CN103063833A - 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
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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
2S) the unmarked electrochemical immunosensor of the fast detecting clenbuterol hydrochloride of core-shell nano compound substance structure belongs to Nano-function thin films and biosensor technology field.
Background technology
The advantages such as electrochemical immunosensor is highly sensitive owing to it, specificity 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 are exactly the raising of the effectively fixing and performances such as sensitivity, reappearance and persistence of immune molecule.
Gold (Au) nano particle since have specific surface area large, be easy to various biomolecules (nucleic acid, protein and biomolecule etc.) in conjunction with, characteristics 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 sulfydryl is rich on its surface, and be easy to combine with biomolecule, but Ag
2It is inhomogeneous that the S nano particle prepares 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, therefore is very beneficial for preparing electrochemica biological sensor.
Clenbuterol hydrochloride is that a class is called beta-stimulants (medicine of β-agonist), rather than a certain specific medicine.This class medicine has the function that realizes promoting lean meat growth, the growth of inhibition fat meat, so be referred to as " clenbuterol hydrochloride ".In China, usually said clenbuterol hydrochloride refers to Clenbuterol (Clenbuterol), and ordinary consumer then is referred to as clenbuterol hydrochloride to this type of medicine.When they are used for its feeding to surpass therapeutic dose 5-10 consumption doubly, significant nutrition " redistribution effects " is namely arranged---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 once be used as growth accelerator, the feed addictive of the livestock and poultry such as ox, sheep, pig.
" clenbuterol hydrochloride " kind catalogue according to office of the food security council of State Council " " clenbuterol hydrochloride " focus efforts on special areas scheme " (the food peace is done (2011) No. 14) regulation is 16 kinds, comprising: 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); West 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 serious nervous symptoms may occur.Poisoning this serious phenomenon that takes place frequently for clenbuterol hydrochloride, Dec 27 calendar year 2001, on February 9th, 2002, April 9, the Ministry of Agriculture issues the documents respectively and forbids that food animal bans use of beta-stimulants as feed addictive (No. 176, No. 193 bulletins of the Ministry of Agriculture, No. 1519 regulations).
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 fit to detect thermally labile and strong polarity, specificity is good, selectivity is strong, it is higher to detect degree of accuracy, can with system's couplings such as mass spectrum (MS), the robotization of Realization analysis process, with the half authenticity method of HPLC method as the detection residual of kelengtelu, the lowest detectable limit scope is 1 ~ 15 ngg in China
-1Shortcoming 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 fast highly sensitive qualitative analysis of separating effect and mass spectrum is altogether organic chromatogram, can in the simultaneous situation of multiple residue, carry out qualitative and quantitative analysis to certain specific residue, compare with the HPLC method, this method detection sensitivity is higher, false positive rate is lower, and China is legal for detecting the authenticity method of Clenbuterol with GC-MS; Shortcoming is that instrument and equipment is complicated, operating process is loaded down with trivial details, and is very high to reviewer's operative skill requirement, and is unsuitable for scene and fast detecting.
3. euzymelinked immunosorbent assay (ELISA) (ELISA): advantage is by utilizing the efficient catalytic effect of the combination of immunology antigen and antibody specific and enzyme, and with the clenbuterol hydrochloride content in ocular estimate or the colorimetric determination sample, so this method selectivity is strong, specificity good; Shortcoming is that step is various, and it is larger to detect error.
In order to address the above problem, the present invention adopts the electrochemical immunosensor that makes up 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 become the preferred material of assembling electrochemical immunosensor because it has the characteristics such as large specific surface area, stable performance and good biocompatibility, electric conductivity.The present invention is by generating nanometer Ag at nanometer Au nuclear
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
2The S nano composite material is used it for the preparation electrochemical immunosensor, has further improved the electronics transmission efficiency of its electrode surface and the adsorbance of antibody, has significantly improved the sensitivity that detects.The present invention designed a kind of simple, fast, the good and highly sensitive electrochemical immunosensor of specificity and corresponding electrochemical immunoanalytical method.
Summary of the invention
One of order of the present invention is that the loaded down with trivial details technical ability that reaches the reviewer of instrument and equipment complexity, operating process of avoiding existing in the existing detection method requires the shortcomings such as high, the preparation method of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride is provided, characteristics highly sensitive, high specificity that prepared sensor has, and prepare simply, easy to operate, the rapid sensitive that can be used for clenbuterol hydrochloride detects;
Two of purpose of the present invention be to provide a kind of simple, special, sensitive, detect the detection method of clenbuterol hydrochloride in pork, beef and the mutton fast and efficiently.
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 is modified with the clenbuterol hydrochloride antibody-solutions again, drips at last to be coated with 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 the Au-Ag nano particle is centrifugal, again be dispersed in 0.1 molL
-1CTAB in, add 100 mL sulphur precursor solutions, behind concussion 1 ~ 3 min, leave standstill 1 ~ 2 h, after the centrifuging, remove supernatant, 50 ℃ of lower vacuum drying, make Au-Ag
2S core-shell nano compound substance.
The preparation method of the unmarked immunosensor of described a kind of fast detecting clenbuterol hydrochloride more specifically with preferred preparation method is:
(1) Au-Ag
2S core-shell nano composite manufacture:
1) at 250 mL, 1 mmolL
-1Add 60 mL, 10 mmolL in the chlorauric acid solution
-1Freezing point-boron hydracid sodium solution behind the stirring 1h, is diluted 10 times, makes nanometer Au seed;
2) at 150 mL, 1 mmolL
-1Drip 0.1 molL in the chlorauric acid solution
-1Ascorbic acid is to colourless, adds 5 in Au seed in the step 1) again, leaves standstill 2 h, makes the Au nano particle;
3) with step 2) in the Au nano particle that makes centrifugal after, again be 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
-1The NaOH aqueous solution, 1 min that vibrates leaves standstill 1 h, makes the Au-Ag nano particle of nucleocapsid structure;
4) after the Au-Ag nano particle that makes in the step 3) is centrifugal, again be dispersed in 300 mL, 0.1 molL
-1CTAB in, add 100 mL sulphur precursor solutions, behind concussion 1 ~ 3 min, leave standstill 1 ~ 2 h, after the centrifuging, remove supernatant, 50 ℃ of lower vacuum drying make 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 the ultrapure water preparation;
Described freezing point-boron hydracid sodium solution, preparation method are that the test tube that will pre-install boron hydracid sodium solution places ice cube to cool off 3-4 min, after the taking-up test tube prepares boron hydracid sodium solution, again test tube are put into ice cube and cool off a period of time;
Described sulphur precursor solution, the preparation method will be in the sodium sulfide solution adds the sulphur powder, places 80 ℃ of heating of reactor 2h all to dissolve to the sulphur powder.
(2) Au-Ag
2The preparation of S core-shell nano compound substance-chitosan solution: shitosan is joined stirring 6 h in the acetic acid that volume fraction is 1 %, make the chitosan solution of massfraction 0.3 ~ 0.7 %; Get 3 mg Au-Ag
2S core-shell nano compound substance is dispersed in the 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 the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 μ m successively, and the ethanol ultrasonic cleaning is rinsed well with ultrapure water again, makes the glass-carbon electrode surface be minute surface.
(4) electrode face finish: be coated with 6 μ L Au-Ag surperficial the dripping of glass-carbon electrode
2S core-shell nano compound substance-chitosan solution is put in 4 ℃ of refrigerators, dries film forming; Ultrapure water cleans, and drips to be coated with 6 μ L clenbuterol hydrochloride antibody-solutions again, is put in 4 ℃ of refrigerators, dries film forming; After ultrapure water cleans, be put in to preserve in 4 ℃ of refrigerators and use;
Described clenbuterol hydrochloride antibody-solutions, the preparation method is: the phosphate buffered solution constant volume with clenbuterol hydrochloride antibody stoste adding pH is 7.4 is put in to preserve in 4 ℃ of refrigerators and uses.
(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 unmarked immunosensor of described fast detecting clenbuterol hydrochloride, be put in to preserve in 4 ℃ of refrigerators and use;
Described bovine serum albumin solution, the preparation method is: the phosphate buffered solution constant volume with bovine serum albumin(BSA) stoste adding pH is 7.4 is put in to preserve in 4 ℃ of refrigerators and uses.
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: with 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 the step (1) dripped respectively be coated onto working electrode surface, be put in to preserve in 4 ℃ of refrigerators and use.
(3) working curve is drawn: as contrast electrode, platinum electrode is modified good working electrode with step (2) and is formed three-electrode system as auxiliary electrode, connects electrochemical workstation, at K with saturated calomel electrode
3[Fe (CN)
6] in the solution, (detect in SWV) Zai – 0.6 ~ 0.2 V voltage range, the response current of blank standard specimen is designated as with square wave voltammetry
I 0, the response current that contains the clenbuterol hydrochloride standard solution of variable concentrations is denoted as
I i, the difference that response current reduces is Δ
I=
I 0 -
I i , Δ
IMass concentration with the clenbuterol hydrochloride standard solution
CBetween linear, draw Δ
I–
CWorking curve.
(4) detection of clenbuterol hydrochloride: replace clenbuterol hydrochloride standard solution in the step (1) with testing sample, detect according to the method in step (2) and (3), the difference DELTA that reduces according to response current
IAnd working curve, obtain the content of clenbuterol hydrochloride in the 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 passes through 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 with 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 stronger 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 can further be applied in the Test paper with obvious blue, has larger development potentiality.
Description of drawings
Fig. 1 is preparation method's synoptic 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 the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 μ m successively, and the ethanol ultrasonic cleaning is rinsed well with ultrapure water again, makes the glass-carbon electrode surface be minute surface.
(2) Au-Ag
2S core-shell nano composite manufacture:
1) at 250 mL, 1 mmolL
-1Add 60 mL, 10 mmolL in the chlorauric acid solution
-1Freezing point-boron hydracid sodium solution behind the stirring 1h, is diluted 10 times, makes nanometer Au seed;
2) at 150 mL, 1 mmolL
-1Drip 0.1 molL in the chlorauric acid solution
-1Ascorbic acid is to colourless, adds 5 in Au seed in the step 1) again, leaves standstill 2 h, makes the Au nano particle;
3) with step 2) in the Au nano particle that makes centrifugal after, again be 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
-1The NaOH aqueous solution, 1 min that vibrates leaves standstill 1 h, makes the Au-Ag nano particle of nucleocapsid structure;
4) after the Au-Ag nano particle that makes in the step 3) is centrifugal, again be dispersed in 300 mL, 0.1 molL
-1CTAB in, add 100 mL sulphur precursor solutions, behind concussion 1 ~ 3 min, leave standstill 1 ~ 2 h, after the centrifuging, remove supernatant, 50 ℃ of lower vacuum drying make 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 the ultrapure water preparation;
Described freezing point-boron hydracid sodium solution, preparation method are that the test tube that will pre-install boron hydracid sodium solution places ice cube to cool off 3-4 min, after the taking-up test tube prepares boron hydracid sodium solution, again test tube are put into ice cube and cool off a period of time;
Described sulphur precursor solution, the preparation method will be in the sodium sulfide solution adds the sulphur powder, places 80 ℃ of heating of reactor 2h all to dissolve to the sulphur powder.
(3) Au-Ag
2The preparation of S core-shell nano compound substance-chitosan solution: shitosan is joined stirring 6 h in the acetic acid that volume fraction is 1 %, make the chitosan solution of massfraction 0.3 %; Get 3 mg Au-Ag
2S core-shell nano compound substance is dispersed in the 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: the phosphate buffered solution constant volume with clenbuterol hydrochloride antibody stoste adding pH is 7.4 is put in to preserve in 4 ℃ of refrigerators and uses.
(5) preparation of bovine serum albumin solution: the phosphate buffered solution constant volume with bovine serum albumin(BSA) stoste adding pH is 7.4 is put in to preserve in 4 ℃ of refrigerators and uses.
(6) electrode face finish: according to step shown in Figure 1, be coated with 6 μ L Au-Ag surperficial the dripping of glass-carbon electrode
2S core-shell nano compound substance-chitosan solution is put in 4 ℃ of refrigerators, dries film forming; Ultrapure water cleans, and drips to be coated with 6 μ L clenbuterol hydrochloride antibody-solutions again, is put in 4 ℃ of refrigerators, dries film forming; After ultrapure water cleans, be put in to preserve in 4 ℃ of refrigerators and use.
(7) the non-specific avtive spot in enclosed-electrode surface: according to step shown in Figure 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 unmarked immunosensor of described fast detecting clenbuterol hydrochloride, be put in to preserve in 4 ℃ of refrigerators 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
2S core-shell nano composite manufacture:
1) at 250 mL, 1 mmolL
-1Add 60 mL, 10 mmolL in the chlorauric acid solution
-1Freezing point-boron hydracid sodium solution behind the stirring 1h, is diluted 10 times, makes nanometer Au seed;
2) at 150 mL, 1 mmolL
-1Drip 0.1 molL in the chlorauric acid solution
-1Ascorbic acid is to colourless, adds 5 in Au seed in the step 1) again, leaves standstill 2 h, makes the Au nano particle;
3) with step 2) in the Au nano particle that makes centrifugal after, again be 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
-1The NaOH aqueous solution, 1 min that vibrates leaves standstill 1 h, makes the Au-Ag nano particle of nucleocapsid structure;
4) after the Au-Ag nano particle that makes in the step 3) is centrifugal, again be dispersed in 300 mL, 0.1 molL
-1CTAB in, add 100 mL sulphur precursor solutions, behind concussion 1 ~ 3 min, leave standstill 1 ~ 2 h, after the centrifuging, remove supernatant, 50 ℃ of lower vacuum drying make 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 stirring 6 h in the acetic acid that volume fraction is 1 %, make the chitosan solution of massfraction 0.5 %; Get 3 mg Au-Ag
2S core-shell nano compound substance is dispersed in the 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 3The preparation method of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride, as shown in Figure 1
(1) processing of glass-carbon electrode: with embodiment 1.
(2) Au-Ag
2S core-shell nano composite manufacture:
1) at 250 mL, 1 mmolL
-1Add 60 mL, 10 mmolL in the chlorauric acid solution
-1Freezing point-boron hydracid sodium solution behind the stirring 1h, is diluted 10 times, makes nanometer Au seed;
2) at 150 mL, 1 mmolL
-1Drip 0.1 molL in the chlorauric acid solution
-1Ascorbic acid is to colourless, adds 5 in Au seed in the step 1) again, leaves standstill 2 h, makes the Au nano particle;
3) with step 2) in the Au nano particle that makes centrifugal after, again be 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
-1The NaOH aqueous solution, 1 min that vibrates leaves standstill 1 h, makes the Au-Ag nano particle of nucleocapsid structure;
4) after the Au-Ag nano particle that makes in the step 3) is centrifugal, again be dispersed in 300 mL, 0.1 molL
-1CTAB in, add 100 mL sulphur precursor solutions, behind concussion 1 ~ 3 min, leave standstill 1 ~ 2 h, after the centrifuging, remove supernatant, 50 ℃ of lower vacuum drying make 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 stirring 6 h in the acetic acid that volume fraction is 1 %, make the chitosan solution of massfraction 0.7 %; Get 3 mg Au-Ag
2S core-shell nano compound substance is dispersed in the 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 is used for the detection of clenbuterol hydrochloride, and 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: with 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 the step (1) dripped respectively be coated onto working electrode surface, be put in to preserve in 4 ℃ of refrigerators and use.
(3) working curve is drawn: as contrast electrode, platinum electrode is modified good working electrode with step (2) and is formed three-electrode system as auxiliary electrode, connects electrochemical workstation, at K with saturated calomel electrode
3[Fe (CN)
6] in the solution, (detect in SWV) Zai – 0.6 ~ 0.2 V voltage range, the response current of blank standard specimen is designated as with square wave voltammetry
I 0, the response current that contains the clenbuterol hydrochloride standard solution of variable concentrations is denoted as
I i, the difference that response current reduces is Δ
I=
I 0 -
I i , Δ
IMass concentration with the clenbuterol hydrochloride standard solution
CBetween linear, draw Δ
I–
CWorking curve.
(4) detection of clenbuterol hydrochloride: replace clenbuterol hydrochloride standard solution in the step (1) with testing sample, detect according to the method in step (2) and (3), the difference DELTA that reduces according to response current
IAnd working curve, obtain the content of clenbuterol hydrochloride in the 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 sees Table 1.
The technical indicator that table 1 detects 16 kinds of clenbuterol hydrochlorides all is better than similar technological means detection technique index.
Embodiment 5The detection of clenbuterol hydrochloride in the pork sample
Accurately take by weighing the pork sample, adopt conventional method to carry out sample preparation, detect according to embodiment 4 described steps, testing result sees Table 2.
Table 2 testing result as can be known, result's relative standard deviation (RSD) is less than 3.5%, average recovery rate is 95.0 ~ 105%, shows that the present invention can be used for the detection of clenbuterol hydrochloride in pork and the pig urine samples, highly sensitive, the high specificity of method, the result accurately and reliably.
Embodiment 6The detection of clenbuterol hydrochloride in the beef sample
Accurately take by weighing beef sample, adopt conventional method to carry out sample preparation, detect according to embodiment 4 described steps, testing result sees Table 3.
Table 3 testing result as can be known, result's relative standard deviation (RSD) is less than 3.3 %, average recovery rate is 90.0 ~ 106%, shows that the present invention can be used for the detection of clenbuterol hydrochloride in the beef sample, highly sensitive, the high specificity of method, the result accurately and reliably.
Embodiment 7The detection of clenbuterol hydrochloride in the meat samples
Accurately take by weighing meat samples, adopt conventional method to carry out sample preparation, detect according to embodiment 4 described steps, testing result sees Table 4.
Table 4 testing result as can be known, result's relative standard deviation (RSD) is less than 3.5 %, average recovery rate is 93.0 ~ 105%, shows that the present invention can be used for the detection of clenbuterol hydrochloride in the meat samples, highly sensitive, the high specificity of method, the result accurately and reliably.
Claims (5)
1. the preparation method of 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 is modified with the clenbuterol hydrochloride antibody-solutions again, drips at last to be coated with bovine serum albumin solution the non-specific avtive spot in enclosed-electrode surface.
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, Au-Ag
2The mass ratio of S core-shell nano compound substance and shitosan 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 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 the Au-Ag nano particle is centrifugal, again be dispersed in 0.1 molL
-1CTAB in, add 100 mL sulphur precursor solutions, behind concussion 1 ~ 3 min, leave standstill 1 ~ 2 h, after the centrifuging, remove supernatant, 50 ℃ of lower vacuum drying, make Au-Ag
2S core-shell nano compound substance.
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 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: with 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 the step (1) dripped respectively be coated onto working electrode surface, preserve in 4 ℃ of refrigerators;
(3) working curve is drawn: as contrast electrode, platinum electrode is modified good working electrode with step (2) and is formed three-electrode system as auxiliary electrode, connects electrochemical workstation, at K with saturated calomel electrode
3[Fe (CN)
6] in the solution, (detect in SWV) Zai – 0.6 ~ 0.2 V voltage range, the response current of blank standard specimen is designated as with square wave voltammetry
I 0, the response current that contains the clenbuterol hydrochloride standard solution of variable concentrations is denoted as
I i, the difference that response current reduces is Δ
I=
I 0-
I i, Δ
IMass concentration with the clenbuterol hydrochloride standard solution
CBetween linear, draw Δ
I-
CWorking curve;
(4) detection of clenbuterol hydrochloride: replace clenbuterol hydrochloride standard solution in the step (1) with testing sample, detect according to the method in step (2) and (3), the difference DELTA that reduces according to response current
IAnd working curve, obtain the content of clenbuterol hydrochloride in the testing sample.
5. the preparation method of the unmarked immunosensor of a kind of fast detecting clenbuterol hydrochloride as claimed in claim 1, it is one of following to it is characterized in 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.
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