CN103063833B - Preparation method and application of unmarked immunosensor for rapidly detecting clenbuterol - Google Patents
Preparation method and application of unmarked immunosensor for rapidly detecting clenbuterol Download PDFInfo
- Publication number
- CN103063833B CN103063833B CN201310026155.8A CN201310026155A CN103063833B CN 103063833 B CN103063833 B CN 103063833B CN 201310026155 A CN201310026155 A CN 201310026155A CN 103063833 B CN103063833 B CN 103063833B
- Authority
- CN
- China
- Prior art keywords
- clenbuterol hydrochloride
- preparation
- clenbuterol
- solution
- electrode
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention relates to a preparation method and application of an unmarked immunosensor for rapidly detecting clenbuterol. Clenbuterol means a medicine of a beta-stimulant type instead of a specific medicine, and has the functions of prompting lean meat growth and suppressing fat meat growth as well as extremely serious side effects, and when a large amount of clenbuterol is taken, the cardiovascular system of people can be damaged and severe neurological symptoms can be caused. According to the invention, a gold-silver-sulfide nano composite material modified electrode with a core-shell structure which combines the excellent characteristics of nano gold and nano silver sulfide is adopted for preparing the unmarked immunosensor, and a clenbuterol antibody is well immobilized, so that the unmarked immunosensor is low in cost, high in sensitivity, good in specificity, rapid in detection and simple to prepare.
Description
Technical field
The present invention relates to a kind of preparation method and application of unmarked immunosensor of fast detecting clenbuterol hydrochloride.Specifically based on gold-silver sulfide (Au-Ag
2the unmarked electrochemical immunosensor of the fast detecting clenbuterol hydrochloride that S) core-shell nano compound substance builds, belongs to Nano-function thin films and biosensor technology field.
Background technology
Electrochemical immunosensor is because the advantages such as it is highly sensitive, specificity is good, easy and simple to handle are widely used in the fields such as clinical diagnosis, environmental analysis, Pharmaceutical Analysis, food safety detection.The electrochemical immunosensor that processability is superior, its most critical technology is exactly the raising of the performances such as effectively fixing and sensitivity, reappearance and persistence of immune molecule.
Gold (Au) nano particle due to have specific surface area large, be easy to various biomolecules (nucleic acid, protein and biomolecule etc.) in conjunction with, the feature such as harmless, be widely used in the research of immunosensor.Silver sulfide (Ag
2s) nano particle is a kind of semiconductor nano material, because its rich surface is containing sulfydryl, and be easy to combine with biomolecule, but Ag
2it is inhomogeneous that S nano particle is prepared separately nucleation.The present invention utilizes layer-by-layer, gold-silver sulfide (Au-Ag of preparation
2s) core-shell nano is evenly distributed, and specific surface area significantly increases, and is easier to combine with biomolecule, is therefore very beneficial for preparing electrochemica biological sensor.
Clenbuterol hydrochloride is the medicine that a class is called beta-stimulants (β-agonist), rather than a certain specific medicine.This class medicine has realizes the function that promotes lean meat growth, suppresses fat meat growth, so be referred to as " clenbuterol hydrochloride ".In China, usually said clenbuterol hydrochloride refers to Clenbuterol (Clenbuterol), and ordinary consumer is referred to as clenbuterol hydrochloride this type of medicine.When they are used for its feeding to surpass therapeutic dose 5-10 consumption doubly, there is significant nutrition " redistribution effects "---promote the proteins deposited and lipolysis of animal body, suppress fat deposition, can significantly improve trunk lean meat percentage, increase weight and improve food conversion ratio, therefore be once used as growth accelerator, the feed addictive of the livestock and poultry such as ox, sheep, pig.
According to " clenbuterol hydrochloride " kind catalogue of the < < of office of the food security council of State Council " clenbuterol hydrochloride " focus efforts on special areas scheme > > (food peace is done (2011) No. 14) regulation, be 16 kinds, comprise: Ractopamine (Ractopamine); Clenbuterol (Clenbuterol); Salbutamol (Salbutamol); Salbutamol sulfate (Salbutamol Sulfate); Dopamine hydrochloride (Dopamine Hydrochloride); Cimaterol (Cimaterol); Bricalin (Terbutaline Sulfate); Phenolethanolamine A(Phenylethanolamine A); Bambuterol (Bambuterol); Hydrochloric acid Zilpaterol (Zilpaterol Hydrochloride); Clorprenaline hydrochloride (Clorprenaline Hydrochloride); Mabuterol (Mabuterol); Western Boot sieve (Cimbuterol); Bromine Boot sieve (Brombuterol); Tartrate Afromoterol (Arformoterol Tartrate); Formoterol fumarate (Formoterol Fumatrate).
Clenbuterol hydrochloride can improve the lean meat percentage of pig, brings more economic worths, but it has the spinoff of danger close, when intake is larger, can cause the damage of cardiovascular system, and may occur serious nervous symptoms.Poisoning this serious phenomenon that takes place frequently for clenbuterol hydrochloride, the ， Ministry of Agriculture issued the documents respectively and forbade that food animal bans use of beta-stimulants as feed addictive (No. 176, No. 193 bulletins of the Ministry of Agriculture, No. 1519 regulations) Dec 27 calendar year 2001, on February 9th, 2002, April 9.
The method that detects clenbuterol hydrochloride has high performance liquid chromatography (HPLC), gas chromatography-mass spectrography (GC-MS) and euzymelinked immunosorbent assay (ELISA) (ELISA).These methods, though have certain sensitivity, there are the following problems, limited the popularization in actual applications of these methods:
1. high performance liquid chromatography (HPLC): advantage is beta-stimulants and the metabolic product thereof that is applicable to detecting thermally labile and strong polarity, specificity is good, selectivity is strong, it is higher to detect degree of accuracy, can with the system couplings such as mass spectrum (MS), the robotization of Realization analysis process, China is using HPLC method as the half authenticity method that detects residual of kelengtelu, and lowest detectable limit scope is 1 ~ 15 ngg
-1; Shortcoming is that the sample preparation time is long, and testing process is loaded down with trivial details, be difficult to operation, and instrument is valuable, is subject to certain restrictions in actual applications.
2. gas chromatography-mass spectrography (GC-MS): advantage is by efficiently the highly sensitive qualitative analysis of separating effect and mass spectrum is fast altogether organic chromatogram, can in the simultaneous situation of multiple residue, to certain specific residue, carry out qualitative and quantitative analysis, compare with HPLC method, this method detection sensitivity is higher, false positive rate Geng Di， China is legal for detecting the authenticity method of Clenbuterol by GC-MS; Shortcoming is that instrument and equipment is complicated, operating process is loaded down with trivial details, very high to reviewer's operative skill requirement, and is unsuitable for scene and fast detecting.
3. euzymelinked immunosorbent assay (ELISA) (ELISA): advantage is that with the clenbuterol hydrochloride content in ocular estimate or colorimetric determination sample, so this method selectivity is strong, specificity good by utilizing the efficient catalytic effect of the combination of immunology antigen and antibody specific and enzyme; Shortcoming is that step is various, detects error larger.
In order to address the above problem, the present invention adopts the electrochemical immunosensor building with nano composite material to detect clenbuterol hydrochloride.In the preparation process of electrochemical immunosensor, key factor is exactly electrode modified material, noble metal nanometer material and noble metal semiconductor nano material have the features such as large specific surface area, stable performance and good biocompatibility, electric conductivity due to it, become the preferred material of assembling electrochemical immunosensor.The present invention by generating nanometer Ag on nanometer Au core
2s semiconductor shell, has prepared the Au-Ag that has the two characteristic concurrently, embodies the nucleocapsid structure of excellent collaborative effect of enhanced sensitivity
2s nano composite material, uses it for and prepares electrochemical immunosensor, has further improved the electronics transmission efficiency of its electrode surface and the adsorbance of antibody, has significantly improved the sensitivity detecting.The present invention designed a kind of simple, fast, the good and highly sensitive electrochemical immunosensor of specificity and electrochemical immunoanalytical method accordingly.
Summary of the invention
One of order of the present invention is to avoid that the instrument and equipment that exists in existing detection method is complicated, operating process is loaded down with trivial details and reviewer's technical ability is required to the shortcomings such as high, the preparation method of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride is provided, feature highly sensitive, high specificity that prepared sensor has, and prepare simple, easy to operately, the rapid sensitive that can be used for clenbuterol hydrochloride detects;
Two of object of the present invention is to provide a kind of simple, special, sensitive, detection method of detecting fast and efficiently clenbuterol hydrochloride in pork, beef and mutton.
The technical solution used in the present invention is as follows:
1. the preparation method of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride of the present invention, its preparation process is: take glass-carbon electrode as working electrode, at electrode face finish Au-Ag
2s core-shell nano compound substance-chitosan solution, then modify with clenbuterol hydrochloride antibody-solutions, finally drip painting bovine serum albumin solution, the non-specific avtive spot in enclosed-electrode surface;
Described Au-Ag
2s core-shell nano compound substance-chitosan solution, Au-Ag
2the mass ratio of S core-shell nano compound substance and shitosan is 1: 4 ~ 9;
Described Au-Ag
2s core-shell nano compound substance, Au and Ag
2the mass ratio of S is 3.4 ~ 8.0: 1, and its preparation process is: after Au-Ag nano particle is centrifugal, be again dispersed in 0.1 molL
-1cTAB in, add 100 mL sulphur precursor solutions, shake after 1 ~ 3 min, standing 1 ~ 2 h, after centrifuging, removes supernatant, vacuum drying at 50 ℃, makes Au-Ag
2s core-shell nano compound substance.
The preparation method of the described unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride, with preferred preparation method is more specifically:
(1) Au-Ag
2the preparation of S core-shell nano compound substance:
1) at 250 mL 1 mmolL
-1in chlorauric acid solution, add 60 mL 10 mmolL
-1freezing point-boron hydracid sodium solution, stirs after 1h, dilutes 10 times, makes nanometer Au seed;
2) at 150 mL 1 mmolL
-1in chlorauric acid solution, drip 0.1 molL
-1ascorbic acid is to colourless, then adds 5, Au seed in step 1), and standing 2 h, make Au nano particle;
3) by step 2) in the Au nano particle that makes centrifugal after, be again dispersed in 300 mL 0.1 molL
-1cTAB in, add successively 30 mL 0.1 molL
-1ascorbic acid, 3 ~ 7 mL 0.01 molL
-1agNO
3, 30 mL 0.1 molL
-1naOH aqueous solution, 1 min that vibrates, standing 1 h, makes the Au-Ag nano particle of nucleocapsid structure;
4) after the Au-Ag nano particle making in step 3) is centrifugal, be again dispersed in 300 mL 0.1 molL
-1cTAB in, add 100 mL sulphur precursor solutions, shake after 1 ~ 3 min, standing 1 ~ 2 h, after centrifuging, removes supernatant, vacuum drying at 50 ℃, makes Au-Ag
2s core-shell nano compound substance, Au and Ag
2the mass ratio of S is 3.4 ~ 8.0: 1;
Described gold chloride, boron hydracid sodium, ascorbic acid, AgNO
3with the aqueous solution of NaOH, all use ultrapure water preparation;
Described freezing point-boron hydracid sodium solution, preparation method is placed in cooling 3-4 min of ice cube by the test tube of prepackage boron hydracid sodium solution, takes out test tube and prepares after boron hydracid sodium solution, then test tube is put into cooling a period of time of ice cube;
Described sulphur precursor solution, preparation method will add sulphur powder in sodium sulfide solution, be placed in 80 ℃ of reactors heating 2h and all dissolve to sulphur powder.
(2) Au-Ag
2the preparation of S core-shell nano compound substance-chitosan solution: shitosan is joined in the acetic acid that volume fraction is 1 % and stirs 6 h, make the chitosan solution of massfraction 0.3 ~ 0.7 %; Get 3 mg Au-Ag
2s core-shell nano compound substance, is dispersed in 4 mL chitosan solutions Au-Ag again
2the mass ratio of S core-shell nano compound substance and shitosan is 1: 4 ~ 9.
(3) processing of glass-carbon electrode: the glass-carbon electrode of diameter 4 mm is used successively to the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 μ m, ethanol ultrasonic cleaning, then rinse well with ultrapure water, make glass-carbon electrode surface be minute surface.
(4) electrode face finish: drip and be coated with 6 μ L Au-Ag on glass-carbon electrode surface
2s core-shell nano compound substance-chitosan solution, is put in 4 ℃ of refrigerators, dries film forming; Ultrapure water cleans, then drips painting 6 μ L clenbuterol hydrochloride antibody-solutions, is put in 4 ℃ of refrigerators, dries film forming; After ultrapure water cleans, be put in 4 ℃ of refrigerators and preserve and use;
Described clenbuterol hydrochloride antibody-solutions, preparation method is: it is 7.4 phosphate buffered solution constant volume that clenbuterol hydrochloride antibody stoste is added to pH, is put in 4 ℃ of refrigerators and preserves and use.
(5) the non-specific avtive spot in enclosed-electrode surface: get 6 μ L bovine serum albumin solutions and drip and be coated in (4) prepared electrode surface, in 4 ℃ of refrigerators, dry film forming, ultrapure water cleans, make the described unmarked immunosensor of fast detecting clenbuterol hydrochloride, be put in 4 ℃ of refrigerators and preserve and use;
Described bovine serum albumin solution, preparation method is: it is 7.4 phosphate buffered solution constant volume that bovine serum albumin(BSA) stoste is added to pH, is put in 4 ℃ of refrigerators and preserves and use.
2. the preparation method of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride of the present invention, described immunosensor is applied to clenbuterol hydrochloride and detects, and its detecting step is:
(1) standard solution preparation: prepare one group of clenbuterol hydrochloride standard solution that comprises the variable concentrations of blank standard specimen, end liquid is the phosphate buffered solution of pH 7.4.
(2) working electrode is modified: using the unmarked immunosensor of fast detecting clenbuterol hydrochloride of the present invention as working electrode, the clenbuterol hydrochloride standard solution of the variable concentrations of preparation in step (1) is dripped respectively and is coated onto working electrode surface, be put in 4 ℃ of refrigerators and preserve and use.
(3) working curve is drawn: using saturated calomel electrode as contrast electrode, platinum electrode is as auxiliary electrode, and the working electrode of having been modified with step (2) forms three-electrode system, connects electrochemical workstation, at K
3[Fe (CN)
6] in solution, with square wave voltammetry, (in SWV) – 0.6 ~ 0.2 V voltage range, detect, the response current of blank standard specimen is designated as
i 0, the response current of the clenbuterol hydrochloride standard solution that contains variable concentrations is denoted as
i i, the difference that response current reduces is Δ
i=
i 0 -
i i , Δ
imass concentration with clenbuterol hydrochloride standard solution
cbetween linear, draw Δ
i–
cworking curve.
(4) detection of clenbuterol hydrochloride: replace the clenbuterol hydrochloride standard solution in step (1) with testing sample, detect according to the method in step (2) and (3), the difference DELTA reducing according to response current
iand working curve, obtain the content of clenbuterol hydrochloride in testing sample.
Described K
3[Fe (CN)
6] concentration of solution is 5 mmolL
-1.
The concentration of the phosphate buffered solution of described pH 7.4 is 67 mmolL
-1.
It is one of following that described clenbuterol hydrochloride is selected from: Ractopamine, Clenbuterol, salbutamol, salbutamol sulfate, Dopamine hydrochloride, Cimaterol, bricalin, phenolethanolamine A, bambuterol, hydrochloric acid Zilpaterol, clorprenaline hydrochloride, Mabuterol, western Boot sieve, bromine Boot sieve, tartrate Afromoterol, formoterol fumarate.
useful achievement of the present invention
(1) immunosensor preparation of the present invention is simple, easy to operate, and by synergy and the sensitization of nano material, can realize quick, sensitive, the high selectivity of actual sample are detected, and has future develop.
(2) the present invention is first by Au-Ag
2s core-shell nano compound substance is applied to the preparation of electrochemical immunosensor, described Au-Ag
2s core-shell nano conductivity of composite material is better than nanometer Au or nanometer Ag, and electric signal obtains sending out more by force greatly; Outer Ag
2s has strengthened the absorption of biomolecule and immobilized, has significantly improved sensitivity, stability and the accuracy of electrochemical immunosensor; And this material, with obvious blue, can further be applied in Test paper, has larger development potentiality.
Accompanying drawing explanation
Fig. 1 is preparation method's schematic diagram of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride.
Embodiment
embodiment 1the preparation method of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride:
(1) processing of glass-carbon electrode: the glass-carbon electrode of diameter 4 mm is used successively to the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 μ m, ethanol ultrasonic cleaning, then rinse well with ultrapure water, make glass-carbon electrode surface be minute surface.
(2) Au-Ag
2the preparation of S core-shell nano compound substance:
1) at 250 mL 1 mmolL
-1in chlorauric acid solution, add 60 mL 10 mmolL
-1freezing point-boron hydracid sodium solution, stirs after 1h, dilutes 10 times, makes nanometer Au seed;
2) at 150 mL 1 mmolL
-1in chlorauric acid solution, drip 0.1 molL
-1ascorbic acid is to colourless, then adds 5, Au seed in step 1), and standing 2 h, make Au nano particle;
3) by step 2) in the Au nano particle that makes centrifugal after, be again dispersed in 300 mL 0.1 molL
-1cTAB in, add successively 30 mL 0.1 molL
-1ascorbic acid, 3 mL 0.01 molL
-1agNO
3, 30 mL 0.1 molL
-1naOH aqueous solution, 1 min that vibrates, standing 1 h, makes the Au-Ag nano particle of nucleocapsid structure;
4) after the Au-Ag nano particle making in step 3) is centrifugal, be again dispersed in 300 mL 0.1 molL
-1cTAB in, add 100 mL sulphur precursor solutions, shake after 1 ~ 3 min, standing 1 ~ 2 h, after centrifuging, removes supernatant, vacuum drying at 50 ℃, makes Au-Ag
2s core-shell nano compound substance, Au and Ag
2the mass ratio of S is 3.4: 1;
Described gold chloride, boron hydracid sodium, ascorbic acid, AgNO
3with the aqueous solution of NaOH, all use ultrapure water preparation;
Described freezing point-boron hydracid sodium solution, preparation method is placed in cooling 3-4 min of ice cube by the test tube of prepackage boron hydracid sodium solution, takes out test tube and prepares after boron hydracid sodium solution, then test tube is put into cooling a period of time of ice cube;
Described sulphur precursor solution, preparation method will add sulphur powder in sodium sulfide solution, be placed in 80 ℃ of reactors heating 2h and all dissolve to sulphur powder.
(3) Au-Ag
2the preparation of S core-shell nano compound substance-chitosan solution: shitosan is joined in the acetic acid that volume fraction is 1 % and stirs 6 h, make the chitosan solution of massfraction 0.3 %; Get 3 mg Au-Ag
2s core-shell nano compound substance, is dispersed in 4 mL chitosan solutions Au-Ag again
2the mass ratio of S core-shell nano compound substance and shitosan is 1: 4.
(4) preparation of clenbuterol hydrochloride antibody-solutions: it is 7.4 phosphate buffered solution constant volume that clenbuterol hydrochloride antibody stoste is added to pH, is put in 4 ℃ of refrigerators and preserves and use.
(5) preparation of bovine serum albumin solution: it is 7.4 phosphate buffered solution constant volume that bovine serum albumin(BSA) stoste is added to pH, is put in 4 ℃ of refrigerators and preserves and use.
(6) electrode face finish: according to step shown in Fig. 1, drip and be coated with 6 μ L Au-Ag on glass-carbon electrode surface
2s core-shell nano compound substance-chitosan solution, is put in 4 ℃ of refrigerators, dries film forming; Ultrapure water cleans, then drips painting 6 μ L clenbuterol hydrochloride antibody-solutions, is put in 4 ℃ of refrigerators, dries film forming; After ultrapure water cleans, be put in 4 ℃ of refrigerators and preserve and use.
(7) the non-specific avtive spot in enclosed-electrode surface: according to step shown in Fig. 1, getting 6 μ L bovine serum albumin solutions drips and is coated in (6) prepared electrode surface, in 4 ℃ of refrigerators, dry film forming, ultrapure water cleans, make the described unmarked immunosensor of fast detecting clenbuterol hydrochloride, be put in 4 ℃ of refrigerators and preserve and use.
embodiment 2the preparation method of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride:
(1) processing of glass-carbon electrode: with embodiment 1.
(2) Au-Ag
2the preparation of S core-shell nano compound substance:
1) at 250 mL 1 mmolL
-1in chlorauric acid solution, add 60 mL 10 mmolL
-1freezing point-boron hydracid sodium solution, stirs after 1h, dilutes 10 times, makes nanometer Au seed;
2) at 150 mL 1 mmolL
-1in chlorauric acid solution, drip 0.1 molL
-1ascorbic acid is to colourless, then adds 5, Au seed in step 1), and standing 2 h, make Au nano particle;
3) by step 2) in the Au nano particle that makes centrifugal after, be again dispersed in 300 mL 0.1 molL
-1cTAB in, add successively 30 mL 0.1 molL
-1ascorbic acid, 5 mL 0.01 molL
-1agNO
3, 30 mL 0.1 molL
-1naOH aqueous solution, 1 min that vibrates, standing 1 h, makes the Au-Ag nano particle of nucleocapsid structure;
4) after the Au-Ag nano particle making in step 3) is centrifugal, be again dispersed in 300 mL 0.1 molL
-1cTAB in, add 100 mL sulphur precursor solutions, shake after 1 ~ 3 min, standing 1 ~ 2 h, after centrifuging, removes supernatant, vacuum drying at 50 ℃, makes Au-Ag
2s core-shell nano compound substance, Au and Ag
2the mass ratio of S is 4.8: 1;
(3) Au-Ag
2the preparation of S core-shell nano compound substance-chitosan solution: shitosan is joined in the acetic acid that volume fraction is 1 % and stirs 6 h, make the chitosan solution of massfraction 0.5 %; Get 3 mg Au-Ag
2s core-shell nano compound substance, is dispersed in 4 mL chitosan solutions Au-Ag again
2the mass ratio of S core-shell nano compound substance and shitosan is 1: 6.
(4) preparation of clenbuterol hydrochloride antibody-solutions: with embodiment 1.
(5) preparation of bovine serum albumin solution: with embodiment 1.
(6) electrode face finish: with embodiment 1.
(7) the non-specific avtive spot in enclosed-electrode surface: with embodiment 1.
embodiment 3a preparation method for the unmarked immunosensor of fast detecting clenbuterol hydrochloride, as shown in Figure 1
(1) processing of glass-carbon electrode: with embodiment 1.
(2) Au-Ag
2the preparation of S core-shell nano compound substance:
1) at 250 mL 1 mmolL
-1in chlorauric acid solution, add 60 mL 10 mmolL
-1freezing point-boron hydracid sodium solution, stirs after 1h, dilutes 10 times, makes nanometer Au seed;
2) at 150 mL 1 mmolL
-1in chlorauric acid solution, drip 0.1 molL
-1ascorbic acid is to colourless, then adds 5, Au seed in step 1), and standing 2 h, make Au nano particle;
3) by step 2) in the Au nano particle that makes centrifugal after, be again dispersed in 300 mL 0.1 molL
-1cTAB in, add successively 30 mL 0.1 molL
-1ascorbic acid, 7 mL 0.01 molL
-1agNO
3, 30 mL 0.1 molL
-1naOH aqueous solution, 1 min that vibrates, standing 1 h, makes the Au-Ag nano particle of nucleocapsid structure;
4) after the Au-Ag nano particle making in step 3) is centrifugal, be again dispersed in 300 mL 0.1 molL
-1cTAB in, add 100 mL sulphur precursor solutions, shake after 1 ~ 3 min, standing 1 ~ 2 h, after centrifuging, removes supernatant, vacuum drying at 50 ℃, makes Au-Ag
2s core-shell nano compound substance, Au and Ag
2the mass ratio of S is 8.0: 1;
(3) Au-Ag
2the preparation of S core-shell nano compound substance-chitosan solution: shitosan is joined in the acetic acid that volume fraction is 1 % and stirs 6 h, make the chitosan solution of massfraction 0.7 %; Get 3 mg Au-Ag
2s core-shell nano compound substance, is dispersed in 4 mL chitosan solutions Au-Ag again
2the mass ratio of S core-shell nano compound substance and shitosan is 1: 9.
(4) preparation of clenbuterol hydrochloride antibody-solutions: with embodiment 1.
(5) preparation of bovine serum albumin solution: with embodiment 1.
(6) electrode face finish: with embodiment 1.
(7) the non-specific avtive spot in enclosed-electrode surface: with embodiment 1.
embodiment 4the immunosensor that above-described embodiment 1-3 is prepared, for the detection of clenbuterol hydrochloride, step is as follows:
(1) standard solution preparation: prepare one group of clenbuterol hydrochloride standard solution that comprises the variable concentrations of blank standard specimen, end liquid is the phosphate buffered solution of pH 7.4.
(2) working electrode is modified: using the unmarked immunosensor of fast detecting clenbuterol hydrochloride of the present invention as working electrode, the clenbuterol hydrochloride standard solution of the variable concentrations of preparation in step (1) is dripped respectively and is coated onto working electrode surface, be put in 4 ℃ of refrigerators and preserve and use.
(3) working curve is drawn: using saturated calomel electrode as contrast electrode, platinum electrode is as auxiliary electrode, and the working electrode of having been modified with step (2) forms three-electrode system, connects electrochemical workstation, at K
3[Fe (CN)
6] in solution, with square wave voltammetry, (in SWV) – 0.6 ~ 0.2 V voltage range, detect, the response current of blank standard specimen is designated as
i 0, the response current of the clenbuterol hydrochloride standard solution that contains variable concentrations is denoted as
i i, the difference that response current reduces is Δ
i=
i 0 -
i i , Δ
imass concentration with clenbuterol hydrochloride standard solution
cbetween linear, draw Δ
i–
cworking curve.
(4) detection of clenbuterol hydrochloride: replace the clenbuterol hydrochloride standard solution in step (1) with testing sample, detect according to the method in step (2) and (3), the difference DELTA reducing according to response current
iand working curve, obtain the content of clenbuterol hydrochloride in testing sample.
Described K
3[Fe (CN)
6] concentration of solution is 5 mmolL
-1.
The concentration of the phosphate buffered solution of described pH 7.4 is 67 mmolL
-1.
It is one of following that described clenbuterol hydrochloride is selected from: Ractopamine, Clenbuterol, salbutamol, salbutamol sulfate, Dopamine hydrochloride, Cimaterol, bricalin, phenolethanolamine A, bambuterol, hydrochloric acid Zilpaterol, clorprenaline hydrochloride, Mabuterol, western Boot sieve, bromine Boot sieve, tartrate Afromoterol, formoterol fumarate.
The technical indicator that the prepared immunosensor of the present invention detects 16 kinds of clenbuterol hydrochlorides is in Table 1.
The technical indicator that table 1 detects 16 kinds of clenbuterol hydrochlorides is all better than similar technological means detection technique index.
embodiment 5the detection of clenbuterol hydrochloride in pork sample
Accurately take pork sample, adopt conventional method to carry out sample preparation, according to the step described in embodiment 4, detect, testing result is in Table 2.
Table 2 testing result is known, and the relative standard deviation of result (RSD) is less than 3.5%, and average recovery rate is 95.0 ~ 105%, shows that the present invention can be used for the detection of clenbuterol hydrochloride in pork and pig urine samples, highly sensitive, the high specificity of method, and result is accurately and reliably.
embodiment 6the detection of clenbuterol hydrochloride in beef sample
Accurately take beef sample, adopt conventional method to carry out sample preparation, according to the step described in embodiment 4, detect, testing result is in Table 3.
Table 3 testing result is known, and the relative standard deviation of result (RSD) is less than 3.3 %, and average recovery rate is 90.0 ~ 106%, shows that the present invention can be used for the detection of clenbuterol hydrochloride in beef sample, highly sensitive, the high specificity of method, and result is accurately and reliably.
embodiment 7the detection of clenbuterol hydrochloride in meat samples
Accurately take meat samples, adopt conventional method to carry out sample preparation, according to the step described in embodiment 4, detect, testing result is in Table 4.
Table 4 testing result is known, and the relative standard deviation of result (RSD) is less than 3.5 %, and average recovery rate is 93.0 ~ 105%, shows that the present invention can be used for the detection of clenbuterol hydrochloride in meat samples, highly sensitive, the high specificity of method, and result is accurately and reliably.
Claims (4)
1. a preparation method for the unmarked immunosensor of fast detecting clenbuterol hydrochloride, is characterized in that, preparation process is: take glass-carbon electrode as working electrode, at electrode face finish Au-Ag
2s core-shell nano compound substance-chitosan solution, then modify with clenbuterol hydrochloride antibody-solutions, finally drip painting bovine serum albumin solution, the non-specific avtive spot in enclosed-electrode surface, described Au-Ag
2s core-shell nano compound substance, Au and Ag
2the mass ratio of S is 3.4 ~ 8.0: 1, and its preparation process is: after Au-Ag nano particle is centrifugal, be again dispersed in 0.1 molL
-1cTAB in, add 100 mL sulphur precursor solutions, shake after 1 ~ 3 min, standing 1 ~ 2 h, after centrifuging, removes supernatant, vacuum drying at 50 ℃, makes Au-Ag
2s core-shell nano compound substance.
2. the preparation method of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride as claimed in claim 1, is characterized in that, described Au-Ag
2s core-shell nano compound substance-chitosan solution is by Au-Ag
2s core-shell nano compound substance is distributed in chitosan solution to be made, described chitosan solution is that shitosan sterling is joined to volume fraction is to be prepared from 1% acetic acid, in described chitosan solution, the massfraction of shitosan sterling is 0.3 ~ 0.7 %, described Au-Ag
2the mass ratio of S core-shell nano compound substance and shitosan sterling is 1: 4 ~ 9.
3. the preparation method of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride as claimed in claim 1, is characterized in that, described immunosensor is applied to the detection of clenbuterol hydrochloride, and its detecting step is:
(1) standard solution preparation: prepare one group of clenbuterol hydrochloride standard solution that comprises the variable concentrations of blank standard specimen, end liquid is the phosphate buffered solution of pH 7.4;
(2) working electrode is modified: using the unmarked immunosensor of fast detecting clenbuterol hydrochloride of the present invention as working electrode, the clenbuterol hydrochloride standard solution of the variable concentrations of preparation in step (1) is dripped respectively and is coated onto working electrode surface, in 4 ℃ of refrigerators, preserve;
(3) working curve is drawn: using saturated calomel electrode as contrast electrode, platinum electrode is as auxiliary electrode, and the working electrode of having been modified with step (2) forms three-electrode system, connects electrochemical workstation, at K
3[Fe (CN)
6] in solution, with square wave voltammetry, (in SWV) – 0.6 ~ 0.2 V voltage range, detect, the response current of blank standard specimen is designated as
i 0, the response current of the clenbuterol hydrochloride standard solution that contains variable concentrations is denoted as
i i, the difference that response current reduces is Δ
i=
i 0-
i i, Δ
imass concentration with clenbuterol hydrochloride standard solution
cbetween linear, draw Δ
i-
cworking curve;
(4) detection of clenbuterol hydrochloride: replace the clenbuterol hydrochloride standard solution in step (1) with testing sample, detect according to the method in step (2) and (3), the difference DELTA reducing according to response current
iand working curve, obtain the content of clenbuterol hydrochloride in testing sample.
4. the preparation method of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride as claimed in claim 1, is characterized in that described clenbuterol hydrochloride is selected from one of following: Ractopamine, Clenbuterol, salbutamol, salbutamol sulfate, Dopamine hydrochloride, Cimaterol, bricalin, phenolethanolamine A, bambuterol, hydrochloric acid Zilpaterol, clorprenaline hydrochloride, Mabuterol, western Boot sieve, bromine Boot sieve, tartrate Afromoterol, formoterol fumarate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310026155.8A CN103063833B (en) | 2013-01-21 | 2013-01-21 | Preparation method and application of unmarked immunosensor for rapidly detecting clenbuterol |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310026155.8A CN103063833B (en) | 2013-01-21 | 2013-01-21 | Preparation method and application of unmarked immunosensor for rapidly detecting clenbuterol |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103063833A CN103063833A (en) | 2013-04-24 |
| CN103063833B true CN103063833B (en) | 2014-02-26 |
Family
ID=48106544
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310026155.8A Expired - Fee Related CN103063833B (en) | 2013-01-21 | 2013-01-21 | Preparation method and application of unmarked immunosensor for rapidly detecting clenbuterol |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103063833B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343126B (en) * | 2013-07-19 | 2015-12-02 | 暨南大学 | Ractopamine hydrochloride aptamers and the aptamers electrochemica biological sensor detecting Ractopamine hydrochloride |
| CN103439394B (en) * | 2013-09-13 | 2015-04-08 | 遵义师范学院 | Electrochemical method for detecting content of brown meat essence |
| CN103808772B (en) * | 2013-12-03 | 2016-08-31 | 大连大学 | Bricalin electrochemical sensor |
| CN104502429B (en) * | 2015-01-25 | 2015-10-21 | 济南大学 | The preparation method and application of unmarked electrogenerated chemiluminescence clenbuterol hydrochloride immunosensor |
| CN105137063B (en) * | 2015-07-09 | 2016-04-20 | 济南大学 | A kind of preparation method of the unmarked electrochemical immunosensor for clenbuterol hydrochloride detection |
| CN105158453B (en) * | 2015-07-09 | 2017-01-25 | 济南大学 | Preparation method of label-free electrochemical immunosensor for nonyl phenol detection |
| CN105241939B (en) * | 2015-09-16 | 2017-08-01 | 山东理工大学 | A kind of preparation method and application based on gold and silver core-shell magnetic graphene Adsorption of Cadmium immunosensor |
| CN105738351B (en) * | 2016-02-25 | 2018-05-25 | 济南大学 | A kind of preparation method and application of the electrogenerated chemiluminescence Clenbuterol sensor based on magnetic two-dimensional nano composite material |
| CN105784993B (en) * | 2016-03-16 | 2017-05-31 | 济南大学 | A kind of preparation method and application of Ractopamine electrochemica biological immunosensor |
| CN105842438B (en) * | 2016-03-28 | 2017-12-29 | 南京邮电大学 | A kind of preparation method of Prussian blue cubic block/molybdenum disulfide nano-composite material |
| CN106442671B (en) * | 2016-09-12 | 2017-12-12 | 济南大学 | One kind is based on BiOBr/Ag2The preparation method of the unmarked insulin sensor of S composites |
| CN107132260B (en) * | 2017-05-16 | 2019-06-18 | 山东省农业科学院农业质量标准与检测技术研究所 | A kind of electrochemical sensor based on nano material detection Ractopamine |
| CN109254060B (en) * | 2018-11-05 | 2021-01-12 | 济南大学 | Clenbuterol electrochemical sensing electrode and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4246337A (en) * | 1978-05-01 | 1981-01-20 | Corning Glass Works | Photosensitive medium for optical information storage |
| CN101980018A (en) * | 2010-10-29 | 2011-02-23 | 上海交通大学 | Method for preparing immuno biosensor for measuring ractopamine (RAC) |
| CN102087283A (en) * | 2009-12-08 | 2011-06-08 | 华中科技大学 | Chitosan-nano-gold enzyme immunosensor for detecting mycobacterium tuberculosis and application thereof |
-
2013
- 2013-01-21 CN CN201310026155.8A patent/CN103063833B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4246337A (en) * | 1978-05-01 | 1981-01-20 | Corning Glass Works | Photosensitive medium for optical information storage |
| CN102087283A (en) * | 2009-12-08 | 2011-06-08 | 华中科技大学 | Chitosan-nano-gold enzyme immunosensor for detecting mycobacterium tuberculosis and application thereof |
| CN101980018A (en) * | 2010-10-29 | 2011-02-23 | 上海交通大学 | Method for preparing immuno biosensor for measuring ractopamine (RAC) |
Non-Patent Citations (2)
| Title |
|---|
| G.W.S. Van Osch et al..The use of a gold-containing membrane for ion-sensitive electrodes and their application in analyzing systems.《Z. Anal. Chem.》.1975,第273卷271-274. |
| The use of a gold-containing membrane for ion-sensitive electrodes and their application in analyzing systems;G.W.S. Van Osch et al.;《Z. Anal. Chem.》;19751231;第273卷;271-274 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103063833A (en) | 2013-04-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103063833B (en) | Preparation method and application of unmarked immunosensor for rapidly detecting clenbuterol | |
| CN102749373B (en) | Preparation method and application of environmental estrogen electrochemical immunosensor | |
| CN104502429B (en) | The preparation method and application of unmarked electrogenerated chemiluminescence clenbuterol hydrochloride immunosensor | |
| CN105137063B (en) | A kind of preparation method of the unmarked electrochemical immunosensor for clenbuterol hydrochloride detection | |
| Kalcher et al. | Electrochemical sensors and biosensors based on heterogeneous carbon materials | |
| Bagheri Hashkavayi et al. | Label‐free electrochemical aptasensor for determination of chloramphenicol based on gold nanocubes‐modified screen‐printed gold electrode | |
| Tüdös et al. | Rapid surface plasmon resonance-based inhibition assay of deoxynivalenol | |
| Duan et al. | Selection and application of ssDNA aptamers against clenbuterol hydrochloride based on ssDNA library immobilized SELEX | |
| CN102749442B (en) | Manufacture method of silver hybridization mesoporous ferroferric oxide antibiotic immunosensor and application thereof | |
| CN104502593B (en) | The preparation method of the unmarked immunosensor of a kind of galvanochemistry stomach neoplasms tumor markers | |
| Zhang et al. | Immunoaffinity chromatography purification and ultrahigh performance liquid chromatography tandem mass spectrometry determination of tetrodotoxin in marine organisms | |
| CN103424448A (en) | Method for detecting trace ochratoxin A (OTA) by adopting electrochemical aptamer sensor | |
| CN107132260B (en) | A kind of electrochemical sensor based on nano material detection Ractopamine | |
| CN104133070A (en) | Preparation method and use of environmental estrogen label-free immunosensor | |
| Vanoosthuyze et al. | Development of a fast and simple method for determination of β-agonists in urine by extraction on empore membranes and detection by a test strip immunoassay | |
| Kabiraz et al. | Highly sensitive detection of clenbuterol in urine sample by using surface plasmon resonance immunosensor | |
| Gu et al. | Detection of six β-agonists by three multiresidue immunosensors based on an anti-bovine serum albumin-ractopamine-clenbuterol-salbutamol antibody | |
| CN103163193B (en) | Preparation method of electrochemical sensor for detecting three clenbuterol hydrochloride and application | |
| CN102520030B (en) | Manufacturing method of label-free electrochemical immunosensor for detecting zearalanol | |
| CN111707721A (en) | Preparation method and application of 17 beta-estradiol electrochemiluminescence aptamer sensor | |
| Oguri et al. | Assay and biological relevance of endogenous histamine and its metabolites: application of microseparation techniques | |
| Attaran et al. | Determination of salbutamol in pharmaceutical and serum samples by adsorptive stripping voltammetry on a carbon paste electrode modified by iron titanate nanopowders | |
| CN101858918A (en) | Microgap array electrode-based electrochemical immunosensor and method for detecting ractopamine in animal-derived food thereof | |
| Li et al. | Quick detection technique for clenbuterol hydrochloride by using surface plasmon resonance biosensor | |
| Li et al. | Immunosensor for rapid detection of gibberellin acid in the rice grain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140226 Termination date: 20210121 |