CN104931571A - Immunosensor and preparation method and 2,4-D detection method thereof - Google Patents
Immunosensor and preparation method and 2,4-D detection method thereof Download PDFInfo
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- CN104931571A CN104931571A CN201510330433.8A CN201510330433A CN104931571A CN 104931571 A CN104931571 A CN 104931571A CN 201510330433 A CN201510330433 A CN 201510330433A CN 104931571 A CN104931571 A CN 104931571A
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Abstract
The invention discloses an immunosensor and a preparation method and 2,4-D detection method thereof. An electrode which is decorated with the 2,4-D serves as the immunosensor. The preparation method includes the hydroxylation step, the amidogen silanization step and the 2,4-D decoration step. The 2,4-D detection method includes the steps of providing the immunosensor; processing to-be-detected liquid, wherein 2,4-D antibodies marked by horse radish peroxidase are added to the to-be-detected liquid; conducting competitive binding, wherein the immunosensor is placed in the to-be-detected liquid for incubation, the 2,4-D in the to-be-detected liquid and the 2,4-D decorated on the immunosensor are combined with the 2,4-D antibodies marked by the horse radish peroxidase in a competitive mode; conducting measurement through the voltammetry, wherein the response current generated by the 2,4-D antibodies which are marked by the horse radish peroxidase and combined with the immunosensor is measured through the voltammetry, and the response current is compared with a standard curve to obtain the content of the 2,4-D in the to-be-detected liquid.
Description
Technical field
The present invention relates to the detection field of 2,4-D, particularly relate to the detection method of a kind of immunosensor and preparation method thereof and 2,4-D.
Background technology
2,4-dichlorophenoxyacetic acid (2,4-D) is a kind of common environmental hormone, through being commonly used for herbicide and plant growth regulator in agricultural production, not easily degrades under natural conditions.Know from experience cause internal system, immune system or nerve problems if 2,4-D enters people by skin, respiratory system or digestive system, can generation dysfunction time serious, the threat mankind multiply existence.
At present, the detection method of 2,4-D comprises: high performance liquid chromatography (HPLC), vapor-phase chromatography (GC) and Liquid Chromatography-Mass Spectrometry (LC-MS).Wherein GC adopts gas to make mobile phase, is applicable to detect volatile matter; HPLC utilizes chemical means separating mixture, is separated the signal peak obtaining not same compound, is judged the content of determinand by signal peak area ratio by post; LC-MS judges determinand content by the signal peak area ratio of different compound, is detected the molecular weight of different compound by MS simultaneously.
Although above-mentioned detection method is highly sensitive, accuracy good, but instrument is expensive, need professional and technical personnel to operate, the pre-service such as sample separation purification loaded down with trivial details consuming time (as: GC need to carry out esterification is derivative could sample introduction, and the boron fluoride toxicity used is larger; The pre-treatment of HPLC needs at substantial organic solvent etc.), therefore traditional instrument analytical approach is difficult to meet the demand detected fast a large amount of sample.
Immunoassay is a kind of important method of toxic pollutant in testing environment, has quick, simple and highly sensitive feature, therefore, is used for immunosensor and the detection method of 2,4-D detection desirable to provide one.
Summary of the invention
The invention provides a kind of immunosensor for detecting 2,4-D; The invention provides a kind of preparation method of immunosensor for obtaining the immunosensor of detection 2,4-D; The detection method that the invention provides a kind of 2,4-D solves prior art detecting instrument costliness, processing procedure very complicated, can not meet the problem detecting screening fast.
According to an aspect of the present invention, provide a kind of immunosensor, the electrode of this immunosensor for modifying 2,4-D.
Alternatively, according to immunosensor of the present invention, described 2,4-D are modified on the electrodes by APTES.
Alternatively, according to immunosensor of the present invention, described electrode is the electrode introducing hydroxyl.
Alternatively, according to immunosensor of the present invention, described electrode is glass-carbon electrode.
According to a further aspect in the invention, provide a kind of preparation method of immunosensor, the method comprises:
Hydroxylation steps: electrode and H
2sO
4solution contacts, and electrode introduces hydroxyl;
Aminosilylation step: hydroxylation electrode contacts with APTES, and electrode introduces amino silane;
2,4-D modification step: amino silane polarizing electrode and 2,4-D contact, electrode introduces 2,4-D.
Alternatively, preparation in accordance with the present invention, described hydroxylation steps is specially:
Electrode is placed in the H of 0.1 ~ 0.3mol/L
2sO
4in solution, under the current potential of 0 ~ 2.0V (vs.SCE), cyclic voltammetry scan 6 ~ 8 encloses.
Alternatively, preparation in accordance with the present invention, described Aminosilylation step is specially:
Hydroxylated electrode is placed in the solution that mass concentration is the APTES of 4 ~ 6%, at 36 ~ 38 DEG C of incubation 13 ~ 17min.
Alternatively, preparation in accordance with the present invention, described 2,4-D modification steps are specially: amino silane polarizing electrode be placed in 2,4-D solution, at 36 ~ 38 DEG C of incubation 8 ~ 12min.
Alternatively, preparation in accordance with the present invention, in described 2,4-D modification steps, described 2,4-D is 2,4-D through carbodiimides and N-hydroxy-succinamide activation.
According to a further aspect in the invention, providing the detection method of a kind of 2,4-D, providing according to immunosensor of the present invention, the electrode of this immunosensor for modifying 2,4-D;
Wherein, the method comprises:
Liquid treatment step to be measured: 2, the 4-D antibody adding horseradish peroxidase-labeled in liquid to be measured;
Competition binding step: described immunosensor is placed in described liquid incubation to be measured, in liquid to be measured 2,4-D and immunosensor on 2,4-D antibody of horseradish peroxidase-labeled described in 2, the 4-D competition binding modified;
Voltammetric determination step: the response current of 2,4-D antibody generations of the horseradish peroxidase-labeled combined with voltammetric determination immunosensor, obtains 2,4-D content in liquid to be measured by response current amount and typical curve comparison.
Beneficial effect of the present invention is as follows:
According to immunosensor of the present invention, electrode is modified 2,4-D, used this immunosensor, utilized the specific reaction of antigen-antibody to carry out 2,4-D in testing environment.
According to the preparation method of immunosensor of the present invention, electrode is introduced hydroxyl, on the electrode introducing hydroxyl, introduces amino, the electrode introducing amino modifies 2,4-D, is modified at 2,4-D on electrode, 2,4-D in liquid to be measured can be detected with this immunosensor.
According to detection method of the present invention, provide a kind of detection method of quick, simple and highly sensitive 2,4-D.
Accompanying drawing explanation
Fig. 1 is the immunosensor preparation method mechanism figure according to one embodiment of the present invention;
Fig. 2 is the detection mechanism figure according to detection method 2,4-D;
Fig. 3 is the x-ray photoelectron energy spectrogram of glass-carbon electrode;
Fig. 4 is the x-ray photoelectron energy spectrogram of the glass-carbon electrode after introducing hydroxyl;
Fig. 5 is the x-ray photoelectron energy spectrogram of the glass-carbon electrode after Aminosilylation;
The x-ray photoelectron energy spectrogram that Fig. 6 is the glass-carbon electrode of modifying 2,4-D;
Fig. 7 is the cyclic voltammogram of the immunosensor combining enzyme labelled antibody;
Fig. 8 is the square wave voltammogram of the immunosensor combining enzyme labelled antibody;
Fig. 9 is the canonical plotting that square wave voltammetry detects 2,4-D.
Embodiment
Concrete embodiment is only explanation of the present invention, and does not form the restriction to content of the present invention, to be further described and to describe below in conjunction with accompanying drawing and concrete embodiment to the present invention.
The electrode of immunosensor according to the present invention for modifying 2,4-D.
According to immunosensor of the present invention, electrode has been modified 2,4-D, the specific reaction of antigen and antibody can be utilized to detect in liquid to be measured 2, the content of 4-D, and make to be combined in enzyme in the enzyme labelled antibody on electrode more firmly, detection signal is more stable, improves the stability that immunosensor detects.
According to the immunosensor of one embodiment of the present invention, 2,4-D is modified on electrode by APTES.
According to immunosensor of the present invention, electrode is modified APTES and namely on electrode, introduces amino, amino with 2,4-D in carboxyl to react generation acid amides, 2,4-D is modified on electrode.
According to the immunosensor of one embodiment of the present invention, electrode is the electrode introducing hydroxyl.
Electrode introduces hydroxyl, and the ethoxy of hydroxyl in APTES is combined, and connects by generating ehter bond, also connects and generate intermolecular silicon ehter bond between the ethoxy between APTES molecule.
According to the immunosensor of one embodiment of the present invention, preferred glass-carbon electrode, also can select graphite electrode, indium-tin oxide electrode, gold electrode and platinum electrode.
According to the preparation method of immunosensor of the present invention, the method comprises:
Hydroxylation steps: electrode and H
2sO
4solution contacts, and electrode introduces hydroxyl;
Aminosilylation step: hydroxylation electrode contacts with APTES, and electrode introduces amino silane;
2,4-D modification step: amino silane polarizing electrode and 2,4-D contact, electrode introduces 2,4-D.
According to the preparation method of immunosensor of the present invention, preferred glass-carbon electrode (GCE), preferably carry out pretreated GCE, GCE is preferably polished to minute surface by preprocessing process in the suspension of the alumina powder of 0.3 ~ 0.6um, then supersound washing 3 ~ 5min in absolute ethyl alcohol and intermediate water successively.
Fig. 1 shows the schematic diagram of the preparation method of this immune sensing electrode, and as can be seen from Figure 1, GCE introduces hydroxyl by hydroxylation at electrode surface; Then hydroxylated electrode contacts with APTES, ethoxy wherein in APTES and hydroxyl react generation ehter bond, be combined between the ethoxy between the APTES molecule on hydroxyl and generate ehter bond connection, define reticulate texture; One deck is defined with-NH in addition on the surface of electrode
2thin polymer film; Electrode surface is with-NH
2, the carboxyl on 2,4-D and-NH
2reaction generates acid amides, thus has been modified on electrode, forms immunosensor.According to the Specific adsorption between antigen and antibody, utilize the immunosensor that the inventive method prepares, the content of 2,4-D can be detected.
According to the preparation method of one embodiment of the present invention, institute's hydroxylation steps is specially:
Electrode is placed in the H of 0.1 ~ 0.3mol/L
2sO
4in solution, under the current potential of 0 ~ 2.0V (vs.SCE), cyclic voltammetry scan 6 ~ 8 encloses.
According to the preparation method of immunosensor of the present invention, hydroxylation steps is preferably at H
2sO
4carry out in solution: the H GCE after cleaning being placed in 0.1 ~ 0.3mol/L
2sO
4in, preferred 0.2mol/L, then activates at 0 ~ 2.0V (vs.SCE) potential range cyclic voltammetry scan 6 ~ 8 circle, introduces hydroxyl on GCE surface, then rinses with intermediate water, N
2dry up.
Preparation in accordance with the present invention, Aminosilylation step is specially:
Hydroxylated electrode is placed in the solution that mass concentration is the APTES of 4 ~ 6%, at 36 ~ 38 DEG C of incubation 13 ~ 17min.
According to the preparation method of immunosensor of the present invention, the electrode introducing hydroxyl is preferably immersed by percentage to the quality 4 ~ 6% by Aminosilylation step, preferably 5%, APTES (APS) acetone soln in, at 36 ~ 38 DEG C, preferably 37 DEG C, constant temperature oven in incubation 13 ~ 17min, preferred 15min, forms one deck band-NH at electrode surface by C-O-Si
2thin polymer film, with intermediate water rinse, N
2dry up, be labeled as APS/GCE.
According to the preparation method of one embodiment of the present invention, 2,4-D modification step is specially: amino silane polarizing electrode be placed in 2,4-D solution, at 36 ~ 38 DEG C of incubation 8 ~ 12min.
According to the preparation method of immunosensor of the present invention, 2,4-D modification step, preferably puts into 2 by APS/GCE, in 4-D solution, at 36 ~ 38 DEG C, preferably 37 DEG C, constant temperature oven in incubation 8 ~ 12min, preferred 10min, 2,4-D are by-the NH on carboxyl and electrode polymer film
2be connected, mark 2,4-D/APS/GCE.
According to the preparation method of immunosensor of the present invention, 2,4-D modification step is specially: amino silane polarizing electrode be placed in 2,4-D solution, at 36 ~ 38 DEG C of incubation 8 ~ 12min.
According to the preparation method of immunosensor of the present invention, in 2,4-D modification step, 2,4-D is through carbodiimides and N-hydroxy-succinamide activation 2,4-D.
At 2,4-D modification step, preferably through 2 after carbodiimides (EDC) and N-hydroxy-succinamide (NHS) activation, 4-D, concrete activation act is, in 2, the 4-D standard solution of the 0.5000g/L of 5ml, add 2.0mgEDC and 3.0mgNHS, vibration 1min, room temperature activation 15min, its consumption can expand according to ratio and reduce, by 2 in reactivation process, the activated carboxylic of 4-D is carbonyl, makes carbonyl and amino be easier to condensation reaction occurs.
According to the detection method of the present invention 2,4-D, provide according to immunosensor of the present invention, the electrode of this immunosensor for modifying 2,4-D;
Wherein, the method comprises:
Liquid treatment step to be measured: 2, the 4-D antibody adding horseradish peroxidase-labeled in liquid to be measured;
Competition binding step: immunosensor is placed in liquid incubation to be measured, in liquid to be measured 2,4-D and immunosensor on 2, the 4-D antibody of 2,4-D competition binding horseradish peroxidase-labeled modified;
Voltammetric determination step: the response current of 2,4-D antibody generations of the horseradish peroxidase-labeled combined with voltammetric determination immunosensor, obtains 2,4-D content in liquid to be measured by response current amount and typical curve comparison.
According to Fig. 2 according to detection mechanism figure of the present invention, can find out, immunosensor combines 2 of horseradish peroxidase-labeled, 4-D antibody (is called for short enzyme labelled antibody, be abbreviated as HRP-anti-2,4-D), be HRP-anti-2 in conjunction with later electrode designations, 4-D/2,4-D/APS/GCE.Voltammetric determination HRP-anti-2 can be used, the response current that 4-D/2,4-D/APS/GCE produce.There is following redox reaction to produce response current in the HRP catalytic substrate be attached on electrode:
HRP(Fe
3+)+H
2O
2→HRP(Ⅰ)+H
2O；
HRP(Ⅰ)+QH
2→HRP(Ⅱ)+Q；
HRP(Ⅱ)+QH
2→HRP(Fe
3+)+Q+H
2O；
Q+2e→QH
2
Wherein, HRP (I) and HRP (II) represents that enzyme is participating in the intermediate state in catalytic process, and HRP (I) is+5 valency oxidation state, and HRP (II) is+4 valency oxidation state; QH
2then p-dihydroxy-benzene and oxidation states 1,4-benzoquinone thereof is represented respectively with Q.
According to above-mentioned mechanism, voltammetric determination HRP-anti-2 can be adopted, the response current of 4-D/2,4-D/APS/GCE.In liquid to be measured, the concentration of 2,4-D is larger within the specific limits, and in liquid to be measured, 2,4-D is more in conjunction with HRP-anti-2,4-D, and electrode is modified 2, the 4-D HRP-anti-2 combined, 4-D will reduce, and the response current of voltammetric determination also can corresponding reduction thereupon.And 2,4-D finite concentration scope, response current and 2, the concentration of 4-D is linear, therefore can preparing standard solution preparation standard curve in this concentration range, the response current then measured according to determinand and typical curve comparison draw the content of in liquid to be measured 2,4-D.
As can be seen here, according to immunosensor of the present invention and preparation method thereof, 2,4-D detection method optional element more, different embodiments can be combined into according to claim of the present invention.Therefore embodiment is only explanation of the present invention, instead of limitation of the scope of the invention, describes further below in conjunction with specific embodiments to the present invention.
Embodiment 1
According to the preparation method of immunosensor of the present invention, select GCE, first pre-service is carried out to GCE: GCE is polished to minute surface in the suspension of the alumina powder of 0.3um, then supersound washing 3min in absolute ethyl alcohol and intermediate water successively; Enter hydroxylation steps afterwards: H electrode being placed in 0.1mol/L
2sO
4in solution, under the current potential of 0 ~ 2.0V (vs.SCE), cyclic voltammetry scan 6 encloses, and rinses, N with intermediate water
2dry up; Enter Aminosilylation step afterwards: hydroxylated electrode is placed in the solution that mass concentration is the APTES of 4%, at 36 DEG C of incubation 13min, rinse with intermediate water, N
2dry up, be labeled as APS/GCE; Finally enter 2,4-D modification step, put into by APS/GCE in 2,4-D solution, incubation 8min in the constant temperature oven of 36 DEG C, wherein 2,4-D is 2,4-D through carbodiimides and N-hydroxy-succinamide activation.
Embodiment 2
According to the preparation method of immunosensor of the present invention, select GCE, first pre-service is carried out to GCE: GCE is polished to minute surface in the suspension of the alumina powder of 0.6um, then supersound washing 5min in absolute ethyl alcohol and intermediate water successively; Enter hydroxylation steps afterwards: H electrode being placed in 0.3mol/L
2sO
4in solution, under the current potential of 0 ~ 2.0V (vs.SCE), cyclic voltammetry scan 8 encloses, and rinses, N with intermediate water
2dry up; Enter Aminosilylation step afterwards: hydroxylated electrode is placed in the solution that mass concentration is the APTES of 6%, at 38 DEG C of incubation 17min, rinse with intermediate water, N
2dry up, be labeled as APS/GCE; Finally enter 2,4-D modification step, put into by APS/GCE in 2,4-D solution, incubation 12min in the constant temperature oven of 38 DEG C, wherein 2,4-D is 2,4-D through carbodiimides and N-hydroxy-succinamide activation.
Embodiment 3
According to the preparation method of immunosensor of the present invention, select GCE, first pre-service is carried out to GCE: GCE is polished to minute surface in the suspension of the alumina powder of 0.4um, then supersound washing 4min in absolute ethyl alcohol and intermediate water successively; Enter hydroxylation steps afterwards: H electrode being placed in 0.2mol/L
2sO
4in solution, under the current potential of 0 ~ 2.0V (vs.SCE), cyclic voltammetry scan 7 encloses, and rinses, N with intermediate water
2dry up; Enter Aminosilylation step afterwards: hydroxylated electrode is placed in the solution that mass concentration is the APTES of 5%, at 37 DEG C of incubation 15min, rinse with intermediate water, N
2dry up, be labeled as APS/GCE; Finally enter 2,4-D modification step, put into by APS/GCE in 2,4-D solution, incubation 10min in the constant temperature oven of 37 DEG C, wherein 2,4-D is 2,4-D through carbodiimides and N-hydroxy-succinamide activation.
Embodiment 4
According to the preparation method of immunosensor of the present invention, select GCE, first pre-service is carried out to GCE: glass-carbon electrode is polished to minute surface in the suspension of the alumina powder of 0.4um, then supersound washing 5min in absolute ethyl alcohol and intermediate water successively; Enter hydroxylation steps afterwards: H electrode being placed in 0.1mol/L
2sO
4in solution, under the current potential of 0 ~ 2.0V (vs.SCE), cyclic voltammetry scan 8 encloses, and rinses, N with intermediate water
2dry up; Enter Aminosilylation step afterwards: hydroxylated electrode is placed in the solution that mass concentration is the APTES of 6%, at 36 DEG C of incubation 14min, rinse with intermediate water, N
2dry up, be labeled as APS/GCE; Finally enter 2,4-D modification step, put into by APS/GCE in 2,4-D solution, incubation 9min in the constant temperature oven of 36 DEG C, wherein 2,4-D is 2,4-D through carbodiimides and N-hydroxy-succinamide activation.
Embodiment 5
According to the preparation method of immunosensor of the present invention, select GCE, first pre-service is carried out to GCE: GCE is polished to minute surface in the suspension of the alumina powder of 0.5um, then supersound washing 5min in absolute ethyl alcohol and intermediate water successively; Enter hydroxylation steps afterwards: H electrode being placed in 0.2mol/L
2sO
4in solution, under the current potential of 0 ~ 2.0V (vs.SCE), cyclic voltammetry scan 6 encloses, and rinses, N with intermediate water
2dry up; Enter Aminosilylation step afterwards: hydroxylated electrode is placed in the solution that mass concentration is the APTES of 4%, at 38 DEG C of incubation 16min, rinse with intermediate water, N
2dry up, be labeled as APS/GCE; Finally enter 2,4-D modification step, put into by APS/GCE in 2,4-D solution, incubation 11min in the constant temperature oven of 37 DEG C, wherein 2,4-D is 2,4-D through carbodiimides and N-hydroxy-succinamide activation.
The embodiment of the present invention 1 ~ 5 has all prepared the immunosensor that can detect 2,4-D.
Applicant, when preparing the immunosensor in embodiment 3, has all done x-ray photoelectron energy spectrogram (XPS) to each step.Its XPS schemes as seen in figures 3-6.
Fig. 3 is the XPS figure of glass-carbon electrode, Fig. 4 is the XPS figure after introducing hydroxyl, Fig. 4 and Fig. 3 compares and can find out that the ratio of glassy carbon electrode surface oxygen element and carbon rises to 24.7% by 13.31%, the photoelectron spectrum line strength of oxygen element obviously increases, and this illustrates that hydroxyl is successfully incorporated on electrode surface.
Fig. 5 is the XPS figure after Aminosilylation step, and Fig. 5 and Fig. 4 compares, and N element and Si element have appearred in electrode surface respectively, and this illustrates that APTES successfully introduces electrode surface.
Fig. 6 is the XPS figure after 2,4-D modification step, and Fig. 6 and Fig. 5 compares, and has occurred chlorine element at electrode surface, and this shows to be modified at electrode surface containing 2,4-D of chlorine atom.
As can be seen here, method according to the present invention has prepared immunosensor of the present invention.
Select the immunosensor that embodiment 3 prepares, 2,4-D in liquid to be measured have been detected.Carrying out detection to 2,4-D in liquid to be measured can adopt cyclic voltammetry also can adopt square wave voltammetry.
The immunosensor obtained according to the preparation method of embodiment 3 is put into 2 of the horseradish peroxidase-labeled of 1.0ug/ml by applicant, in 4-D antibody, incubation 30min in the constant temperature oven of 37 DEG C, carry out cyclic voltammetry respectively with the immunosensor after above-mentioned process to detect and square wave voltammetry detection, its testing conditions is containing 1.0mmol/L p-dihydroxy-benzene, 1.6mmol/LH
2o
20.01mol/L buffer solution (PBS, pH7.4) in; Blank is the buffer solution (PBS, pH7.4) of 0.01mol/L, and scanning result as shown in Figure 7 and Figure 8.
Fig. 7 shows electrode HRP-anti-2, the cyclic voltammogram of 4-D/2,4-D/APS/GCE.Wherein, a is for containing 1.0mmol/L p-dihydroxy-benzene and 1.6mmol/L H
2o
20.01mol/L buffer solution (PBS, pH 7.4), b is blank 0.01mol/L PBS (pH 7.4), and as seen in Figure 6, a creates obvious response current, does not then produce response current in b.
Fig. 8 shows electrode HRP-anti-2, the square wave anode voltammogram of 4-D/2,4-D/APS/GCE, and a is for containing 1.0mmol/L HQ
2with 1.6mmol/L H
2o
20.01mol/L PBS (pH 7.4), b is blank 0.01mol/L PBS (pH 7.4).As seen in Figure 7, create obvious response current in a, in b, then do not produce response current.
Can find out according to Fig. 7 and Fig. 8, two kinds of methods all may be used for the detection of 2,4-D, and the sensitivity of square wave voltammetry can be better.Therefore in following description, square wave voltammetry is adopted.
Before employing square wave voltammetry detects 2,4-D in liquid to be measured, first prepare typical curve.The condition of preparation standard curve is as follows: immunosensor embodiment 3 prepared is placed in the standard solution of 0ug/ml, 0.1ug/ml, 0.5ug/ml, 4.8ug/ml, 10ug/ml, 15ug/ml respectively, be all 2 of the horseradish peroxidase mark of 1.0ug/ml containing concentration in described standard solution, 4-D, utilizes square wave voltammetry to detect the response current that immunosensor produces after the constant temperature oven incubation 30min of 37 DEG C by the immunosensor being placed in solution.Linear relationship is as shown in Figure 8 there is according to the concentration detecting in response current and titer 2,4-D.As shown in Figure 9, within the scope of 0.10 ~ 15.0ug/ml, response current and 2, the concentration of 4-D is good linear relationship, equation of linear regression is ip (μ A)=-1.267c (μ g/mL)+75.193, correlation coefficient r=0.9973, detects and is limited to 0.01ug/ml (S/N=3), restriction concentrations 0.03ug/ml in " drinking water quality criterion " that the detection limit of this immunosensor revise lower than the World Health Organization (WHO).
Applicant's river got respectively in river A, river B, river C carries out the detection of 2,4-D.The immunosensor that embodiment 3 prepares is adopted in this testing process.First, add 2,4-D antibody of horseradish peroxidase-labeled in river sample after the pre-treatment, make 2,4-D antibody concentration of horseradish peroxidase-labeled be 1.00mg/ml; Then the immunosensor prepared according to embodiment 3 is placed in water sample, incubation 30min in the constant temperature oven of 37 DEG C, in water sample to be measured 2,4-D and immunosensor on 2, the 4-D antibody of 2,4-D competition binding horseradish peroxidase-labeled modified; The response current of 2,4-D antibody generations of the horseradish peroxidase-labeled combined with the above-mentioned immunosensor of voltammetric determination, response current amount and typical curve comparison are obtained 2,4-D content in liquid to be measured, and result is as shown in table 1:
Table 1
| Sample | Response current (μ A) | Concentration (μ g/mL) |
| River A | 75 | 0 |
| River B | 67.5 | 6 |
| River C | 61.5 | 10 |
By the immunosensor that embodiment 3 prepares, be placed in river A and carry out recovery testu, measurement result is in table 2.
Table 2
Experimental result according to table 2 can be found out, the recovery of the method is between 96.4% ~ 98.7%, and the recovery is much larger than 95%, and therefore this detection method is applicable to the detection of 2,4-D, and the Stability and veracity of detection method is high.
Obviously, those skilled in the art can carry out various change and modification to the present invention and not depart from the spirit and scope of the present invention.Like this, if these amendments of the present invention and modification belong within the scope of the claims in the present invention and equivalent technologies thereof, then the present invention is also intended to comprise these change and modification.
Claims (10)
1. an immunosensor, is characterized in that, the electrode of this immunosensor for modifying 2,4-D.
2. immunosensor as claimed in claim 1, it is characterized in that, described 2,4-D are modified on the electrodes by APTES.
3. immunosensor as claimed in claim 2, it is characterized in that, described electrode is the electrode introducing hydroxyl.
4. immunosensor as claimed in claim 3, it is characterized in that, described electrode is glass-carbon electrode.
5. a preparation method for immunosensor, is characterized in that, the method comprises:
Hydroxylation steps: electrode and H
2sO
4solution contacts, and electrode introduces hydroxyl;
Aminosilylation step: hydroxylation electrode contacts with APTES, and electrode introduces amino silane;
2,4-D modification step: amino silane polarizing electrode and 2,4-D contact, electrode introduces 2,4-D.
6. preparation method as claimed in claim 5, it is characterized in that, described hydroxylation steps is specially:
Electrode is placed in the H of 0.1 ~ 0.3mol/L
2sO
4in solution, under the current potential of 0 ~ 2.0V (vs.SCE), cyclic voltammetry scan 6 ~ 8 encloses.
7. preparation method as claimed in claim 5, it is characterized in that, described Aminosilylation step is specially:
Hydroxylated electrode is placed in the solution that mass concentration is the APTES of 4 ~ 6%, at 36 ~ 38 DEG C of incubation 13 ~ 17min.
8. preparation method as claimed in claim 5, it is characterized in that, described 2,4-D modification steps are specially: amino silane polarizing electrode be placed in 2,4-D solution, at 36 ~ 38 DEG C of incubation 8 ~ 12min.
9. preparation method as claimed in claim 5, is characterized in that, in described 2,4-D modification steps, described 2,4-D is 2,4-D through carbodiimides and N-hydroxy-succinamide activation.
10. the detection method of a kind 2,4-D, is characterized in that, provides Claims 1 to 5 arbitrary described immunosensor, the electrode of this immunosensor for modifying 2,4-D;
Wherein, the method comprises:
Liquid treatment step to be measured: 2, the 4-D antibody adding horseradish peroxidase-labeled in liquid to be measured;
Competition binding step: described immunosensor is placed in described liquid incubation to be measured, in liquid to be measured 2,4-D and immunosensor on 2,4-D antibody of horseradish peroxidase-labeled described in 2, the 4-D competition binding modified;
Voltammetric determination step: the response current of 2,4-D antibody generations of the horseradish peroxidase-labeled combined with voltammetric determination immunosensor, obtains 2,4-D content in liquid to be measured by response current amount and typical curve comparison.
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| Title |
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| 薛瑞: "纳米材料电化学传感界面的构建及农药残留检测应用", 《中国博士学位论文全文数据库 工程科技I辑》 * |
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