CN1603809A - Disposable ampere type immunosensor for detecting two or four drops and preparing and using method thereof - Google Patents
Disposable ampere type immunosensor for detecting two or four drops and preparing and using method thereof Download PDFInfo
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- CN1603809A CN1603809A CN 200410096073 CN200410096073A CN1603809A CN 1603809 A CN1603809 A CN 1603809A CN 200410096073 CN200410096073 CN 200410096073 CN 200410096073 A CN200410096073 A CN 200410096073A CN 1603809 A CN1603809 A CN 1603809A
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Abstract
This invention relates to a disposable ampere immune sensor and its process method, which belongs to environment measurement field. The sensor adopts thick-film electrode made with wire net printing process, which is characterized by the following: PVC base plate, orderly distributed carbon round disc, reference electrode, pair electrode and connection ends with each electrode, isolation layer on the base plate, biology molecule film layer fixed on the carbon round electrode.
Description
Technical field
The invention belongs to the environmental monitoring field, but particularly fast detecting 2, the new technology that 4-drips.This disposable immunosensor can be used for the pollution fast monitored of lake, reservoir, river, sewage treatment plant, insecticide factory etc., also can be used for food 2 such as vegetables, and 4-drips the fast detecting of residues of pesticides.
Background technology
Since the forties, human a large amount of synthetic organic chemistry pesticide control farmland disease insect pest of using, 2, it is 2 that 4-drips, and (2,4-drips ichlorophenoxyacetic Aci and drips the 4-dichlorphenoxyacetic acid, Chinese is abbreviated as 2,4-drips) be widely used herbicide and plant growth regulator in a kind of world wide, be that whole world annual sales amount surpasses one of agricultural chemicals of hundred million yuan, also be 20th century whole world use one of 22 kinds of agricultural chemicals of pesticide species the most extensive and that global agricultural is had the greatest impact.Studies show that in a large number at present, 2,4-drips and is easy to enter human body by skin and breathing, has potential carcinogenicity (bringing out soft tissue malignant tumour and He Jiejin lymphomas), mutagenicity, and the human normal fertility of influence, the harm that human health and ecologic environment are caused can not be ignored.With 2, the Cmax that 4-drips is defined as 0.03mg/L in the unconventional test item of " People's Republic of China's standard drinking water quality hygienic practice ".At present, commonly used 2, it is leaching (Liqui drip-Soli drip Extraction) and gas chromatography-mass spectrum technology (GC-MS) that 4-drips detection method, need complicated pretreatment and expensive equipment, testing process complicated (extraction, reduction, derivatization and purifying), length consuming time have limited the routine monitoring number of times.
Immuno analytical method also is to detect 2, one of important analysis method that 4-drips.Mainly be to utilize material marks 2 such as radioactive isotope, enzyme, 4-drips, and to be measured 2,4-drips and mark 2, and 4-drips common competition in conjunction with 2, after 4-drips antibody, reflects in the water sample to be measured 2, the concentration that 4-drips by measuring label content.In recent years, the ampere-type immunosensor of testing environment organic contaminant also more and more causes people's attention.This electrochemical device combines with traditional immunochemical analyses method, and existing excellent specificity has lower detectability again.And compare with other technologies, this sensor to instrument and equipment require low, simple to operate.Adopt the disposable thick-film type electrochemical immunosensor of silk-screen printing technique preparation further to reduce the cost that detects, make quick on a large scale, on-the-spot testing environment organic contaminant become possibility.
In recent years, adopt silk-screen printing technique to develop the immunosensor of three-electrode structure in succession, primary structure is the disc type of bar formula and symmetry.Its discal patch formula structure is not considered the characteristics of electrode surface solution diffusion direction center of circle symmetry, needs the extra structure that increases quantitative pond.Though the symmetry disc type has been improved this point, its contrast electrode and to the electrode symmetry separate from the working electrode both sides, its Electric Field Distribution and electric current trend are monolateral distribution, cause the input instability.
Present 2,4-D immunosensor technology of preparing mainly is to rest on the laboratory study stage, use mainly be glass-carbon electrode.The electrode of minority integrated circuit technology preparation also all is based on the direct competitive experiment.And the research of indirect immune competition law also because the abutment effect of conjugate is obvious, will adopt 2 in therefore detecting, and 4-D and its antiserum be the method for incubation in advance.The detection time of this method and detection step are longer.
Summary of the invention
The present invention seeks to overcoming the weak point of prior art, a kind of detection 2 is proposed, disposable ampere-type immunosensor and preparation and using method that 4-drips, this sensor construction is simple, compare with other symmetrical expression three-electrode structures, it is even to have Electric Field Distribution, ringwise symmetry, consistent with the dispersal direction of electron mediator, can suppress current noise preferably, eliminate and disturb.This preparation and using method are easy to operate, and cost is low, can carry out 2 fast, and the field that 4-drips is analyzed.
Detection 2 of the present invention, the disposable ampere-type immunosensor that 4-drips, for adopting the thick membrane electrode of silk-screen printing technique preparation, it is characterized in that, comprise the PVC substrate, and the carbon disk electrode that on this substrate, distributes with one heart successively, contrast electrode, to electrode and the terminals that link to each other with each electrode, insulation course, and the biomolecule rete of on the carbon disk electrode, fixing.
Of the present invention above-mentioned 2, the preparation method of the disposable ampere-type immunosensor of 4-D is as follows:
1) silk-screen printing technique prepares sensor: earlier the printing silver slurry layer forms three electrodes of concentric distribution on the PVC substrate, the subregion printing carbon slurry layer on the silver slurry layer, and (carbon dust diameter can at 10～100 μ m) republishes insulation course; (electrode that prints can spend the night under 50 ± 3 ℃ for the carbon disk electrode at center in work; Measure the charge transfer coefficient of electrode with sampling in the carbon disk electrode of a collection of preparation);
2) 2,4-drips-PLL synthetic: adopt the carbodlimide method Synthetic 2,4-drips-the PLL conjugate, and coupling ratio is between 10～20;
3) 2,4-drips sero-fast selecting for use: select rabbit source 2 for use, 4-drips antiserum, and it is tired greater than 2000;
4) 2,4-drips the immobilization of responsive biomolecule at sensor surface: at first adopt fixedly poly-D-lysine-2 of glutaraldehyde cross-linking method, 4-drips, oralbumin (OVA), glutaraldehyde, and wherein OVA is used to block nonspecific binding site; Carry out the precincubation of inhibitory reaction then, drip normal concentration 2 at sensor surface, 4-drips with the antiserum mixed solution and solidifies back formation biomolecule rete.
The present invention uses above-mentioned immunosensor to measure 2, and the method that 4-drips may further comprise the steps:
(1) indirect competition immunity: (since the immunosensor surface in advance incubation 2,4-drips standard sample and antiserum) directly on above-mentioned immunosensor, drip 2 when measuring, 4-D and the electrode surface antigen immunity back (each sensor can drip sample 4～40 μ l) that is at war with, peroxidase is marked positive anti-rabbit (IGG) be added drop-wise to electrode surface, with the polyclonal antibody (being the biomolecule rete) on the sensor immunity takes place, (background solution can be H to add substrate solution then
2O
2With OPD);
(2) amperometric determination: after dripping substrate solution afterreaction 120 ± 10s, be under-100 to-150mV the condition in polarizing voltage, use potentiostat to detect time current curve under the constant potential; Can check in the sample 2 from electric current-concentration standard curve, the concentration that 4-drips.
Characteristics technique effect of the present invention:
The present invention adopts how anti-technology preparation 2, and 4-drips antiserum, adopts silk-screen printing technique to prepare thick membrane electrode, and adopts the cross-linking method fixing biological molecules.2,4-drips the conjugate with poly-D-lysine (PLL), and with 2,4-drips antiserum and the HRP ELIAS secondary antibody is carried out indirect immune competitive reaction.Under reduction potential control, at the HRP enzyme at o-phenylenediamine and H
2O
2Acting in conjunction enzymatic reaction takes place down produces reduction current, detect reduction current and can reflect 2, the concentration that 4-drips.
In order to overcome long shortcoming detection time that precincubation causes, the present invention is 2, and the direct incubation of 4-D and antiserum is at sensor surface, thereby reduces detection time, simplification detection step
The present invention has highly sensitive, and it is low to detect lower limit, and detection speed is fast, and is with low cost, simple operation and other advantages.
Description of drawings
Fig. 1 is of the present invention 2, and 4-drips the structural drawing of immunosensor.
Fig. 2 is the process drawing that the present invention adopts the sensor of silk-screen printing technique preparation.
Fig. 3 is the process flow diagram of indirect immune competitive reaction of the present invention.
Fig. 4 is 2, and 4-drips the calibration curve of detection.
Embodiment
A kind of detection 2 that the present invention proposes, disposable ampere-type immunosensor that 4-drips and preparation thereof and using method reach embodiment in conjunction with the accompanying drawings and further specify as follows:
A kind of detection 2 of the present invention, the disposable ampere-type immunosensor that 4-drips be as shown in Figure 1:
This sensor adopts the thick membrane electrode of silk-screen printing technique preparation, adopt the concentric type three-electrode structure, electrode structure, comprise PVC substrate 6, and the carbon disk electrode 1 that distributes with one heart successively that serigraphy becomes on this substrate 6, contrast electrode 2, to electrode 3 and the terminals 5 that link to each other with each electrode, insulation course 4, and the biomolecule rete of on the carbon disk electrode, fixing.
Carbon disk electrode 1 diameter of embodiments of the invention is 2mm, and the radius of contrast electrode 2 is 2mm, is 3mm to the radius of electrode 3.
Of the present invention 2, the preparation method of the disposable ampere-type immunosensor of 4-D as shown in Figure 2:
1) silk-screen printing technique prepares sensor: printing forms three electrodes that distribute with one heart on the PVC substrate earlier, shown in Fig. 2 (1), subregion printing carbon slurry layer on the silver slurry layer, shown in Fig. 2 (2), the carbon dust diameter of present embodiment republishes insulation course at 10～100 μ m, shown in Fig. 2 (3), electrode after the printing is shown in Fig. 2 (4); (the carbon disk electrode of present embodiment is diameter 2mm for the carbon disk electrode at center in work.The electrode that prints spends the night under 50 ± 3 ℃; Measure the charge transfer coefficient of homogeneous prepared in batches carbon electrode);
2) 2,4-drips-PLL synthetic: adopt the carbodlimide method Synthetic 2,4-drips-the PLL conjugate, and coupling ratio is between 10～20;
3) 2,4-drips sero-fast selecting for use: select rabbit source 2 for use, 4-drips antiserum, and it is tired should be greater than 2000;
4) 2,4-drips the immobilization of responsive biomolecule at sensor surface: at first adopt fixedly poly-D-lysine-2 of glutaraldehyde cross-linking method, 4-drips, OVA, glutaraldehyde, and wherein OVA is used to block nonspecific binding site; Carry out the precincubation of inhibitory reaction then, drip normal concentration 2,4-drips with the antiserum mixed solution and solidifies back formation biomolecule rete.
The present invention uses above-mentioned immunosensor to measure 2, and the method that 4-drips may further comprise the steps as shown in Figure 3:
(1) indirect competition immunity: since the immunosensor surface in advance incubation 2,4-drips standard sample and antiserum, directly on above-mentioned immunosensor, drip during mensuration and to be measured contain 2, the immunity that is at war with of the liquid of 4-D and electrode surface antigen, after each sensor drips sample 4～40 μ l, peroxidase is marked positive anti-rabbit (IGG) be added drop-wise to electrode surface, with polyclonal antibody (being the biomolecule rete) the generation immunity on the sensor, (present embodiment is H to add substrate solution then
2O
2With o-o-phenylenediamine (OPD));
(2) amperometric determination: when dripping substrate solution afterreaction 120 ± 10s, be in polarizing voltage-100mV to-150mV condition, use potentiostat to detect time current curve under the constant potential.Can check in 2 from electric current-concentration curve (as shown in Figure 4), the concentration that 4-drips.
Embodiments of the invention are described in detail as follows:
1. the preparation of sensor
(1) preparation 2,4-D-PLL conjugate step is as follows:
Take by weighing 2 of 18mg, 4-D dissolves in the PBS damping fluid of 1.5mL 0.05M, adds 19mg EDC and stirs.The PLL that adds 200 μ L 10mg/mL then also stirs in solution.Mixed solution at room temperature left standstill 1 hour.4 ± 1 ℃ of dialysis 24h (0.1M NaHCO
3, change PBS solution 4 times).
(2) preparation antiserum step is as follows:
The employing female New Zealand white rabbits is carried out.The immune coupling ratio that adopts water-soluble carbodiimide (EDC) coupling test to obtain is 16: 1 a synthetic antigen immune mouse.The immunizing dose of New Zealand white rabbit adopts identical initial immunity and booster immunization dosage.Immunogene dosage is 0.8mg, and haptens dosage is 40 μ g.The Freund's complete adjuvant emulsification of the immunogene of initial immunity.The immunogene of booster immunization adopts incomplete Freund emulsification.New Zealand white rabbit adopts the subcutaneous multi-point injection in back.Behind the initial immunity 10 days or 14 days, carry out booster immunization.Took a blood sample to measure in 7 days subsequently or 10 days and tire.Repeat booster immunization, antiserum up to obtain to tire higher (greater than 2000).New Zealand white rabbit adopts the auricular vein blood taking method.
(3) structure of employing sensor of the present invention.The material that silk-screen printing technique prepares sensor is: the PVC film that 0.2mm is thick, and the silver slurry is the 427SS of Acheson company, and the carbon slurry is Japanese Jujo Ch01, and the printing blue color ink is as insulation course.After finishing printing at every turn, electrode all in baking oven with 40 ℃ of dry 2h.Before sessile antibody, the electrode for preparing at room temperature aging 7 days.
(4) carrying out biomolecule fixes.Carry out the deionized water ultrasonic cleaning earlier, nitrogen dries up; The back is at sensor surface coating buffer poly-D-lysine-2, and 4-drips, OVA, glutaraldehyde; Method for coating: each electrode drips sample 4 μ l, 37 ℃ of incubation 2hr, and after cleaning dries up, 4 ℃ of preservations.Drip at last normal concentration 2,4-drips, the antiserum mixed solution, and 37 ℃ of reaction 1hr, cleaning dries up.
2. the use of sensor
Directly drip 2 when (1) detecting, the immunity that is at war with of 4-D and electrode surface antigen, each sensor dropping sample 4～40 μ l, 37 ± 1 ℃ are reacted 1hr, and cleaning dries up.Then peroxidase is marked positive anti-rabbit (IGG) and be added drop-wise to electrode surface, immunity takes place with polyclonal antibody on the sensor (being first antibody), each sensor point sample 4 μ l, 37 ± 1 ℃ of reaction 0.5hr, cleaning dries up.
(2) detection of enzyme activity: substrate solution is H
2O
2Be respectively 1mM, 5mM with OPD concentration.The substrate solution of amperometric determination is with the dilution of PBS damping fluid, OPD and H
2O
2Concentration is respectively 5mM, 1mM.Each sensor drips 40 μ L substrate solutions, behind reaction 120 ± 10s, uses the time current curve under the CHI660A electrochemical workstation detection constant potential.Wherein polarizing voltage be-100mV is to-150mV, detection time 120 ± 10s.(Fig. 4) can check in 2 from electric current-concentration curve, the concentration that 4-drips.
(3) making of normalized current-concentration curve: adopt above-mentioned immunosensor and detection method, with 2,4-drips the PBS solution of standard sample preparation 600mg/L.And be diluted to 1.0ng/mL.Measure its typical curve.The result is as shown in Figure 4: detect lower limit: 1ng/mL; Between linear zone: 1ng/mL-30 μ g/mL; The linearity: R=97.4%
Claims (3)
1, a kind of detection 2, the disposable ampere-type immunosensor that 4-drips, for adopting the thick membrane electrode of silk-screen printing technique preparation, it is characterized in that, comprise the PVC substrate, and the carbon disk electrode that on this substrate, distributes with one heart successively, contrast electrode, to electrode and the terminals that link to each other with each electrode, insulation course, and the biomolecule rete of on the carbon disk electrode, fixing.
2, a kind of 2, the preparation method of the disposable ampere-type immunosensor of 4-D is characterized in that, may further comprise the steps:
1) silk-screen printing technique prepares sensor: print three electrodes that silver slurry layer forms concentric distribution earlier on the PVC substrate, the subregion printing carbon slurry layer on the silver slurry layer republishes insulation course; Work is the carbon disk electrode at center;
2) 2,4-drips-PLL synthetic: adopt the carbodlimide method Synthetic 2,4-drips-the PLL conjugate, and coupling ratio is between 10～20;
3) 2,4-drips sero-fast selecting for use: select rabbit source 2 for use, 4-drips antiserum, and it is tired greater than 2000;
4) 2,4-drips the immobilization of responsive biomolecule at sensor surface: at first adopt fixedly poly-D-lysine-2 of glutaraldehyde cross-linking method, 4-drips, oralbumin, glutaraldehyde, and wherein oralbumin is used to block nonspecific binding site; Carry out the precincubation of inhibitory reaction then, drip normal concentration 2 at sensor surface, 4-drips with the antiserum mixed solution and solidifies back formation biomolecule rete.
3, the disposable ampere-type immunosensor of a kind of use measures 2, the method that 4-drips, it is characterized in that, sensor comprises the PVC substrate, and the carbon disk electrode that on this substrate, distributes with one heart successively, contrast electrode, to electrode and the terminals that link to each other with each electrode, insulation course, and the biomolecule rete of on the carbon disk electrode, fixing;
Measure 2,4-drips and may further comprise the steps:
(1) indirect competition immunity: directly on described immunosensor, drip sample 2, the immunity that is at war with of 4-D and electrode surface antigen, peroxidase is marked positive anti-rabbit be added drop-wise to electrode surface, the molecule film generation immunity with on this sensor adds substrate solution then;
(2) amperometric determination: after dripping substrate solution afterreaction 120 ± 10s, be under-100 to-150mV the condition in polarizing voltage, use potentiostat to detect time current curve under the constant potential; Can check in the sample 2 from electric current-concentration standard curve, the concentration that 4-drips.
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| CN101216451B (en) * | 2008-01-18 | 2012-01-04 | 华东理工大学 | DNA biosensor electrode manufacture method and uses thereof |
| CN101915847A (en) * | 2010-06-13 | 2010-12-15 | 黑龙江八一农垦大学 | Biosensor for detecting milk progesterone of cow |
| CN102262112B (en) * | 2011-04-02 | 2014-03-26 | 中国科学院烟台海岸带研究所 | Alloy electrode electrochemical sensor for detecting trace heavy metals |
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| CN103308675A (en) * | 2013-05-08 | 2013-09-18 | 北京工业大学 | Preparation and detection methods of screen-printed electrode immunosensor for rapidly detecting microcystin |
| CN103940868A (en) * | 2014-04-15 | 2014-07-23 | 山东理工大学 | Fast detector of pesticide residues based on enzyme immunosensor |
| CN103940868B (en) * | 2014-04-15 | 2016-02-24 | 山东理工大学 | A kind of rapid detector for pesticide residue based on enzyme immunosensor |
| CN105277400A (en) * | 2014-07-07 | 2016-01-27 | 闳伟科技股份有限公司 | Electrochemical biosensing test piece and batch manufacture method thereof |
| CN104931692A (en) * | 2015-06-04 | 2015-09-23 | 塔里木大学 | Electrochemical immunosensor for chlorpyrifos detection, and preparation method and application thereof |
| CN109239406A (en) * | 2018-10-08 | 2019-01-18 | 西安交通大学 | One kind is for organelle drug locating delivering detection device and dispensing detection method |
| CN109521078A (en) * | 2018-10-30 | 2019-03-26 | 上海大学 | Alloy nanometer crystals/vulcanization molybdenum composite material, its jettisonable electrochemical sensor and its manufacturing method |
| CN109521078B (en) * | 2018-10-30 | 2021-08-10 | 上海大学 | Alloy nanocrystal/molybdenum sulfide composite material, disposable electrochemical sensor thereof and manufacturing method thereof |
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| CN100374852C (en) | 2008-03-12 |
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