of single 6-methoxyl; The chromatographic purifying enzyme is cut product, collects the enzymatic activity peak component in the chromatographic process, gets the two furocoumarin oxidase of 6-methoxyl after concentrating; Be mixed with protein concentration and be the enzyme liquid of 2mg/mL, subsequent use;
The preparation of the biology sensor that e, the two furocoumarin oxidase of 6-methoxyl are modified:
Get SWCN prepared among the step c-shitosan mixed liquor and drip on the basal electrode that poly-o-phenylenediamine is modified in step a, dry film forming under the room temperature; And then prepared enzyme liquid in the dropping steps d, 4 ℃ of assemblings are spent the night, and promptly get the biology sensor that the two furocoumarin oxidase of 6-methoxyl are modified.
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| CN102175736A (en) * | 2011-01-20 | 2011-09-07 | 暨南大学 | Enzyme electrode for detecting sterigmatocystin and preparation and application thereof |
| CN103512938B (en) * | 2012-06-15 | 2016-02-17 | 中国科学院理化技术研究所 | A kind of electrochemical sensor and device thereof that can be used for at-once monitor and detect aqueous bio toxicity |
| CN108562751A (en) * | 2018-05-04 | 2018-09-21 | 深圳市西尔曼科技有限公司 | The detection method of enzyme membrane and preparation method thereof, glycosylated hemoglobin |
| CN111505293B (en) * | 2020-04-03 | 2022-11-18 | 北京望尔生物技术有限公司 | Test strip for detecting variegated aspergillin and application thereof |
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