CN101980018B - Method for preparing immuno biosensor for measuring ractopamine (RAC) - Google Patents
Method for preparing immuno biosensor for measuring ractopamine (RAC) Download PDFInfo
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Abstract
The invention discloses a method for preparing an immuno biosensor for measuring ractopamine (RAC) in the technical field of chemical detection. Nanogold, a carbon nano tube having conjugated bonding coupled RAC-bovine serum albumin, and prussian blue are modified on a glassy carbon electrode sequentially to obtain the electrochemical immuno biosensor. By the method, the electrochemical immuno biosensor which has high sensitivity, high stability and can quickly test the ractopamine can be prepared.
Description
Technical field
What the present invention relates to is the apparatus and method in a kind of chemical detection technique field, and specifically a kind of employing tri compound nano material is applied to measure immunity biosensor of beta-stimulants hormone Ractopamine (RAC) in the solution and preparation method thereof.
Background technology
Ractopamine (ractopamine, RAC), belong to beta-stimulants, nutritional labeling in the animal body is shifted to muscle by fat, show the nutrition redistribution effects, and then the metabolism of regulation and control animal body, lipolysis strengthened, promote protein synthetic, significantly improve carcass lean meat percentage and the price of deed.But, after the mankind have eaten the residual animal foodstuff that Ractopamine arranged, intoxicating phenomenon in various degree can occur, people's health and lives safety in their abuse and the residual serious harm in animal food, and has had a strong impact on the export trade of China's animal products.So China and European Union and in the world most countries all expressly forbid that Ractopamine is used as feed addictive.Country has set up " detection method liquid chromatography one mass spectrum of Clenbuterol, Ractopamine, salbutamol, Terbutaline residual quantity/mass spectroscopy in the Imported and exported animals derived food, SN/T 1924-2007 " standard.But compare with the detection of Clenbuterol (clenbuterol hydrochloride), the detection of Ractopamine also shows deficiency, need demand urgently abundant and raising.Therefore, seek Ractopamine fast and accurately determination techniques be still people's urgent expectation, Research Significance is great.
Use at present maximum Ractopamine assay methods and mainly contain liquid phase chromatography, gas chromatography mass spectrometry method, high performance liquid chromatography and euzymelinked immunosorbent assay (ELISA) etc.But these analytical approachs need large-sized analytic instrument, and operating process is loaded down with trivial details, can not realize Site Detection.The present invention can promote the electronics transmission of bio-electrical activity molecule well in conjunction with characteristic electron and the surface micro-structure of carbon nano-tube uniqueness, and is easy to the large molecule of fixed biologically and can keeps its active characteristics; Has large, the bioaffinity advantages of higher of specific surface area with nanogold particle; And the electro catalytic activity of Prussian blue excellence, and the characteristics such as high stability, easy preparation, set up the Direct Electrochemistry biology sensor that Ractopamine is detected, obtain lower detection limit and the wider range of linearity.
Summary of the invention
The present invention is directed to the prior art above shortcomings, a kind of preparation method of the immunity biosensor for measuring Ractopamine is provided, can promote well the electronics transmission of bio-electrical activity molecule, and be easy to the large molecule of fixed biologically and can keep its active characteristics; Has large, the bioaffinity advantages of higher of specific surface area with nanogold particle; And the electro catalytic activity of Prussian blue excellence, and the characteristics such as high stability, easy preparation, set up the immune electrochemica biological sensor that Ractopamine is detected, have the characteristics of high selectivity, low detection limit and the wide range of linearity.
The present invention is achieved by the following technical solutions, the present invention obtains electrochemical immunosensor by with nm of gold, the carbon nano-tube with conjugation bonding coupling Ractopamine-bovine serum albumin(BSA) (BSA) and Prussian blue the modification successively on the glass-carbon electrode.
Described carbon nano-tube with conjugation bonding coupling Ractopamine-bovine serum albumin(BSA) (BSA) prepares in the following manner:
The first step, with the H of multi-walled carbon nano-tubes at 70 ℃
2SO
4: HNO
3Volume ratio is ultrasonic concussion 6 hours in 3: 1 the solution, obtains the functionalization multi-walled carbon nano-tubes, and to be dispersed in the pH value be in 7.4 the phosphate buffer solution and sonic oscillation disperses with the functionalization multi-walled carbon nano-tubes, obtains carbon nano tube dispersion liquid;
Second step, above-mentioned dispersion and 2ml are contained the 1-ethyl of 6mM-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution mix, then at room temperature stirred 20 minutes.Consequent potpourri centrifugal 15 minutes at 13000r/m abandons supernatant, repeats the second step several times to eliminate excessive EDC;
The 3rd step, Ractopamine antigen-BSA of 0.1mg/mL mixed with volume ratio with mentioned solution at 1: 1 and stir after, placed under the 13000r/m centrifugal 15 minutes and shift supernatant, obtain the Ractopamine antigen of conjugation-BSA-multi-walled carbon nano-tubes bond;
The 4th step, the bond that the 3rd step was obtained place the immersion of 1mL5%BSA solution after 30 minutes, use PBS buffer solution centrifugal more than 3 times to eliminate free Ractopamine antigen and BSA, and will obtain the again dissolving in the PBS of 1mL buffer solution of Ractopamine antigen-BSA-multi-walled carbon nano-tubes, stir and form homodisperse carbon nano-tube with conjugation bonding coupling Ractopamine-bovine serum albumin(BSA) (BSA).
Described glass-carbon electrode refers to: diameter is 3mm and respectively through the Al of 1.0 and 0.3 μ m
2O
3Behind the powder emulsion sanding and polishing, the glass-carbon electrode of each ultrasonic 3min in the second alcohol and water;
The described modification successively refers to: the HAuCl that glass-carbon electrode elder generation deposited gold nano particle is placed on 100mg/L
4In the solution under-0.2V electromotive force potentiostatic scanning 60s; Then drip at electrode surface and be coated with 4 μ L Ractopamine antigen-BSA-multi-walled carbon nano-tubes bonds and dry 5h at room temperature; Again electrode is inserted and contain 2.5mM FeCl
3, 2.5mM K
3[Fe (CN)
6], in the Prussian blue solution of 0.1M KCl and 0.1M HCl under the 0.4V electromotive force potentiostatic scanning 40s, again electrode is inserted in the solution contain 0.1M KCl and 0.1M HCl in-0.2 to 1.1V potential range, with the speed of sweeping of 100mv/s, cyclic voltammetry scan 10 circles; Place at last and be immersed in 5% BSA solution 30min under 37 ℃.
The present invention realizes the test of Ractopamine in the following manner: will modify good electrode immersion cumulative volume is the free Ractopamine that contains variable concentrations of 50 μ L, in the phosphate buffer solution of the Ractopamine polyclonal antibody of 8 μ g/mL, under 37 ℃, hatch 40min, rear in the K of 2mM with the phosphate buffer solution flushing
3[Fe (CN)
6] carry out differential pulse voltammetry (DPV) scanning in the solution.
Experimental result shows that along with the increase of Ractopamine concentration in Incubating Solution, the DPV peak current increases.Being defined in the modified electrode DPV peak current of hatching in the Incubating Solution that only contains Anti-ractopamine antibody is I
0, the modified electrode DPV peak current after hatching is I
X, and calculate Δ I=I
X-I
0, mapping can obtain linearity curve to Ractopamine concentration (C) with Δ I.Ractopamine concentration is directly proportional with Δ I in the 1-1000ng/mL scope, and slope is 0.00301, and linearly dependent coefficient is 0.9954.
The present invention has following beneficial effect: because carbon nano-tube and nanogold particle can promote the electronics transmission of bio-electrical activity molecule well, and be easy to the large molecule of fixed biologically and can keep its activity, Ractopamine is fixed on carbon nano-tube-poly-thionine-In Glassy Carbon Electrode Modified With Nano-gold surface effectively, and keeps greater activity.Therefore, the immune-electrochemistry sensor of fixedly Anti-ractopamine antibody of the present invention can at normal temperatures, hand over hand detect the concentration of residual Ractopamine.
Description of drawings
Fig. 1 is the competition mechanism principle of work synoptic diagram of immunosensor;
Among the figure: (a) reaction of antibody in free Ractopamine and the Ractopamine competition and the solution that are fixed on the electrode in the solution, (b) electrode clean is placed on the K of 2mM
3[Fe (CN)
6] in the solution, (c) Electrochemical Detection.
Fig. 2 is the cyclic voltammogram under the different condition.
Among the figure: (a) naked glass-carbon electrode, (b) MWCNT-Ractopamine antigen-BSA modified electrode, (c) MWCNT-Ractopamine antigen-BSA/ golden nanometer particle compound modified electrode, (d) Prussian blue/MWCNT-Ractopamine antigen-BSA/ golden nanometer particle compound modified electrode, (e) after Prussian blue/MWCNT-Ractopamine antigen-BSA/ golden nanometer particle compound modified electrode is hatched 40min in the solution of the Anti-ractopamine antibody that contains 8 μ g/mL, at the K of 2mM
3[Fe (CN)
6] solution in cyclic voltammogram, sweep speed for 50mv/s.
Fig. 3 is the electron scanning micrograph of nano particle.
Among the figure: (a) golden nanometer particle, (b) MWCNT-golden nanometer particle compound, (c) electron scanning micrograph of Prussian blue-MWCNT-golden nanometer particle compound.
Fig. 4 is modified electrode at the Anti-ractopamine antibody that contains 8 μ g/mL and (a) 0ng/mL, (b) 1ng/mL, (c) 5ng/mL, (d) 25ng/mL, (e) 100ng/mL, (f) 500ng/mL, (g) hatch in the PBS solution of the free Ractopamine of 1000ng/mL after, in the K of 2mM
3[Fe (CN)
6] solution in the differential pulse voltammetry curve, pulse-response amplitude 50mV, pulse width 50ms.Inserting figure is that free Ractopamine concentration is to the working curve of Δ I.
Embodiment
The below elaborates to embodiments of the invention, and present embodiment is implemented under take technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
Step 1, the structure of immunosensor: first that multi-walled carbon nano-tubes is carboxylated, again with coupling the Ractopamine antigen of BSA be fixed on the carbon nano-tube of functionalization with the form of conjugation bonding, make carbon nano-tube-Ractopamine antigen-bsa conjugate thing.The glass-carbon electrode polishing grinding, clean after, successively depositing nano gold, carbon nano-tube-Ractopamine antigen-bsa conjugate thing, Prussian blue are immersed in 30min in 5% the BSA solution with modifying good electrode under 37 ℃.
Step 2, immunosensor is to the detection of Ractopamine: will modifying good electrode, to immerse cumulative volume be the free Ractopamine that contains variable concentrations of 50 μ L, in the phosphate buffer solution of the Ractopamine polyclonal antibody of 8 μ g/mL, under 37 ℃, hatch 40min, rear in the K of 2mM with the phosphate buffer solution flushing
3[Fe (CN)
6] carry out differential pulse voltammetry (DPV) scanning in the solution.Experimental result shows that along with the increase of Ractopamine concentration in Incubating Solution, the DPV peak current increases.
Such as Fig. 1, detect the schematic diagram of Ractopamine for immunosensor, when modified electrode immerses in the Incubating Solution that contains Anti-ractopamine antibody, Anti-ractopamine antibody reaction in the Incubating Solution in free Ractopamine and the Ractopamine competition and the solution that are fixed on the electrode, antibody is attached on the electrode and causes with K
3[Fe (CN)
6] be the variation of the electrochemical signals of probe, thus realize the detection to Ractopamine in the solution.
1 cyclic voltammetric result:
Such as Fig. 2, bare electrode (2a) is at the K3[Fe of 2mM (CN) 6] cyclic voltammetry curve in the solution shows a pair of obvious Fe (CN)
6 3-/4-The redox peak, after having modified MWCNT-Ractopamine antigen-BSA on the electrode (2b), redox peak electric potential difference broadens, and peak current descends to some extent simultaneously, may be because the antigen-BSA of electrode surface has hindered the electronics transmission; After having modified MWCNT-Ractopamine antigen-BSA/ golden nanometer particle compound on the electrode (2c), peak current increases, and illustrates that golden nanometer particle has the function that strengthens the electronics transmission; Modified on the electrode (2d) behind Prussian blue/MWCNT-Ractopamine antigen-BSA/ golden nanometer particle compound, peak current is larger, illustrates that Prussian blue electro-catalysis effect is better.
2 scanning electron microscope results
Such as Fig. 3, Fig. 3 a shows that golden nanometer particle is deposited on electrode surface, and Fig. 3 b shows that golden nanometer particle has been attached on the multi-walled carbon nano-tubes, and Fig. 3 c shows Prussian blue in electrode surface deposition film forming.
The drafting of 3 working curves
Such as Fig. 4, under the concentration of different Ractopamines, sensor immerses and contains K
3[Fe (CN)
6] the buffer solution of PBS get DPV figure.As expected, along with the increase of Ractopamine concentration in Incubating Solution, the DPV peak current increases.That is to say that free Ractopamine concentration is higher, the antibody that is fixed on the Ractopamine molecule combination on the electrode is fewer.Being defined in the modified electrode DPV peak current of hatching in the Incubating Solution that only contains Anti-ractopamine antibody is I
0, the modified electrode DPV peak current after hatching is I
X, and calculate Δ I=I
X-I
0, mapping can obtain linearity curve to Ractopamine concentration (C) with Δ I.Ractopamine concentration is directly proportional with Δ I in the 1-1000ng/mL scope, and slope is 0.00301, and linearly dependent coefficient is 0.9954.
Embodiment 2
The mensuration of mark-on Ractopamine in the animal feed actual sample
Step 1, the processing of animal feed sample: it is levigate to get respectively 1g pig feed sample, accurate weighing is in the sample hose of 10mL, the blank sample that does not contain Ractopamine with the standard addition method preparation, with 3 mark-on samples that contain different Ractopamine concentration, add the fresh phosphoric acid-methanol extract liquid (0.2M) of 8mL, potpourri sonic oscillation 30min, centrifugal 10min under 3000r/m, supernatant is transferred in the volumetric flask that volume is 25mL, residue repeats to extract 3 times with the identical extract of 8mL, 5mL and 4mL, and supernatant is incorporated in the volumetric flask.The supernatant of getting 1mL is transferred in the sample hose of 5mL at nitrogen and is blown under the condition concentration and evaporation under 55 ℃ of temperature, and the pH that concentrate adds 1mL is used for electrochemical analysis after the dissolving of 7.4 phosphate buffer solutions.
Step 2, the structure of immunosensor: first that multi-walled carbon nano-tubes is carboxylated, again with coupling the Ractopamine antigen of BSA be fixed on the carbon nano-tube of functionalization with the form of conjugation bonding, make carbon nano-tube-Ractopamine antigen-bsa conjugate thing.The glass-carbon electrode polishing grinding, clean after, successively depositing nano gold, carbon nano-tube-Ractopamine antigen-bsa conjugate thing, Prussian blue are immersed in 30min in 5% the BSA solution with modifying good electrode under 37 ℃.
Table 1 detects the recovery and the error result of the Ractopamine concentration in the mark-on feed for immunosensor.
The mensuration of mark-on Ractopamine in the pork actual sample
Step 1, the processing of pork actual sample: get respectively 2g pork sample and smash, accurate weighing is in the sample hose of 10mL, the blank sample that does not contain Ractopamine with the standard addition method preparation, with 3 mark-on samples that contain different Ractopamine concentration, add 7mL ethyl acetate, potpourri sonic oscillation 30min, centrifugal 15min under 4000r/m, supernatant is transferred in the volumetric flask that volume is 25mL, residue repeats to extract 3 times with the ethyl acetate of 6mL respectively, and supernatant is incorporated in the volumetric flask.The supernatant of getting 1mL is transferred in the sample hose of 5mL at nitrogen and is blown under the condition concentration and evaporation under 55 ℃ of temperature, and the pH that concentrate adds 1mL is used for electrochemical analysis after the dissolving of 7.4 phosphate buffer solutions.
Step 2, the structure of immunosensor: first that multi-walled carbon nano-tubes is carboxylated, again with coupling the Ractopamine antigen of BSA be fixed on the carbon nano-tube of functionalization with the form of conjugation bonding, make carbon nano-tube-Ractopamine antigen-bsa conjugate thing.The glass-carbon electrode polishing grinding, clean after, successively depositing nano gold, carbon nano-tube-Ractopamine antigen-bsa conjugate thing, Prussian blue are immersed in 30min in 5% the BSA solution with modifying good electrode under 37 ℃.
Table 2 detects the recovery and the error result of the Ractopamine concentration in the mark-on pork for immunosensor.
Claims (3)
1. preparation method who be used for to measure the immunity biosensor of Ractopamine, it is characterized in that, by with nm of gold, the carbon nano-tube with conjugation bonding coupling Ractopamine-bovine serum albumin(BSA) (BSA) and Prussian blue the modification successively on the glass-carbon electrode, obtain electrochemical immunosensor; The described modification successively refers to: the HAuCl that glass-carbon electrode elder generation deposited gold nano particle is placed on 100mg/L
4In the solution under-0.2V electromotive force potentiostatic scanning 60s; Then drip at electrode surface and be coated with 4 μ L Ractopamine antigen-BSA-multi-walled carbon nano-tubes bonds and dry 5h at room temperature; Again electrode is inserted and contain 2.5 mM FeCl
3, 2.5 mM K
3[Fe (CN)
6], in the Prussian blue solution of 0.1 M KCl and 0.1 M HCl under the 0.4V electromotive force potentiostatic scanning 40s, again electrode is inserted in the solution contain 0.1 M KCl and 0.1 M HCl in-0.2 to 1.1V potential range, with the speed of sweeping of 100mv/s, cyclic voltammetry scan 10 circles; Place at last and be immersed in 5% BSA solution 30min under 37 ℃.
2. the preparation method of the immunity biosensor for measuring Ractopamine according to claim 1 is characterized in that described carbon nano-tube with conjugation bonding coupling Ractopamine-bovine serum albumin(BSA) (BSA) prepares in the following manner:
The first step, with the H of multi-walled carbon nano-tubes at 70 ℃
2SO
4: HNO
3Volume ratio is ultrasonic concussion 6 hours in the solution of 3:1, obtains the functionalization multi-walled carbon nano-tubes, and to be dispersed in the pH value be in 7.4 the phosphate buffer solution and sonic oscillation disperses with the functionalization multi-walled carbon nano-tubes, obtains carbon nano tube dispersion liquid;
Second step, above-mentioned dispersion liquid and 2ml are contained the 1-ethyl of 6mM-(3-dimethylaminopropyl) carbodiimide hydrochloride solution mix, then at room temperature stirred 20 minutes.Consequent potpourri centrifugal 15 minutes at 13000 r/m abandons supernatant, repeats the second step several times to eliminate excessive 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride;
The 3rd step, Ractopamine antigen-BSA of 0.1mg/mL mixed with volume ratio 1:1 with mentioned solution and stir after, placed under 13000 r/m centrifugal 15 minutes and shift supernatant, obtain the Ractopamine antigen of conjugation-BSA-multi-walled carbon nano-tubes bond;
The 4th step, the bond that the 3rd step was obtained place the immersion of 1mL5%BSA solution after 30 minutes, use PBS buffer solution centrifugal more than 3 times to eliminate free Ractopamine antigen and BSA, and will obtain the again dissolving in the PBS of 1mL buffer solution of Ractopamine antigen-BSA-multi-walled carbon nano-tubes, stir and form homodisperse carbon nano-tube with conjugation bonding coupling Ractopamine-bovine serum albumin(BSA).
3. the preparation method of the immunity biosensor for measuring Ractopamine according to claim 1 is characterized in that described glass-carbon electrode refers to: diameter is 3mm and respectively through the Al of 1.0 and 0.3 μ m
2O
3Behind the powder emulsion sanding and polishing, the glass-carbon electrode of each ultrasonic 3min in the second alcohol and water.
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