CN101216450B - Biosensor electrode for detecting aspergillus flavus toxin B1 and method for making same - Google Patents
Biosensor electrode for detecting aspergillus flavus toxin B1 and method for making same Download PDFInfo
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- CN101216450B CN101216450B CN2008100258253A CN200810025825A CN101216450B CN 101216450 B CN101216450 B CN 101216450B CN 2008100258253 A CN2008100258253 A CN 2008100258253A CN 200810025825 A CN200810025825 A CN 200810025825A CN 101216450 B CN101216450 B CN 101216450B
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Abstract
The invention discloses a biosensor electrode for detecting aflatoxin B1 and a preparation method thereof. The biosensor electrode comprises a substrate layer and a reaction layer provided on the substrate layer. The reaction layer includes an electronic mediator, a sol-gel film and aflatoxin oxidase, wherein the electronic mediator is fixed on the substrate layer, and the sol-gel film embeds theaflatoxin oxidase on the substrate modified by the electronic mediator to form a film, thus obtaining aflatoxin oxidase modified electrode. The inventive biosensor electrode has the advantages of fast response, high signal sensitivity up to 1.5 ppm to 0.125 ppb, high specificity, convenient usage, quantitative detection, long service life and good stability.
Description
Technical field
The invention belongs to field of biosensors, relate to a kind of AFB that is used to detect
1Biological sensor electrode and preparation method thereof.
Background technology
AFB
1(AF B
1) be one of at present known strong carcinogenic substance, their normal animal feed and human food of polluting, (International Agency for Research on Cancer IARC) classifies aflatoxin AFB1 as human carcinogenic substance to IARC in 1988.Can cause the pollution of meat, breast and goods thereof with the forage feed fowl poultry that pollutes.If people eat contaminated food, can cause acute poisoning, cause that hepatic necrosis is hemorrhage, slow poisoning can cause liver cancer, lung cancer.Simultaneously, raise the fowl poultry with contaminated feed fowl dirty swine productive rate is reduced, weightening finish is slowed down, and causes heavy economic losses.All there is the strict regulation of limiting the quantity of various countries to the aflatoxin in food and the feed.
At present, to AFB in food and the feed
1Detection method several (AFBs such as TLC (thin-layered chromatography), HPLC (high performance liquid chromatography), immunochemistry (microplate reader method) method are roughly arranged
1The analysis of detection method, 2003 10 phases of food and fermentation industries), and enzyme biologic sensor.The TLC method is comparatively simple, do not need the high instrument and equipment of price, but the pre-treatment of sample is loaded down with trivial details, and is consuming time, poor accuracy, and a large amount of poisonous organic solvent that uses endangers greatly the experimenter.
The HPLC method needs high cost of equipment, and technical merit requires high, and sample preparation is still loaded down with trivial details, and the precision of experimental result is subject to the influence of reagent difference, is not suitable for fast detecting; Immunochemistry (microplate reader method, comprise enzyme linked immunosorbent assay, radioimmunology, affinity chromatography): (1) enzyme linked immunosorbent assay, high specificity, highly sensitive, cost is low, be suitable for batch detection, food and feed, still, the instability of enzyme itself, complex sample is disturbed, and accuracy in detection is not high; (2) radioimmunology, radioimmunology and enzyme immunoassay are similar, but radioimmunology deposits and penetrates pollution, is eliminated; (3) affinity chromatography, developed recently is detection method faster, but costs an arm and a leg, and is difficult to promote.The enzyme biologic sensor method of having reported as Chinese patent ZL 200410028018.9 (another patent of inventor herein's application), prepares sensor AFB by Covalent Immobilization in gold electrode surfaces with Aflatoxin-detofizyme
1, sensing range is at 1ppm～10ppb, and detection sensitivity is not high enough.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of AFB that is used to detect is provided
1Biological sensor electrode, this electrode can detect AFB in the detection architecture of pH4～7,20 ℃～55 ℃
1, sensitivity is higher, and sensing range expands 1.5ppm～0.125ppb to, and detection speed is fast and simple to operation.
The enzyme modified electrode that is used to detect mycotoxin of the present invention, comprise basalis and the responding layer that on basalis, forms, described responding layer comprises electron mediator, collosol and gel film and aflatoxin are separated oxidase, electron mediator is fixed on the basalis, collosol and gel is embedded in electron mediator with the aflatoxin oxidase and modifies in the substrate, forms the biological sensor electrode that the aflatoxin oxidase is modified.
Described electron mediator can be selected organic molecule electron mediator and macromolecule electron mediator for use.The organic molecule electron mediator mainly contains ferrocene and derivant, organic dyestuff, quinone and derivant thereof, tetrathiafulvalene; The macromolecule amboceptor mainly comprises the redox polymerses such as transition metal ion type and organic oxidation reduced form that appraise at the current rate.Macromolecule amboceptor compound normally by low molecule amboceptor compound and macromolecular chain with reactive group react immobilized generation.
The preferred electron mediator of the present invention is made of the multi-walled carbon nano-tubes that activates under the acid oxidase environment, and it is fixed up the embedding of aspergillus flavus poison oxidase by the network-like three dimensions microenvironment that collosol and gel forms.Multi-walled carbon nano-tubes after the activation has played an enhanced sensitivity, i.e. the sensitivity of intensifier electrode, and simultaneously, the multi-walled carbon nano-tubes after the activation has relatively large carboxyl, also very helps fixing to protein.Described multi-walled carbon nano-tubes also can be the carboxylated multi-walled carbon nano-tubes of other strong acid or strong oxidizer activation processing gained preferably with the carboxylated multi-walled carbon nano-tubes of nitration oxidation mode activation processing gained.The presoma of described collosol and gel (Sol-gel) solution is TEOS (tetraethyl orthosilicate, ethyl orthosilicate), presoma also can be selected the methyl silicate (TMOS) of low relative molecular mass, metal or semimetal alkoxide precursors such as butyl titanate for use.
When selecting other different electron mediators for use, can select the different connecting method that is fit to this electron mediator for use.For example, when selecting ferrocene derivatives for use, utilize earlier two isothiocyanates and enzyme molecule to carry out alternating copolymerization and make enzyme membrane as electron mediator, and then the ferrocene derivatives that will have a carboxyl by with the enzyme molecule on amino reaction key be incorporated into the enzyme molecule and get on.
Another object of the present invention is to provide a kind of AFB that is used to detect
1The preparation method of biological sensor electrode.
A kind of AFB that is used to detect of the present invention
1The preparation method of biological sensor electrode, may further comprise the steps:
A, multi-walled carbon nano-tubes carried out carboxylated activation processing after, join mixing in the pure water that contains 0.05%～0.15% surfactant, promptly get carboxylated multi-walled carbon nano-tubes solution;
B, preparation aflatoxin oxidase;
C, under sour environment, ethyl orthosilicate is as presoma, hydroxylated product and corresponding alcohol by catalyzing hydrolysis becomes promptly obtain A solution.
D, working electrode is modified film forming with carboxylated multi-walled carbon nano-tubes solution, the oxidasic enzyme liquid of aflatoxin mixes with above-mentioned A solution, transfer pH of mixed to 7.2 with reductive agent, behind the mixing, drip in electrode surface, after the collosol and gel polycondensation ageing film forming, promptly get the biological sensor electrode that the aflatoxin oxidase is modified.
Among the described step C, can also be dripping an amount of anti-cracking agent of adding in the TEOS of electrode surface sol gel solution, as Triton-100, polyvinylidene etc.
Preferred manufacturing procedure may further comprise the steps:
The preparation of A, carboxylated multi-walled carbon nano-tubes solution:
The effective chloroazotic acid of multi-wall carbon nano-tube was refluxed 8～12 hours, centrifugal, abandon supernatant, dry back is carboxylated multi-walled carbon nano-tubes; Take by weighing carboxylated multi-walled carbon nano-tubes and be dissolved in the pure water that contains 0.05%～0.15% surfactant, after stirring, be made into the carboxylated multi-walled carbon nano-tubes solution of 0.01～1.0mg/ml;
B, the oxidasic preparation of aflatoxin:
(1) 4 week of Armillariella tabescen fungal culture back collection thalline, (every 1000ml contains: pH6.0, PBS 0.05M to add the extraction damping fluid, EDTA 0.5mM, DTT 0.1mM, NaCl 50mM, all the other are redistilled water, pH6.0), smash to pieces in high-speed tissue mashing machine, centrifugal, remove bacterial chip, leave and take supernatant, in supernatant, add solid ammonium sulfate, make it to form 40% ammonium sulfate saturation degree, leave standstill, centrifugal, abandon precipitation; In supernatant, slowly add solid ammonium sulfate, make it form 75% ammonium sulfate saturation degree, leave standstill, centrifugal, abandon supernatant, preserve precipitation.To precipitate with 1: 4 (weight: volume) be dissolved in pH6.5, among the 0.02MPBS, behind the mixing as last all product of next step chromatographic resolution.
(2) advance one one purifying with Phenyl Sepharose 6 Fast Flow (high sub) (phenyl sepharose, hydrophobic chromatoghaphy medium 6FastFlow model)
Get above-mentioned all product of, with sample on the flow velocity of 0.5ml/min.With level pad (pH5.5,0.02MPBS, contain 30% saturation degree ammonium sulfate) carry out wash-out, and then use instead successively and contain 10% saturation degree ammonium sulfate elution buffer (pH6.3,0.02M PBS), and contain 0% saturation degree ammonium sulfate elution buffer (pH7.0,0.02MPBS), wash-out is collected the enzymatic activity peak component in the chromatographic process respectively, gets the aflatoxin oxidase after concentrating.
(3) enzyme activity determination
After having added all ingredients shown in the table 1,30 ℃ of water-baths 30 minutes, wherein the operation of deactivation control group is that the deactivation in 140 ℃ of boiling water baths of enzyme liquid adds in the reaction system identical with the reaction group after 10 minutes again.
Table 1
| The Quality Control group | Blank group | The deactivation control group | Enzyme liquid group | |
| 6.5pH, 0.1mol/LPBS (μ l) enzyme liquid (μ l) 0.01mg/ml AFB 1(μl) | 100 - 2 | 90 10 - | 90102 | 90 10 2 |
Above-mentioned each group is all made 3 parallel pipes, and after reaction was finished, each pipe added 1ml precooling chromatogram methyl alcohol, 12000rpm/ minute, centrifugal 10 minutes, gets supernatant and carry out fluorescent scanning under the wavelength of 435nm.
The definition of enzyme activity unit: per minute makes substrate A FB
1Fluorescent value (435nm) the needed enzyme amount of unit value that descends be defined as the enzyme unit that lives.
C, ethyl orthosilicate are presoma, allow its catalyzing hydrolysis under acid condition, and add anti-cracking agent in this hydrolyzation system, and making the anti-cracking agent volume fraction is 2.5%～7.5% (as Triton-100, polyvinylidene etc.), mixing, ultrasonic evenly.
The preparation of D, multi-walled carbon nano-tubes modification enzyme electrode:
With the platinum electrode is basalis, and polishing is to polishing, and successively at acetone, and strong base solution, ultrasonic cleaning in strong acid solution and the distilled water is added in electrode surface, the oven dry film forming with the carboxylated multi-walled carbon nano-tubes drips of solution of steps A gained; Again with the A solution of the enzyme liquid for preparing among the step B and step C preparation, (volume: ratio mixing volume), and with the pH value to 7.2 that low concentration alkalescence solution is regulated mixed liquor, dripping then in electrode surface ,-4 ℃ of placements be more than 24 hours, ageing with 1: 11; Complete until collosol and gel film polycondensation ageing, promptly get the biological sensor electrode that the aflatoxin oxidase is modified.
More preferred manufacturing procedure may further comprise the steps:
The preparation of A, carboxylated multi-walled carbon nano-tubes solution:
The effective chloroazotic acid of multi-wall carbon nano-tube was refluxed 8～10 hours, and centrifugal 15 minutes of 10000g abandons supernatant, uses the distilled water washing and precipitating, centrifugal 15 minutes of 10000g, and washing precipitation repeats this step twice; Place baking oven to dry precipitation, be carboxylated multi-walled carbon nano-tubes; Take by weighing the carboxylated multi-walled carbon nano-tubes of above-mentioned 1mg and be dissolved in the pure water of adding 0.05%～0.15% surfactant of 10ml, after stirring, be made into the solution of 0.1mg/ml;
B, the oxidasic preparation of aflatoxin:
(1) 4 week of Armillariella tabescen fungal culture back collection thalline, in w: v=12: 1 ratio adds to be extracted damping fluid (every 1000ml contains: the PBS 0.05M of pH6.0, EDTA 0.5mM, DTT 0.1mM, NaCl 50mM, surplus is a redistilled water, the final pH of this extraction damping fluid is 6.0), in high-speed tissue mashing machine, smash to pieces, volume ratio according to 1: 2 in the homogenate adds the mixing of said extracted damping fluid, 4 ℃, 4000g, centrifugal 10 minutes, remove bacterial chip, leave and take supernatant, in supernatant, add solid ammonium sulfate, make it to form 40% ammonium sulfate saturation degree, put in 4 ℃ and spend the night.4 ℃, 10000g, 20 minutes are centrifugal, abandon precipitation; In supernatant, slowly add solid ammonium sulfate, make it form 75% ammonium sulfate saturation degree, put in 4 ℃ and spend the night.4 ℃, 10000g, 20 minutes are centrifugal, abandon supernatant, preserve precipitation.Use pH6.0,0.02mol/L PBS is diluted to 40% ammonium sulfate precipitation liquid with 75% ammonium sulfate precipitation thing, 8000g leaves and takes supernatant after centrifugal 10 minutes, and precipitation adds solid ammonium sulfate makes it into 70% ammonium sulfate saturation degree, put in 4 ℃ and spend the night, leave and take sediment after 8000g is centrifugal, get an amount of above-mentioned sediment, with 1: 4 (weight: volume) be dissolved in pH6.5,0.02M among the PBS, 4 ℃, abundant mixing becomes last all product of next step purifying.
(2) be further purified with Phenyl Sepharose 6 FastFlow (high sub) (being Sweden Pharmacia company product, phenyl sepharose hydrophobic chromatoghaphy medium 6 Fast Flow models)
Wash Phenyl Sepharose 6 Fast Flow (high sub) post with level pad (pH5.5,0.02M PBS contain 30% saturation degree ammonium sulfate) with the flow velocity of 2ml/min, to eluent detection baseline stability.It is an amount of to get on step (1) gained all product, with sample on the flow velocity of 0.5ml/min.When sample when entering the post bed fully, flow velocity level pad (pH5.5 with 2ml/min, 0.02M PBS, contain 30% saturation degree ammonium sulfate), carry out wash-out, baseline is replied when steady, use instead successively and contain 10% saturation degree ammonium sulfate elution buffer (pH6.3,0.02M PBS) and contain 0% saturation degree ammonium sulfate elution buffer (pH7.0,0.02M PBS), being eluted to baseline respectively replys steadily.Collect the enzymatic activity peak component in the chromatographic process, get the aflatoxin oxidase after concentrating.
(3) enzyme activity determination:
After having added all ingredients shown in the table 2,30 ℃ of water-baths 30 minutes, wherein the operation of deactivation control group is that the deactivation in 140 ℃ of boiling water baths of enzyme liquid adds in the reaction system identical with the reaction group after 10 minutes again.
Table 2
| The Quality Control group | Blank group | The deactivation control group | Enzyme liquid group | |
| 6.5pH，0.1mol/L PBS(μl) | 100 | 90 | 90 | 90 |
| Enzyme liquid (μ l) | - | 10 | 10 | 10 |
| 0.01mg/ml AFB 1(μl) | 2 | - | 2 | 2 |
Above-mentioned each group is all made 3 parallel pipes, and after reaction was finished, each pipe added 1ml precooling chromatogram methyl alcohol, 12000rpm/ minute, centrifugal 10 minutes, gets supernatant and carry out fluorescent scanning under the wavelength of 435nm.
The definition of enzyme activity unit: per minute makes substrate A FB
1Fluorescent value (435nm) the needed enzyme amount of unit value that descends be defined as the enzyme unit that lives.
C, be presoma with the ethyl orthosilicate, use the hydrochloric acid catalysis presoma hydrolysis of 0.2M, and add anti-cracking agent in this hydrolyzation system that making the anti-cracking agent volume fraction is 5%, behind the mixing, ultrasonic 1 hour, room temperature leaves standstill 1～2 hour, and was standby.
The preparation of D, multi-walled carbon nano-tubes modification enzyme electrode:
With platinum electrode by 4 volume 98%H
2SO
4With 1 volume 30%H
2O
2Soak 30min in the solution of forming, use acetone, absolute ethyl alcohol, redistilled water ultrasonic cleaning 5min successively, use particle diameter 0.05 μ mAl then
2O
3The polishing of mill pad.Use redistilled water ultrasonic cleaning 5min at last.Then electrode is soaked in 0.5mol/LH
2SO
4, operating potential scope :-500mV-+1500mV scans till electrode signal is stable with cyclic voltammetry, and the carboxylated multi-walled carbon nano-tubes solution of steps A gained is dripped in right amount at electrode surface, dries film forming; Again with the A solution of the enzyme liquid for preparing among the step B and step C preparation with 1: 11 (volume: ratio mixing volume), the ammoniacal liquor with 6.5% is regulated the pH value to 7.2 of mixed liquor, drips then in electrode surface ,-4 ℃ of placements are more than 24 hours; Become transparent membrane until collosol and gel polycondensation ageing, promptly get the biological sensor electrode that the aflatoxin oxidase is modified.
Among the described step C, add the anti-cracking agent Triton-100 of 5 μ l in per 100 μ l TEOS liquid, mixing, ultrasonic, leave standstill, drip in electrode surface then.
The potential application on enzyme biologic sensor based on carbon nano-tube, the present invention is with nitration oxidation mode activation processing multi-walled carbon nano-tubes, with MWNT-COOH as the enzyme reaction electron mediator, the collosol and gel solid film that with TEOS is presoma is fixed up the embedding of aspergillus flavus poison oxidase, thereby is prepared into the immobilized enzyme biological sensor electrode.
Biological sensor electrode of the present invention mainly is to utilize contained aflatoxin oxidase to detect AFB
1, its principle is: AFB
1In the oxidase catalyzed oxidation reaction that takes place down of aflatoxin, producing in course of reaction can detected electric signal.Therefore, biological sensor electrode of the present invention is for AFB
1Detection be high specific, and the sensitivity of its detection is very high.
Compared with prior art, the present invention has following beneficial effect: 1. utilize biological sensor electrode of the present invention, only need sample is carried out simple pre-service, can be used for online detection, response time is very fast, and highly sensitive single sample or batch detection all can be used easily; 2. biological sensor electrode of the present invention can detect AF B in the detection architecture of pH4～7,20 ℃～55 ℃
1, sensitivity is higher, and sensing range expands 1.5ppm～0.125ppb to; 3. biological sensor electrode of the present invention uses and saves time, laborsaving, detection speed is fast and simple to operation, need not train and can apply.
Description of drawings
Fig. 1 is solidificated in synoptic diagram on the MWNT platinum electrode for the aflatoxin oxidase by sol-gal process;
Fig. 2 is variable concentrations AFB
1Cyclic voltammogram (Scan rate:100mv/s);
Fig. 3 is variable concentrations AFB
1Differentiated pulse figure;
Fig. 4 is variable concentrations AFB
1Graph of a relation with peak current;
Wherein, A is the AFB of variable concentrations
1Solution, B are blank PBS.
Embodiment
Embodiment 1 is used to detect AFB
1Biological sensor electrode
Of the present inventionly be used to detect AFB
1Biological sensor electrode, comprise basalis and the responding layer that on basalis, forms, described responding layer comprises electron mediator, collosol and gel film and aflatoxin oxidase, electron mediator is fixed on the basalis, collosol and gel is embedded in electron mediator with the aflatoxin oxidase and modifies film forming in the substrate, is formed for detecting AFB
1Enzyme modified electrode.
In the present embodiment, the electron mediator that is adopted is to be made of the multi-walled carbon nano-tubes that activates under the acid oxidase environment, specifically be the carboxylated multi-walled carbon nano-tubes with nitration oxidation mode activation processing gained, it passes through fixedly aflatoxin oxidase of sol-gal process embedding.
Embodiment 2 is used to detect AFB
1The preparation of biological sensor electrode
(1), the preparation of material
(1) working electrode: select platinum electrode for use, available from Tianjin Lan Li scientific ﹠ technical corporation.
(2) multi-walled carbon nano-tubes (MWNT): available from nanometer port, Shenzhen.
(3) TEOS (tetraethyl orthosilicate, ethyl orthosilicate) is available from Sigma company.
(2), the preparation of carboxylated multi-walled carbon nano-tubes solution
Multi-walled carbon nano-tubes (MWNT) was refluxed 8～10 hours with chloroazotic acid, and centrifugal 15 minutes of 10000g abandons supernatant, uses the distilled water washing and precipitating, centrifugal 15 minutes of 10000g, washing precipitation.Place baking oven to dry precipitation, promptly get carboxylated multi-walled carbon nano-tubes.
Take by weighing the carboxylated MWNT of above-mentioned 1mg and be dissolved in the pure water that 10ml contains 0.05%～0.15% surfactant, after stirring, be made into the solution of 0.1mg/ml.
(3), the oxidasic preparation of aflatoxin:
Armillariella tabescen fungal culture 4 week, the back collected thalline, and (weight: ratio volume) adds to be extracted damping fluid (every 1000ml contains: pH6.0, PBS 0.05M in 1: 2 to get an amount of thalline, EDTA 0.5mM, DTT0.1mM, NaCl 50mM, surplus is redistilled water, pH6.0), smashs to pieces in high-speed tissue mashing machine, centrifugal, remove bacterial chip, leave and take supernatant, add solid ammonium sulfate again, make it to form 40% ammonium sulfate saturation degree, leave standstill, centrifugal, abandon precipitation; In supernatant, slowly add solid ammonium sulfate, make it form 75% ammonium sulfate saturation degree, leave standstill, centrifugal, abandon supernatant, preserve precipitation.(weight: volume) be dissolved in pH6.5, among the 0.02MPBS, mixing became last all product of next chromatographic resolution with 1: 4 with above-mentioned precipitation.
Advance one one purifying with Phenyl Sepharose 6 Fast Flow (high sub).Get above-mentioned an amount of all product of, with sample on the flow velocity of 0.5ml/min.With level pad (pH5.5,0.02M PBS, contain 30% saturation degree ammonium sulfate) carry out wash-out, and then use instead successively and contain 10% saturation degree ammonium sulfate elution buffer (pH:6.3,0.02M PBS), and contain 0% saturation degree ammonium sulfate elution buffer (pH7.0,0.02M PBS), wash-out is collected the enzymatic activity peak component in the chromatographic process respectively, gets the aflatoxin oxidase after concentrating.
(4), the preparation of the sol gel solution of TEOS: in the EP of 2ml cleaning pipe, add 405 μ l pure water successively, 10 μ l 0.2M HCl behind the mixing, add 60 μ l TEOS, 25 μ l Triton-100.Vibration is even, and room temperature leaves standstill 2～3 hours, and is standby.
(5), the preparation of multi-walled carbon nano-tubes modification enzyme electrode
With the platinum electrode is basalis, with platinum electrode at Piranha (by 4 volume 98%H
2SO
4With 1 volume 30%H
2O
2Composition) soaks 30min in the solution, use acetone, absolute ethyl alcohol, redistilled water ultrasonic cleaning 5min successively, use particle diameter 0.05 μ mAl then
2O
3The polishing of mill pad, last redistilled water ultrasonic cleaning 5min, dry platinum surface.Then electrode is soaked in 0.5mol/LH
2SO
4, operating potential scope :-500mV-+1500mV scans till the electrode information stability with cyclic voltammetry, and the MWNT solution of the 0.1mg/mL of step (two) gained is dripped 10 μ l at electrode surface, 40～80 ℃ of oven dry film forming; Behind the enzyme liquid and 55 μ l above-mentioned steps (four) gained sol gel solution mixings with 5 μ l above-mentioned steps (three) gained, behind 6.5% ammoniacal liquor adjust pH to 7.2, get this mixed liquor 10 μ l and drip the electrode surface of modifying in above-mentioned MWNT (multi-walled carbon nano-tubes).Put into 4 ℃ of refrigerators and preserve, place more than the 24h, become transparent membrane, promptly get the biological sensor electrode that is used to detect aflatoxin B1, as shown in Figure 1 until collosol and gel polycondensation ageing.
In the present embodiment, select for use aflatoxin B1 as substrate.
Present embodiment is a preferred version, and the Triton-100 of adding 5% is as anti-cracking agent, even do not add the enzyme modified electrode that anti-cracking agent also can be prepared function in this step in the sol gel solution of TEOS.
Embodiment 3 is used to detect the experimental analysis of the biological sensor electrode of aflatoxin B1
The platinum electrode that aflatoxin oxidase of the present invention is modified is formed an electrode system with general to electrode and optional contrast electrode as working electrode.
In the present embodiment, selecting the Ag/AgCl electrode for use is contrast electrode, and Pt silk electrode is that electrode is formed three-electrode system, and phosphate buffer is an electrolytic solution.
(1) processing of sample:
Solid sample: the 100g sample is pulverized with comminutor, uses methyl alcohol: the volume ratio of water=45: 55 is fully dissolved concussion, and with isopyknic chloroform extraction 3 times, by containing the funnel of anhydrous sodium sulfate, 65 ℃ of following nitrogen blowing volatilize.
Liquid sample: (volume: chloroform extraction volume) 3 times, by containing the funnel of anhydrous sodium sulfate, 65 ℃ of following nitrogen blowing volatilized the 100ml sample with 0.5: 1.
(2) MWNT modifies the aflatoxin oxidase electrode to AFB
1Electrochemical response
Potential range-400mV～+ 1250mV, at the 0.1MNa that contains of 10ml
2SO
40.01M phosphate buffer (pH7.0) in, carry out cyclic voltammetric (CV) research and differential pulse volt-ampere (DPV) is studied, chronoamperometry, sweep speed 100mV/s with BAS-100 Electrochemical analyzer.Detect MWNT respectively and modify sol-gal process curing ADTZ modified electrode AFB
1Response condition.
One, cyclic voltammetry detects
Adopt BAS-100 electrochemical analyser three-electrode system (contrast electrode: Ag/AgCl electrode, to electrode: Pt silk electrode, working electrode: modify the Au electrode), select the cyclic voltammetry scan pattern, it is as follows that electrochemical parameter is set:
Operating potential scope :-400mv～+ 1250mv
Sweep speed: 100mv/s; Sensitivity: 10 μ A/V; The tranquillization time: 2s
After setting parameter, use different modification working electrodes respectively, add the AFB of variable concentrations in the end liquid of pH6.60PBS (containing 1g/lkcl) and the liquid system of the identical end 10ml0.2M
1Ethanolic solution or above-mentioned sample carry out cyclic voltammetry scan and detect.
The test effect: with the sweep speed of 100mv/s ,-400mv～+ 1250mv operating potential scope in the AFB of cyclic voltammetry to variable concentrations
1Solution is measured, and as shown in Figures 2 and 3, peak current is with AFB
1The increase of concentration and increasing.
Two, the differentiated pulse voltammetry detects
Select differentiated pulse volt-ampere scan pattern, it is as follows that electrochemical parameter is set:
Pulse-response amplitude: 50mv; Image data potential region :-400mv～+ 1250mv; Sampling width: 20ms;
Sweep velocity: 100mv/s; The tranquillization time: 10s
Test effect: with the AFB of sol-gal process immobilization aflatoxin oxidase modified electrode to variable concentrations
1Solution carries out the scanning of differentiated pulse volt-ampere, and measured result shows this electrode pair AFB as shown in Figure 3
1Sensitivity produces different big or small pulse signals (signal magnitude and concentration have proportional relationship) under the different concentration.It is AFB
1Content can show by pulse signal.
As shown in Figure 3, with the immobilized aflatoxin oxidase of sol-gal process modified electrode in+30mv position with the differentiated pulse voltammetry to AFB
1Carry out quantitative test, obtain 5.00 * 10
-5～5.00 * 10
-4In the g/L concentration range, peak current is along with AFB
1The increase of concentration and rise AFB
1Linear detection range be 5.00 * 10
-5～5.00 * 10
-4G/L, linear equation are Ip=7.609+6.613 C, R
2=0.96941, P＜0.05.
Fig. 4 shows that biological sensor electrode of the present invention is to AF B
1Response is sensitive, the peak current size and the AFB of generation
1Content relation in direct ratio, promptly can represent AFB in the sample by peak current
1Content.
As shown in Figure 4, at AFB
1Concentration range 5 μ g/L～25 μ g/L, the signal of electrode detection (strength of current) changes more sensitive.The analytical characteristics of electrode is to AFB
1The detection range of linearity have two: range of linearity 1:5 μ g/L～25 μ g/L, its equation of linear regression is Ip=24.03879+7.14296*C, coefficient R
2=0.9792P＜0.0001.Line range of linearity 2:30 μ g/～70 μ g/L, its equation of linear regression is Ip=185.35+1.44143*C, coefficient R
2=0.9865 P＜0.0001.
Claims (9)
1. one kind is used to detect AFB
1Biological sensor electrode, comprise basalis and the responding layer that on basalis, forms, it is characterized in that: described responding layer is made up of electron mediator, collosol and gel and aflatoxin oxidase, electron mediator is fixed on the basalis, collosol and gel is embedded in film forming on the basalis that electron mediator modifies with the aflatoxin oxidase, described electron mediator is made of the multi-walled carbon nano-tubes that activates under the acid oxidase environment, and the presoma of described collosol and gel is an ethyl orthosilicate.
2. biological sensor electrode according to claim 1 is characterized in that: the mass percent of ethyl orthosilicate is 10%～15% in the described collosol and gel.
3. biological sensor electrode according to claim 1 is characterized in that: described multi-walled carbon nano-tubes is the carboxylated multi-walled carbon nano-tubes with nitration oxidation mode activation processing gained.
4. the preparation method of the described biological sensor electrode of one of claim 1-3 is characterized in that may further comprise the steps:
(1), multi-walled carbon nano-tubes carried out carboxylated activation processing after, join mixing in the pure water of the surfactant that contains 0.05 quality %～0.15 quality %, promptly get carboxylated multi-walled carbon nano-tubes solution;
(2), preparation aflatoxin oxidase;
(3), under sour environment, ethyl orthosilicate is as presoma, hydroxylated product and corresponding alcohol by catalyzing hydrolysis becomes promptly obtain A solution;
(4), working electrode is modified film forming, the oxidasic enzyme liquid of aflatoxin mixes with above-mentioned A solution, transfers pH of mixed to 7.2 with ammoniacal liquor with carboxylated multi-walled carbon nano-tubes solution, behind the mixing, drip in electrode surface, after the collosol and gel polycondensation ageing film forming, must be used to detect AFB
1Biological sensor electrode.
5. the preparation method of the described biological sensor electrode of one of claim 1-3 is characterized in that may further comprise the steps:
(1), the preparation of carboxylated multi-walled carbon nano-tubes solution: the effective chloroazotic acid of multi-wall carbon nano-tube was refluxed 8～12 hours, centrifugal, abandon supernatant, dry back is carboxylated multi-walled carbon nano-tubes; Carboxylated multi-walled carbon nano-tubes is dissolved in the pure water of the surfactant that contains 0.05 quality %～0.15 quality %, after ultrasound wave stirs, is made into the carboxylated multi-walled carbon nano-tubes solution of 0.01～1.0mg/ml;
(2), the oxidasic preparation of aflatoxin: collect Armillariella tabescen mycothallus, add and extract damping fluid, broken, centrifugal, get supernatant, obtain sediment with ammonium sulfate precipitation method, to be splined on Phenyl Sepharose 6 Fast Flow behind the PBS mixing, be further purified, collect the enzymatic activity peak component in the chromatographic process, promptly get the aflatoxin oxidase after concentrating;
(3), be presoma with the ethyl orthosilicate, under acid condition, allow its catalyzing hydrolysis, and in this hydrolyzation system, add anti-cracking agent, making the anti-cracking agent volume fraction is 2.5%～7.5%, mixing promptly obtains A solution, and described anti-cracking agent is Triton-100 or polyvinylidene;
(4), the preparation of electrode: with the platinum electrode is basalis, and polishing is to polishing, and successively at acetone, strong base solution, ultrasonic cleaning in strong acid solution and the distilled water is added in electrode surface with the carboxylated multi-walled carbon nano-tubes drips of solution of step (1) gained, the oven dry film forming; With the enzyme liquid of step (2) preparation and the A solution of step (3) preparation,, and, drip then again, place more than 24 hours ageing for-4 ℃ in electrode surface with the pH value to 7.2 that ammoniacal liquor is regulated mixed liquor with 1: 11 volume ratio mixing; Complete until collosol and gel film polycondensation ageing, must be used to detect AFB
1Biological sensor electrode.
6. the preparation method of the described biological sensor electrode of one of claim 1-3 is characterized in that may further comprise the steps:
(1), the preparation of carboxylated multi-walled carbon nano-tubes solution: the effective chloroazotic acid of multi-wall carbon nano-tube was refluxed 8～12 hours, and centrifugal 15 minutes of 10000g abandons supernatant, uses the distilled water washing and precipitating, centrifugal 15 minutes of 10000g, washing precipitation repeats this step twice; Place baking oven to dry precipitation, be carboxylated multi-walled carbon nano-tubes; Take by weighing the carboxylated multi-walled carbon nano-tubes of above-mentioned 1mg and be dissolved in the distilled water of adding surfactant of 10ml, mixing is made into the solution of 0.1mg/ml;
(2), the oxidasic preparation of aflatoxin: collect the mycothallus after Armillariella tabescen cultivated for 4 weeks, by weight, volume ratio is that 12: 1 ratio adds in the extraction damping fluid, in high-speed tissue mashing machine, smash to pieces, in the homogenate according to 1: 2 volume ratio, adding the said extracted damping fluid mixes, 4 ℃, 4000g, centrifugal 10 minutes, get supernatant, be that 40% and 75% ammonium sulfate precipitation method obtains sediment with ammonium sulfate concentrations successively, the gained precipitation is dissolved in pH6.5 with 1: 4 volume ratio, 0.02M among the PBS, 4 ℃, fully be splined on PhenylSepharose 6 Fast Flow behind the mixing, carry out purifying, with containing 10% saturation degree ammonium sulfate elution buffer and containing 0% saturation degree ammonium sulfate elution buffer wash-out respectively, collect the enzymatic activity peak component in the chromatographic process successively, promptly get the aflatoxin oxidase after concentrating;
(3), be presoma with the ethyl orthosilicate, use the hydrochloric acid catalysis presoma hydrolysis of 0.2M, and in this hydrolyzation system, add anti-cracking agent, making the anti-cracking agent volume fraction is 5%, and behind the mixing, room temperature left standstill 1～2 hour, standby, described anti-cracking agent is Triton-100 or polyvinylidene;
(4), the preparation of electrode: with platinum electrode by 4 volume 98%H
2SO
4With 1 volume 30%H
2O
2Soak 30min in the solution of forming, use acetone, absolute ethyl alcohol, redistilled water ultrasonic cleaning 5min successively, use particle diameter 0.05 μ mAl then
2O
3Redistilled water ultrasonic cleaning 5min is used in the polishing of mill pad at last, then electrode is soaked in 0.5mol/LH
2SO
4, operating potential scope :-500mV-+1500mV scans till electrode signal is stable with cyclic voltammetry, and the carboxylated multi-walled carbon nano-tubes drips of solution of step (1) gained is added in electrode surface, the oven dry film forming; The A solution of enzyme liquid that step (2) is prepared and step (3) preparation was with 1: 11 volume ratio mixing again, and the pH value to 7.2 with the ammoniacal liquor of 6.5 quality % is regulated mixed liquor drips then in electrode surface, placed more than 24 hours for-4 ℃; Become transparent membrane until collosol and gel polycondensation ageing, must be used to detect AFB
1Biological sensor electrode.
7. the preparation method of biological sensor electrode according to claim 6, it is characterized in that, extraction damping fluid described in the step (1) is the PBS that contains 0.05M pH6.0 in every 1000ml solution, 0.5mM EDTA, 0.1mM DTT, the NaCl of 50mM, surplus is that secondary steams water, and the pH of this extraction damping fluid is 6.0.
8. the preparation method of biological sensor electrode according to claim 6 is characterized in that, in the step (2), described to contain 10% saturation degree ammonium sulfate elution buffer be the PBS of 0.02M, and its pH is 6.3; Described to contain 0% saturation degree ammonium sulfate elution buffer be the PBS of 0.02M, and its pH is 7.0.
9. any one biological sensor electrode detects AFB in preparation among the claim 1-3
1Biology sensor in application.
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| CN101726521B (en) * | 2009-12-01 | 2012-07-04 | 暨南大学 | Biosensor for rapidly detecting sterigmatocystin and assembling method thereof |
| CN102520168B (en) * | 2011-11-25 | 2014-05-14 | 江南大学 | Immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and preparing method thereof |
| CN103196984B (en) * | 2013-03-13 | 2014-04-23 | 济南大学 | Preparation method and application of sensor for simultaneous detection of multiple aflatoxins |
| CN103196973B (en) * | 2013-03-13 | 2014-06-04 | 济南大学 | Method for preparing seven-channel aflatoxin immunosensor and application |
| RU2534732C1 (en) * | 2013-07-26 | 2014-12-10 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Национальный исследовательский Томский политехнический университет" | Method for quantitative determination of aflatoxin b1 by differential voltammetry |
| CN104407132B (en) * | 2014-10-24 | 2016-05-25 | 济南大学 | A kind of colibacillary electrochemical sensor and preparation method thereof that detects |
| CN104730133B (en) * | 2015-03-19 | 2017-07-07 | 哈尔滨工业大学（威海） | The preparation method of AFB1 immune response electrode |
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