CN104133070B - Preparation method and use of environmental estrogen label-free immunosensor - Google Patents
Preparation method and use of environmental estrogen label-free immunosensor Download PDFInfo
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- CN104133070B CN104133070B CN201410338509.7A CN201410338509A CN104133070B CN 104133070 B CN104133070 B CN 104133070B CN 201410338509 A CN201410338509 A CN 201410338509A CN 104133070 B CN104133070 B CN 104133070B
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Abstract
The invention discloses a preparation method of an environmental estrogen label-free immunosensor and a use of the environmental estrogen label-free immunosensor in environmental estrogen detection, and belongs to the technical field of novel functional nano-materials and environment detection. The preparation method utilizes the two novel functional nano-materials to modify working electrodes, utilizes synergetic catalysis effects and antibody sorption effects of the two novel functional nano-materials, and prepares the environmental estrogen label-free immunosensor which has high sensitivity, good specificity and fast detection rate, is prepared by simple processes and is used for environmental estrogen detection.
Description
Technical field
The present invention relates to preparation method and the application of the unmarked immunosensor of a kind of environmental estrogens.The estrogenic unmarked electrochemical immunosensor of testing environment specifically adopting the novel nanocomposite materials with concerted catalysis effect to build, belongs to Nano-function thin films and technical field of environmental detection.
Background technology
Environmental estrogens is the chemical substance that a class is present in environment, there is the activity of similar biosome inner estrogen, there is after entering human body the estrogenic effect of simulation, by upset the system such as human endocrine, immunity, nerve normal function and produce as carcinogenic, damage the various diseases such as reproductive development system, immune system, nervous system, be the environmental toxin of a class serious harm human health.Common environmental estrogens source comprises as agricultural chemicals (as organo-chlorine pesticide), second-hand-cigarette, waste gas (as the toxic gas that burning plastic garbage produces), food additives etc.
Detection method for environmental estrogens mainly contains high performance liquid chromatography, vapor-phase chromatography, chromatograph-mass spectrometer coupling method, euzymelinked immunosorbent assay (ELISA), radioimmunology etc.But most of detection method instrument is valuable, complicated operation, after reviewer has to pass through and screws up discipline, can detect.Some easy methods as electrochemical methods, though simple to operate, detect sensitive, quick, poor specificity.Therefore, the electrochemical immunosensor of environmental estrogens in quick, sensitive, high specific testing environment can seem particularly outstanding.
Electrochemical immunosensor is widely used in clinical analysis and environment measuring field due to advantages such as it are highly sensitive, specificity is good, easy and simple to handle, generally can be divided into and have mark and unmarked two kinds, due to the characteristic of label, the former requires stricter than the latter to testing environment, operate also relatively loaded down with trivial details, therefore, wherein in testing environment estrogen, unmarked electrochemical immunosensor more has application prospect.The unmarked immunosensor of constructing environment estrogen, crucial technology be improve modified electrode antagonist fixed amount and to the signal response speed of test end liquid and size.The Au@Pd nanometer rod composite material of nucleocapsid structure is a kind of novel noble metal nano compound substance, due to its good biocompatibility and nano-grade size, adds the adsorbance of antibody; And in nucleocapsid structure, Au is core Pd is shell, decreases the use amount of Pd, reduces cost; The Pd of its special Rod-like shape imparting simultaneously exposes more catalysis crystal face, better can improve the catalytic response speed to the hydrogen peroxide as test end liquid and current responsing signal size.In addition, CoFe
2o
4/ rGo mono-aspect adds electrode specific surface area, intensifier electrode conductive capability, and can produce concerted catalysis effect with Au Pd on the other hand, larger enhancing is to the catalytic response speed of hydrogen peroxide and current responsing signal size.Therefore, by the two conbined usage, the structure of unmarked electrochemical immunosensor can be used successfully to.
The present invention successively uses CoFe
2o
4/ rGo and Au@Pd-Ab modifies electrode, then adding of environmental estrogens antigen is utilized, the current responsing signal of electrode pair hydrogen peroxide is reduced, thus achieve the structure of unmarked electrochemical immunosensor, and be applied to simple, that quick, specificity is good and highly sensitive testing environment estrogen.
Summary of the invention
An object of the present invention is that the instrument and equipment avoiding existing in existing detection method is complicated, operating process is loaded down with trivial details and the deficiency such as poor specificity, the preparation method of the unmarked immunosensor of a kind of environmental estrogens is provided, prepared sensor has feature that is highly sensitive, high specificity, and prepare simple, easy to operate, can be used for quick, the Sensitive Detection of environmental estrogens;
Two of object of the present invention is prepared unmarked immunosensor to be applied in the detection of environmental estrogens, realizes quick, Sensitive Detection to multiple environmental estrogens.
The technical solution used in the present invention is as follows:
1. a preparation method for the unmarked immunosensor of environmental estrogens, the unmarked immunosensor of described environmental estrogens comprises: working electrode, contrast electrode and to electrode, the basal electrode of described working electrode is glass-carbon electrode, and CoFe is modified successively in its surface
2o
4/ rGo, glutaraldehyde, Au@PdNRs-Ab, bovine serum albumin (BSA), described contrast electrode is saturated calomel electrode, and described is platinum electrode to electrode, described CoFe
2o
4/ rGo is cobalt ferrite/reduced graphene compound substance, and described Au PtNRs-Ab is the environmental estrogens antibody of golden palladium core-shell nano rod composite material hatching;
It is characterized in that, described preparation method comprises the following steps:
A, preparation CoFe
2o
4/ rGo;
B, preparation Au@PdNRs-Ab;
C, prepare the unmarked immunosensor working electrode of environmental estrogens;
Wherein, step a prepares CoFe
2o
4the concrete steps of/rGo are as follows:
By 8 ~ 12 mg graphene oxide ultrasonic disperse in 25 ~ 35 ml absolute ethyl alcohols, add the Fe (NO of 0.5 ~ 1.5 ml 0.2 mol/mL successively
3)
3aqueous solution, 0.25 ~ 0.75 ml of 0.2 mol/L Co (NO
3)
2aqueous solution and 2.0 ~ 3.0 ml ammoniacal liquor, stir 10 ~ 14h at 80 DEG C, be then transferred in autoclave, reacts 4 ~ 6h at 180 DEG C; After being cooled to room temperature, centrifuging, dries, obtains CoFe in 50 DEG C of vacuum drying chambers
2o
4/ rGo;
The concrete steps that step b prepares Au@PdNRs-Ab are as follows:
Cetyl trimethyl ammonium bromide (CTAB) solution of 20mL gold nanorods solution and 20mL 0.05 ~ 0.2 mol/L is mixed, stir at 30 ~ 45 DEG C, and order adds L-AA (AA) solution of 3 ~ 6 mL 0.1 mol/L, the palladium bichloride (PdCl of 0.5 ~ 1 mL 0.03 mol/L
2) NaOH (NaOH) solution of solution and 3 ~ 6 mL 0.1 mol/L, stop stirring, leave standstill 1 ~ 3h, centrifuging, adds the PBS buffer solution of 20mL pH7.4, and then adds environmental estrogens antibody (Ab), at 4 DEG C, shake 24h, obtain Au@PdNRs-Ab;
The concrete steps that step c prepares environmental estrogens unmarked immunosensor working electrode are as follows:
1. pre-service is carried out to working electrode, make its any surface finish;
2. the electrode surface obtained in 1. drips the CoFe of 5 ~ 10 μ L 1 ~ 5 mg/mL
2o
4/ rGo aqueous solution, dries under room temperature naturally;
3. the electrode surface obtained in 2. drips the glutaraldehyde of 5 ~ 10 μ L, naturally dries under room temperature;
4. the electrode surface obtained in 3. drips the Au@PdNRs-Ab solution of 5 ~ 8 μ L 10 μ g/mL, preserves and dry in 4 DEG C of refrigerators, and ultrapure water cleans, and dries film forming, 4 DEG C of preservations;
5. the electrode surface obtained in 4. drips the BSA solution of 4 ~ 6 μ L 100 μ g/mL, in order to the nonspecific activity site on enclosed-electrode, preserve and dry in 4 DEG C of refrigerators, ultrapure water cleans, dry film forming, namely obtain the working electrode of the unmarked immunosensor of environmental estrogens.
2. the unmarked immunosensor of environmental estrogens of the present invention, for the detection of environmental estrogens, step is as follows:
1) the environmental estrogens antigen standard solution of concentration known is joined in the PBS buffer solution of 40 ~ 60 μ L, pH=7.0 ~ 8.0, obtained antigen mixed solution, get 5 ~ 10 μ L antigen mixed solutions drip be coated onto prepared environmental estrogen immunosensor working electrode on, preserve in 4 DEG C of refrigerators and dry, after the PBS buffer solution cleaning of pH=7.0 ~ 8.0, dry, 4 DEG C of preservations;
2) mercurous chloride electrode will be obtained, as obtaining platinum electrode to electrode, with 1 as contrast electrode) in the working electrode assembled form three-electrode system, be connected on electrochemical workstation; Successively add the PBS buffer solution of 10 ~ 25mL pH=7.0 ~ 8.0 and the hydrogen peroxide (H of 1mL 3 ~ 6 mmol/L in a cell
2o
2) solution; The working electrode of assembling is detected to H by chronoamperometry
2o
2response; According to the relation between the current-responsive of gained and environmental estrogens antigen concentration of standard solution, drawing curve;
3) testing sample solution is replaced the standard solution of environmental estrogens antigen, detect according to the method for drafting of the working curve of described environmental estrogens antigen.
3. the preparation method and application of the unmarked immunosensor of environmental estrogens of the present invention, is characterized in that described environmental estrogens is selected from one of following: estradiol, estriol, diethylstilbestrol, bisphenol-A or nonylphenol.
useful achievement of the present invention
(1) preparation of environmental estrogens of the present invention unmarked immunosensor is simple, easy to operate, achieves and detects quick, sensitive, the high selectivity of multiple environmental estrogens, have market development prospect.
(2) the present invention adopts CoFe first
2o
4/ rGo and the common modified electrode of Au@PdNRs-Ab, and the synergic catalytic effect both utilizing, significantly improve electric current corresponding speed and size, further enhancing the adsorbance of antibody, thus construct unmarked electrochemical immunosensor, there is important scientific meaning and using value.
Embodiment
embodiment 1coFe
2o
4the preparation of/rGo
By 8 mg graphene oxide ultrasonic disperse in 25 ml absolute ethyl alcohols, add the Fe (NO of 0.5 ml 0.2 mol/mL successively
3)
3aqueous solution, 0.25 ml of 0.2 mol/L Co (NO
3)
2aqueous solution and 2.0 ml ammoniacal liquor, stir 10h at 80 DEG C, be then transferred in autoclave, react 4h at 180 DEG C; After being cooled to room temperature, centrifuging, dries, obtains CoFe in 50 DEG C of vacuum drying chambers
2o
4/ rGo.
embodiment 2coFe
2o
4the preparation of/rGo
By 10 mg graphene oxide ultrasonic disperse in 30 ml absolute ethyl alcohols, add the Fe (NO of 1 ml 0.2 mol/mL successively
3)
3aqueous solution, 0.5 ml of 0.2 M Co (NO
3)
2aqueous solution and 2.50 ml ammoniacal liquor, stir 12h at 80 DEG C, be then transferred in autoclave, react 5h at 180 DEG C; After being cooled to room temperature, centrifuging, dries, obtains CoFe in 50 DEG C of vacuum drying chambers
2o
4/ rGo.
embodiment 3coFe
2o
4the preparation of/rGo
By 12 mg graphene oxide ultrasonic disperse in 35 ml absolute ethyl alcohols, add the Fe (NO of 1.5 ml 0.2 mol/mL successively
3)
3aqueous solution, 0.75 ml of 0.2 mol/L Co (NO
3)
2aqueous solution and 3.0 ml ammoniacal liquor, stir 14h at 80 DEG C, be then transferred in autoclave, react 6h at 180 DEG C; After being cooled to room temperature, centrifuging, dries, obtains CoFe in 50 DEG C of vacuum drying chambers
2o
4/ rGo.
embodiment 4the preparation of Au@PdNRs-Ab
Cetyl trimethyl ammonium bromide (CTAB) solution of 20mL gold nanorods solution and 20mL 0.05 mol/L is mixed, stir at 30 DEG C, and order adds L-AA (AA) solution of 3 mL 0.1 mol/L, the palladium bichloride (PdCl of 0.5 mL 0.03 mol/L
2) NaOH (NaOH) solution of solution and 3 mL 0.1 mol/L, stop stirring, leave standstill 1h, centrifuging, adds the PBS buffer solution of 20mL pH7.4, and then adds environmental estrogens antibody (Ab), at 4 DEG C, shake 24h, obtain Au@PdNRs-Ab.
embodiment 5the preparation of Au@PdNRs-Ab
The CTAB solution of 20mL gold nanorods solution and 20mL 0.1 mol/L is mixed, stirs at 35 DEG C, and order adds the AA solution of 4 mL 0.1 mol/L, the PdCl of 0.8 mL 0.03 mol/L
2the NaOH solution of solution and 4 mL 0.1 mol/L, stop stirring, leave standstill 2h, centrifuging, adds the PBS buffer solution of 20mL pH7.4, and then adds environmental estrogens antibody (Ab), at 4 DEG C, shake 24h, obtains Au@PdNRs-Ab.
embodiment 6the preparation of Au@PdNRs-Ab
The CTAB solution of 20mL gold nanorods solution and 20mL 0.2 mol/L is mixed, stirs at 45 DEG C, and order adds the AA solution of 6 mL 0.1 mol/L, the PdCl of 1 mL 0.03 mol/L
2the NaOH solution of solution and 6 mL 0.1 mol/L, stop stirring, leave standstill 3h, centrifuging, adds the PBS buffer solution of 20mL pH7.4, and then adds environmental estrogens antibody (Ab), at 4 DEG C, shake 24h, obtains Au@PdNRs-Ab.
embodiment 7the preparation method of the unmarked immunosensor working electrode of environmental estrogens
(1) pre-service is carried out to working electrode, make its any surface finish;
(2) electrode surface obtained in (1) drips the CoFe of 5 μ L 1 mg/mL
2o
4/ rGo aqueous solution, dries under room temperature naturally;
(3) electrode surface obtained in (2) drips the glutaraldehyde of 5 μ L, naturally dries under room temperature;
(4) electrode surface obtained in (3) drips the Au@PdNRs-Ab solution of 5 μ L 10 μ g/mL, preserves and dry in 4 DEG C of refrigerators, and ultrapure water cleans, and dries film forming, 4 DEG C of preservations;
(5) electrode surface obtained in (4) drips the BSA solution of 4 μ L 100 μ g/mL, in order to the nonspecific activity site on enclosed-electrode, preserve and dry in 4 DEG C of refrigerators, ultrapure water cleans, dry film forming, namely obtain the working electrode of the unmarked immunosensor of environmental estrogens.
embodiment 8the preparation method of the unmarked immunosensor working electrode of environmental estrogens
(1) pre-service is carried out to working electrode, make its any surface finish;
(2) electrode surface obtained in (1) drips the CoFe of 8 μ L 3 mg/mL
2o
4/ rGo aqueous solution, dries under room temperature naturally;
(3) electrode surface obtained in (2) drips the glutaraldehyde of 8 μ L, naturally dries under room temperature;
(4) electrode surface obtained in (3) drips the Au@PdNRs-Ab solution of 6 μ L 10 μ g/mL, preserves and dry in 4 DEG C of refrigerators, and ultrapure water cleans, and dries film forming, 4 DEG C of preservations;
(5) electrode surface obtained in (4) drips the BSA solution of 5 μ L 100 μ g/mL, in order to the nonspecific activity site on enclosed-electrode, preserve and dry in 4 DEG C of refrigerators, ultrapure water cleans, dry film forming, namely obtain the working electrode of the unmarked immunosensor of environmental estrogens.
embodiment 9the preparation method of the unmarked immunosensor working electrode of environmental estrogens
(1) pre-service is carried out to working electrode, make its any surface finish;
(2) electrode surface obtained in (1) drips the CoFe of 10 μ L 5 mg/mL
2o
4/ rGo aqueous solution, dries under room temperature naturally;
(3) electrode surface obtained in (2) drips the glutaraldehyde of 10 μ L, naturally dries under room temperature;
(4) electrode surface obtained in (3) drips the Au@PdNRs-Ab solution of 8 μ L 10 μ g/mL, preserves and dry in 4 DEG C of refrigerators, and ultrapure water cleans, and dries film forming, 4 DEG C of preservations;
(5) electrode surface obtained in (4) drips the BSA solution of 6 μ L 100 μ g/mL, in order to the nonspecific activity site on enclosed-electrode, preserve and dry in 4 DEG C of refrigerators, ultrapure water cleans, dry film forming, namely obtain the working electrode of the unmarked immunosensor of environmental estrogens.
embodiment 10the unmarked immunosensor of environmental estrogens prepared by embodiment 1 ~ 9, for the detection of environmental estrogens, step is as follows
(1) the environmental estrogens antigen standard solution of concentration known is joined in the PBS buffer solution of 40 μ L, pH=7.0, obtained antigen mixed solution, get 5 μ L antigen mixed solutions drip be coated onto prepared environmental estrogen immunosensor working electrode on, preserve in 4 DEG C of refrigerators and dry, after the PBS buffer solution cleaning of pH=7.0, dry, 4 DEG C of preservations;
(2) mercurous chloride electrode will be obtained as contrast electrode, and as obtaining platinum electrode to electrode, form three-electrode system with the working electrode assembled in (1), being connected on electrochemical workstation; Successively add the PBS buffer solution of 10mL pH=7.0 and the H of 1mL 3 mmol/L in a cell
2o
2solution; The working electrode of assembling is detected to H by chronoamperometry
2o
2response; According to the relation between the current-responsive of gained and environmental estrogens antigen concentration of standard solution, drawing curve;
(3) testing sample solution is replaced the standard solution of environmental estrogens antigen, detect according to the method for drafting of the working curve of described environmental estrogens antigen.
embodiment 11the unmarked immunosensor of environmental estrogens prepared by embodiment 1 ~ 9, for the detection of environmental estrogens, step is as follows
(1) the environmental estrogens antigen standard solution of concentration known is joined in the PBS buffer solution of 50 μ L, pH=7.4, obtained antigen mixed solution, get 7 μ L antigen mixed solutions drip be coated onto prepared environmental estrogen immunosensor working electrode on, preserve in 4 DEG C of refrigerators and dry, after the PBS buffer solution cleaning of pH=7.4, dry, 4 DEG C of preservations;
(2) mercurous chloride electrode will be obtained as contrast electrode, and as obtaining platinum electrode to electrode, form three-electrode system with the working electrode assembled in (1), being connected on electrochemical workstation; Successively add the PBS buffer solution of 20mL pH=7.4 and the H of 1mL 4 mmol/L in a cell
2o
2solution; The working electrode of assembling is detected to H by chronoamperometry
2o
2response; According to the relation between the current-responsive of gained and environmental estrogens antigen concentration of standard solution, drawing curve;
(3) testing sample solution is replaced the standard solution of environmental estrogens antigen, detect according to the method for drafting of the working curve of described environmental estrogens antigen.
embodiment 12the unmarked immunosensor of environmental estrogens prepared by embodiment 1 ~ 9, for the detection of environmental estrogens, step is as follows
(1) the environmental estrogens antigen standard solution of concentration known is joined in the PBS buffer solution of 60 μ L, pH=8.0, obtained antigen mixed solution, get 10 μ L antigen mixed solutions drip be coated onto prepared environmental estrogen immunosensor working electrode on, preserve in 4 DEG C of refrigerators and dry, after the PBS buffer solution cleaning of pH=8.0, dry, 4 DEG C of preservations;
(2) mercurous chloride electrode will be obtained as contrast electrode, and as obtaining platinum electrode to electrode, form three-electrode system with the working electrode assembled in (1), being connected on electrochemical workstation; Successively add the PBS buffer solution of 25mL pH=8.0 and the H of 1mL 6 mmol/L in a cell
2o
2solution; The working electrode of assembling is detected to H by chronoamperometry
2o
2response; According to the relation between the current-responsive of gained and environmental estrogens antigen concentration of standard solution, drawing curve;
(3) testing sample solution is replaced the standard solution of environmental estrogens antigen, detect according to the method for drafting of the working curve of described environmental estrogens antigen.
embodiment 13environmental estrogens: the detection of estradiol, estriol, diethylstilbestrol, bisphenol-A or nonylphenol, comprises the following steps
(1) a kind of environmental estrogens is selected, according to the unmarked immunosensor of step constructing environment estrogen described in embodiment 1 ~ 9;
(2) detect according to the step described in embodiment 10 ~ 12, the detection technique index of environmental estrogens is in table 1.
The detection technique index of table 1 environmental estrogens
embodiment 14the detection of environmental estrogens in milk
Accurately pipette milk sample, add the environmental estrogens antigen standard solution of certain mass concentration, not add the milk of environmental estrogens antigen for blank, carry out recovery testu, detect according to the step of embodiment 13, measure the recovery of environmental estrogens in milk sample, testing result is in table 2.
The testing result of multiple environmental estrogens in table 2 milk
Table 2 testing result is known, and the relative standard deviation (RSD) of result is less than 3.0 %, and average recovery rate is 96.0 ~ 102%, shows that the present invention can be used for the detection of multiple environmental estrogens in milk, and highly sensitive, the high specificity of method, result accurately and reliably.
embodiment 15the detection of environmental estrogens in water sample
Accurately pipette certain water sample, add the environmental estrogens antigen standard solution of certain mass concentration, not add the water sample of environmental estrogens antigen for blank, carry out recovery testu, detect according to the step of embodiment 13, measure the recovery of environmental estrogens in water sample, testing result is in table 3.
The testing result of multiple environmental estrogens in table 3 water sample
Table 3 testing result is known, and the relative standard deviation (RSD) of result is less than 3.3 %, and average recovery rate is 97.2 ~ 102%, shows that the present invention can be used for the detection of multiple environmental estrogens in water sample, and highly sensitive, the high specificity of method, result accurately and reliably.
Claims (2)
1. a preparation method for the unmarked immunosensor of environmental estrogens, the unmarked immunosensor of described environmental estrogens comprises: working electrode, contrast electrode and to electrode, the basal electrode of described working electrode is glass-carbon electrode, and CoFe is modified successively in its surface
2o
4/ rGo, glutaraldehyde, Au@PdNRs-Ab, bovine serum albumin BSA, described contrast electrode is saturated calomel electrode, and described is platinum electrode to electrode, described CoFe
2o
4/ rGo is cobalt ferrite/reduced graphene compound substance, and described Au PtNRs-Ab is the environmental estrogens antibody of golden palladium core-shell nano rod composite material hatching;
It is characterized in that, described preparation method comprises the following steps:
A, preparation CoFe
2o
4/ rGo;
B, preparation Au@PdNRs-Ab;
C, prepare the unmarked immunosensor working electrode of environmental estrogens;
Wherein, step a prepares CoFe
2o
4the concrete steps of/rGo are as follows:
By 8 ~ 12 mg graphene oxide ultrasonic disperse in 25 ~ 35 ml absolute ethyl alcohols, add the Fe (NO of 0.5 ~ 1.5 ml 0.2 mol/mL successively
3)
3aqueous solution, 0.25 ~ 0.75 ml of 0.2 mol/L Co (NO
3)
2aqueous solution and 2.0 ~ 3.0 ml ammoniacal liquor, stir 10 ~ 14h at 80 DEG C, be then transferred in autoclave, reacts 4 ~ 6h at 180 DEG C; After being cooled to room temperature, centrifuging, dries, obtains CoFe in 50 DEG C of vacuum drying chambers
2o
4/ rGo;
The concrete steps that step b prepares Au@PdNRs-Ab are as follows:
The cetyl trimethyl ammonium bromide solution of 20mL gold nanorods solution and 20mL 0.05 ~ 0.2 mol/L is mixed, stir at 30 ~ 45 DEG C, and order adds the sodium hydroxide solution of the L-AA solution of 3 ~ 6 mL 0.1 mol/L, the palladium chloride solution of 0.5 ~ 1 mL 0.03 mol/L and 3 ~ 6 mL 0.1 mol/L, stop stirring, leave standstill 1 ~ 3h, centrifuging, add the PBS buffer solution of 20mL pH7.4, and then add environmental estrogens antibody A b, at 4 DEG C, shake 24h, obtain Au@PdNRs-Ab;
The concrete steps that step c prepares environmental estrogens unmarked immunosensor working electrode are as follows:
1. pre-service is carried out to working electrode, make its any surface finish;
2. the electrode surface obtained in 1. drips the CoFe of 5 ~ 10 μ L 1 ~ 5 mg/mL
2o
4/ rGo aqueous solution, dries under room temperature naturally;
3. the electrode surface obtained in 2. drips the glutaraldehyde of 5 ~ 10 μ L, naturally dries under room temperature;
4. the electrode surface obtained in 3. drips the Au@PdNRs-Ab solution of 5 ~ 8 μ L 10 μ g/mL, preserves and dry in 4 DEG C of refrigerators, and ultrapure water cleans, and dries film forming, 4 DEG C of preservations;
5. the electrode surface obtained in 4. drips the BSA solution of 4 ~ 6 μ L 100 μ g/mL, in order to the nonspecific activity site on enclosed-electrode, preserve and dry in 4 DEG C of refrigerators, ultrapure water cleans, dry film forming, namely obtain the working electrode of the unmarked immunosensor of environmental estrogens.
2. the unmarked immunosensor of environmental estrogens as claimed in claim 1, for the detection of environmental estrogens, step is as follows:
1) the environmental estrogens antigen standard solution of concentration known is joined in the PBS buffer solution of 40 ~ 60 μ L, pH=7.0 ~ 8.0, obtained antigen mixed solution, get 5 ~ 10 μ L antigen mixed solutions drip be coated onto prepared environmental estrogen immunosensor working electrode on, preserve in 4 DEG C of refrigerators and dry, after the PBS buffer solution cleaning of pH=7.0 ~ 8.0, dry, 4 DEG C of preservations;
2) mercurous chloride electrode will be obtained, as obtaining platinum electrode to electrode, with 1 as contrast electrode) in the working electrode assembled form three-electrode system, be connected on electrochemical workstation; Successively add the PBS buffer solution of 10 ~ 25mL pH=7.0 ~ 8.0 and the hydrogen peroxide H of 1mL 3 ~ 6 mmol/L in a cell
2o
2solution; The working electrode of assembling is detected to H by chronoamperometry
2o
2response; According to the relation between the current-responsive of gained and environmental estrogens antigen concentration of standard solution, drawing curve;
3) testing sample solution is replaced the standard solution of environmental estrogens antigen, detect according to the method for drafting of the working curve of described environmental estrogens antigen.
3
.the preparation method of the unmarked immunosensor of environmental estrogens as claimed in claim 1, is characterized in that described environmental estrogens is selected from one of following: estradiol, estriol, diethylstilbestrol, bisphenol-A or nonylphenol.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410338509.7A CN104133070B (en) | 2014-07-17 | 2014-07-17 | Preparation method and use of environmental estrogen label-free immunosensor |
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|---|---|---|---|
| CN201410338509.7A CN104133070B (en) | 2014-07-17 | 2014-07-17 | Preparation method and use of environmental estrogen label-free immunosensor |
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| Publication Number | Publication Date |
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| CN104133070A CN104133070A (en) | 2014-11-05 |
| CN104133070B true CN104133070B (en) | 2015-07-08 |
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| CN105717180B (en) * | 2016-02-25 | 2018-03-27 | 济南大学 | A kind of preparation method and application of the optical electro-chemistry aflatoxin biology sensor based on two-dimensional nano composite |
| CN105717179B (en) * | 2016-02-25 | 2018-03-23 | 济南大学 | A kind of preparation method and application of optical electro-chemistry organo-chlorine pesticide biology sensor |
| CN107192751B (en) * | 2017-07-06 | 2019-03-05 | 衡阳师范学院 | A kind of electrochemical sensor and its preparation method and application for detecting estradiol |
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