CN102262115A - Electrochemical immunosensor for melamine content determination, and preparation method and application thereof - Google Patents
Electrochemical immunosensor for melamine content determination, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a label-free electrochemical immunosensor for melamine content determination, and a preparation method and application thereof. The immunosensor comprises a substrate electrode, wherein the surface of the substrate electrode is modified by a graphene-melamine-chitosan composite, and nonspecific active sites are sealed by bovine serum proteins. The method for preparing the electrochemical immunosensor is implemented by fixing melamine antigens on the surface of a glass carbon electrode with graphene/chitosan composite materials. Based on an immunoreactive competition mode, the electrochemical immunosensor monitors immune reaction through cyclic voltammetry and differential pulse voltammetry by taking K3Fe(CN)6 as a probe,, and can be used for melamine content detection. The immunosensor disclosed by the invention is high in sensitivity and specificity, simple in detection method and wide in application range; the limit of detection can reach 0.2 ng/ml, and the linear range is 5-1500 ng/ml; and the immunosensor has the characteristics of high speed, high efficiency, high sensitivity, good specificity, simple operation, low cost and the like.
Description
Technical field
The invention belongs to food safety detection and technical field of analytical chemistry, relate to a kind of melamine detection apparatus and method, specifically a kind of electrochemical immunosensor that is applied to measure violated additive melamine in food and the feed and preparation method thereof, and the method for utilizing this sensor detection by quantitative content of melamine.
Background technology
Melamine (Melamine) is a kind of important chemical material, is usually used in making the applied fireproofing melamine resin, also can be used as water reducer and formaldehyde detersive etc., in part Asian countries, also is used to make chemical fertilizer.Because the protein content detection method " Kjeldahl " that milk and feed industry are commonly used, be to estimate protein content by measuring nitrogen content, requiring the average nitrogen content of protein is about 16 %, and the nitrogen content of melamine is about 66 %, therefore melamine often is used as adjuvant by illegal businessman, to promote the protein content index in milk and the feed, make milk inferior and feed link cross the detection of inspection body.
Studies show that melamine can bring out animal kidney failure and cause death.Melamine metabolism in vivo belongs to torpescence metabolism or inertia metabolism, and promptly the metabolic alterations of any kind can not take place rapidly in vivo for it.Nonruminant is discharged melamine with substance form or homolog form, rather than the form of metabolic product.The melamine poison milk powder case that took place in 2008 makes the analytical chemistry field start the upsurge of research melamine detection method, begins both at home and abroad melamine and detection generation thereof are shown great attention to.
The traditional detection method of melamine has gravimetric method, sublimed method etc., but is only limited to the detection to the melamine raw material.The method that grew up in recent years has high performance liquid chromatography, liquid chromatography mass coupling method, combined gas chromatography mass spectrometry, enzyme linked immunosorbent assay etc.But these analytical approachs need large-sized analytic instrument, and operating process is loaded down with trivial details, and pre-treatment process complexity can not realize on-the-spot the detection.
Also comprise electrochemical detection method in the method for melamine detection, disclose a kind of modified electrode that is used for the fast detecting melamine, comprising: basal electrode as CN201020299766.1; Be arranged at the PDDA film of basal electrode outside surface; And the FDU film that is arranged at the PDDA film outer surface.CN201010235974.X discloses a kind of preparation method of carbon nano tube modified electrode that melamine detects that is used for, carbon nano-tube is dispersed in DMF, tetrahydrofuran, methyl alcohol, ethanol, the water equal solvent, again bonding agents such as this nanotube solution and shitosan, Nafion, polyvinyl alcohol (PVA) are mixed, after being uniformly dispersed it is coated in electrode surface and makes carbon nano tube modified electrode.This electrode can detect melamine, and lowest detection is limited to 10-13mol/L.CN200810234859.3 has disclosed the electrochemical fast detecting method of melamine in a kind of dairy produce, it is characterized in that adopting electrochemical process, with material containing carbon as working electrode, dairy produce is dissolved in the acidic electrolyte solution, under the current potential of calibration, did the cyclic voltammetry scan electrolysis 1～10 minute, according to prepared volt-ampere curve, carry out preliminary qualitative analysis and quantitative test to whether containing melamine in the dairy produce.Described detection method is being still waiting further raising aspect sensitivity and the specificity.
Since the eighties in last century, Study on Biosensor presents the situation of advancing by leaps and bounds with exploitation, and various kinds of sensors is arisen at the historic moment.Wherein, relevant with measuring antigen-antibody reaction scales is an immunosensor.Antigen-antibody can cause the change of multiple signal in conjunction with front and back, as at aspects such as weight, optics, calorifics, galvanochemistry.Immunosensor is used widely detecting on the melamine, as various based on antibody with antigen reactively be used for the immunoreagent (box) that melamine detects.
Since following characteristics and advantage that electrochemical analysis has, as can be implemented in the health check-up survey, be not subjected to the influence (be that sample can be not treated, need do not separate) of color sample, turbidity, and required instrument and equipment is simple relatively, so application prospect is good.
The present invention is intended to the preparation of the Direct Electrochemistry biological immune sensing device that detects by a kind of melamine, sets up that a kind of detection limit is low, the range of linearity is wide, sensitivity and specificity height, the method for on-site measurement content of melamine fast.
Summary of the invention
The object of the present invention is to provide a kind of electrochemical immunosensor and preparation method thereof, described sensor has very high sensitivity and selectivity, can be used for melamine and detects.
Another object of the present invention also is to provide a kind of detection method of simply, fast, efficiently measuring the additive melamine residual amount of violating a ban in the food such as milk.
The technical scheme that realizes the object of the invention employing is as follows:
A kind of electrochemical immunosensor that detects melamine comprises basal electrode, it is characterized in that described basal electrode finishing Graphene-melamine-chitosan complexes, and seals non-specific avtive spot with bovine serum albumin.
The preferred glass-carbon electrode of described basal electrode.
Another object of the present invention provides a kind of preparation method of described immunosensor, and technical scheme is as follows:
A kind of preparation method who detects the electrochemical immunosensor of melamine, it is characterized in that: the ultrasonic dispersion of the shitosan suspending liquid of Graphene back is mixed with melamine, to mix drop and be coated in electrode surface, utilize shitosan with Graphene and melamine bag by and be fixed in electrode surface, and electrode is immersed in bovine serum albumin (BSA) solution nonspecific avtive spot on enclosed-electrode surface.
The preferred glass-carbon electrode of described basal electrode.
The preparation of described immunosensor more specifically comprises the steps: with preferred method
1) preparation of Graphene-shitosan suspending liquid: the HCl of 0.05M is heated to 80～90 ℃, adds load weighted shitosan, stirring and dissolving, it is the NaOH of 0.1M that cooling back adds concentration, regulates pH to 5, join the chitosan solution of 1mg/mL;
Accurately take by weighing 5g Graphene sample in the 2mL chitosan solution, ultrasonic dispersion 1h makes Graphene-shitosan suspending liquid;
2) glass-carbon electrode is handled: diameter is the glass-carbon electrode Al that uses 1.0 and 0.3 μ m respectively of 3mm
2O
3Behind the powder emulsion sanding and polishing, each ultrasonic cleaning 3min in the second alcohol and water respectively;
3) electrode face finish: with 4 μ L Graphene-shitosan suspending liquid and 2 μ L concentration is the melamine aqueous solution of 0.2mg/mL, drips to be applied to step 2) the glass-carbon electrode surface that makes, 40 ℃ of oven dry 30min down;
4) nonspecific avtive spot sealing: the electrode after the finishing is immersed in 30min in 5% the BSA solution under 37 ℃, to seal remaining avtive spot, make described electrochemical immunosensor.
The preparation of described Graphene, can graphite oxide (GO) as presoma, the reduction by graphite oxide and the functionalization of Graphene obtain the Graphene of excellent performance.
Above-mentioned immunosensor can be used for measuring the content of melamine.When electrode is hatched in the solution of the diethylstilbestrol that contains melamine antibody and dissociate, in the solution in free melamine and the melamine that is fixed on electrode surface and the solution melamine antibody generation competitive immunization react, after the fixing melamine of melamine antibody and electrode surface reacted, antibody was adsorbed on electrode surface.Free melamine concentration is high more, and the antibody that is fixed on the melamine combination on the electrode is few more.With K
3Fe (CN)
6Be probe, carry out differential pulse voltammetry (DPV) scanning, antibody can make Fe (CN) in the absorption of electrode surface
6 3-/4-The redox peak point current reduces.Therefore utilize the competitive immunization reaction mechanism, the electrode after the melamine of variable concentrations is hatched is at K
3Fe (CN)
6The curve peak current difference that differential pulse voltammetry in the solution (DPV) obtains.Experimental result shows that along with the increase of melamine concentration in Incubating Solution, the DPV peak current increases, and peak current increase value is linear with melamine concentration, thereby realization is to the detection by quantitative of melamine.
Therefore, the invention still further relates to a kind of method, may further comprise the steps based on described immunosensor detection melamine:
1) standard solution preparation: preparing one group of pH that contains the free melamine of variable concentrations that comprises blank standard specimen and be 7.4 phosphate buffer solution is standard solution, wherein contains the melamine antibody of same concentrations;
2) set up working curve: immunosensor is immersed in the standard solution respectively hatch, hatch the back and wash immunosensor, at K with phosphate buffer solution
3[Fe (CN)
6] carry out differential pulse voltammetry (DPV) scanning, recording responses electric current in the solution; The response current of blank standard specimen is I
0, the response current that contains the standard specimen of free melamine is I
x, the increase Δ I(Δ I=I of response current
x-I
0) be directly proportional with melamine concentration C in the standard solution, draw Δ I-C typical curve, or adopt linear regression method to obtain Δ I-C equation of linear regression;
3) melamine is measured: testing sample is formulated as the phosphate buffer solution that contains with step 1) same concentrations melamine antibody, according to step 2) identical method hatches and differential pulse voltammetry scanning the recording responses electric current to immunosensor; Increase Δ I and typical curve according to response current obtain content of melamine.
Described K
3[Fe (CN)
6] concentration of solution is preferably 2 mM.
The immune response condition optimization is hatched 60 min under 37 ℃.
The preferred 4 μ g/mL of the concentration of described melamine antibody.
Described step 3) preferably adopts standard addition method to measure.
Said method detects content of melamine, and lowest detection is limited to 0.2 ng/ml, the range of linearity 5 ~ 1500 ng/ml.
Immunosensor of the present invention and content of melamine detection method thereof are based on following principle.
Because Graphene has excellent electric property, one of its important use is can be used for preparing high performance nano composite material, but Graphene is insoluble in water and organic solvent commonly used.There are some researches show that when Graphene disperses in shitosan, effectively load transfer will take place between the two, interaction between the two makes Graphene be the molecular level dispersion in shitosan, has good dispersion effect.The present invention with the ultrasonic dispersion of the chitosan solution of Graphene after, mix with melamine and drip and be coated on the electrode, the viscosity of utilizing shitosan with Graphene and melamine bag by and be fixed on the electrode, and electrode is immersed in the BSA solution, to seal nonspecific avtive spot.The electrode face finish of immunosensor is seen Fig. 1.
Simultaneously, the present invention is based on the competitive mode of specific reaction between antibody and the antigen to melamine detection.(a) when electrode immerses in the solution contain melamine antibody and free melamine, be fixed on the melamine and the melamine antibody generation competitive reaction that dissociate in melamine antigen and the solution on the electrode.(b) based on the competitive mode of specific reaction between melamine antibody and the antigen, when electrode was immersed in the solution that contains specific antibodies, the idiosyncrasy of melamine antibody and antigen was adsorbed on the electrode antibody.(c) antibody-antigenic compound causes electrode to produce steric hindrance in the formation of electrode surface, has reduced the electrode zone of action, has hindered probe ion and has arrived electrode, and peak point current descends.Therefore, can melamine free in the solution be detected according to the difference of each stage electrochemical signals of immunosensor.The principle of immunosensor detection melamine of the present invention as shown in Figure 2.
The present invention by Graphene/shitosan composite nano materials fixedly melamine antigen on basal electrode, prepare a kind of non-marked electrochemical immunosensor of melamine, utilize K
3Fe (CN)
6Be probe,, realize that melamine is carried out qualitative and quantitative to be detected with cyclic voltammetry and differential pulse voltammetry monitoring immune response.Sensor of the present invention has the sensitivity and the selectivity of height, detection method is simple, efficient, the scope of application is extensive, detectability can reach 0.2 ng/ml, the range of linearity 5 ~ 1500 ng/ml are a kind of detection methods of simply, fast, efficiently measuring the additive melamine residual amount of violating a ban in the food such as milk.
Description of drawings
Fig. 1 immunosensor electrode face finish of the present invention synoptic diagram.
Fig. 2 immunosensor of the present invention detects the principle of melamine, (a) will modify good electrode and immerse in the solution that contains dissociate melamine and melamine antibody and hatch, (b) with [Fe (CN)
6]
3-/4-For probe carries out Electrochemical Detection, (c) by monitoring the change-detection melamine concentration of electric signal.
The SEM electromicroscopic photograph of Fig. 3 Graphene.
Fig. 4 different modifying electrode (a) bare electrode, (b) melamine/chitosan-modified electrode, (c) Graphene/melamine/chitosan-modified electrode is after (d) Graphene/melamine/chitosan-modified electrode is hatched in containing the antibody of melamine, at the K of 2mM
3[Fe (CN)
6] PBS solution in the CV curve map, sweep speed for 50mv/s.
The incubation time of Fig. 5 modified electrode in Incubating Solution is to the influence of DPV peak current.
Antibody content is to the influence of DPV peak current in Fig. 6 Incubating Solution.
Fig. 7 (a) Graphene/melamine/chitosan-modified electrode, and at free melamine (b) 1500ng/mL of melamine antibody that contains same concentrations and variable concentrations, (c) 1000ng/mL, (d) 500ng/mL, (e) 100ng/mL, (f) 20ng/mL, (g) 5ng/mL, (h) hatch in the Incubating Solution of 0ng/mL after, at the K of 2mM
3[Fe (CN)
6] PBS solution in the DPV curve map.
Fig. 8 is the changes delta I of response current and the canonical plotting of melamine concentration.
Embodiment
Describe the present invention below in conjunction with specific embodiment, described embodiment helps the understanding of the present invention and enforcement, is not to be construed as limiting the invention.Protection scope of the present invention is not exceeded with embodiment, and is limited by claim.
Using the electrochemical workstation (CHI660D) of Shanghai occasion China company in following examples, is working electrode with the modified glassy carbon, and saturated calomel electrode is a contrast electrode, and platinum electrode is that auxiliary electrode carries out electrochemical measurement.Employed melamine antibody is the melamine monoclonal antibody that Beijing Bo Aosen Bioisystech Co., Ltd produces.
The preparation of embodiment 1 immunosensor
A kind of electrochemical immunosensor that detects melamine is to get at glass-carbon electrode finishing Graphene-melamine-chitosan complexes.
Its preparation method is as follows:
1) preparation of Graphene mainly with graphite oxide (GO) as presoma, the reduction by graphite oxide and the functionalization of Graphene obtain the Graphene of excellent performance.
The preparation of graphite oxide: the dense H of 50 mL
2SO
4+ 10 g K
2S
2O
8+ 10 gP
2O
5Mix, 12 g dags are joined in the above-mentioned mixed liquor, react 6 h.Add 2 L water, spend the night.The dense H of 460 mL
2SO
4(the freezing processing of refrigerator) joins dense H with the dag of oxidation
2SO
4The middle stirring.Temperature is controlled at below 10 ℃, slowly adds 60 g KMnO
4, potpourri slowly adds 920 mL deionized waters at 35 ℃ of reaction 2 h, keeps below 50 ℃ stirring reaction 2 h.Add 2.8 L water and 50 mL, 30 % H
2O
2, stirring reaction one day.Use the HCl flushing of 5 L 10%, centrifugal.And then, be neutral to solution with 5 L washing.
The preparation of Graphene: take by weighing above-mentioned graphite oxide 0.05 g, join in the NaOH solution of 100 mL pH=11; Ultrasonic 90 min prepare the graphene oxide dispersion liquid under 150 W; Centrifugal 3 min remove the unstripped graphite oxide of minute quantity under 4000 rpm; Add 0.1 mL hydrazine hydrate in the graphene oxide dispersion liquid after centrifugal,, obtain the Graphene dispersion liquid at 90 ℃ of stirring reaction 2 h.
Be illustrated in figure 3 as the stereoscan photograph of Graphene, Graphene is very thin sheet as can be seen from Figure.
2) preparation of Graphene-shitosan suspending liquid: the HCl of 0.05 M is heated to 80～90 ℃, adds load weighted shitosan, stirring and dissolving, it is the NaOH of 0.1M that the cooling back adds concentration, regulate pH to 5, join the chitosan solution of 1 mg/mL, place 4 ℃ of refrigerators standby.
Accurately take by weighing 5 mg Graphene samples in 2 mL chitosan solutions, ultrasonic dispersion 1 h makes Graphene-shitosan suspending liquid.
3) electrode modification: diameter is the glass-carbon electrode Al that uses 1.0 and 0.3 μ m respectively of 3mm
2O
3Behind the powder emulsion sanding and polishing, each ultrasonic 3min in the second alcohol and water respectively.After the above-mentioned suspending liquid of 4 μ L and 2 μ L concentration are the melamine aqueous solution of 0.2 mg/mL, drip and be applied to the glass-carbon electrode surface of handling, dry 30 min 40 ℃ times.Electrode after the finishing is immersed in 30 min in the BSA solution of 5 % under 37 ℃, to seal remaining avtive spot.
Embodiment 2 modified electrode cyclic voltammetry scans
The different modifying electrode carried out cyclic voltammetric research, its result such as Fig. 4.
(curve is a) at the K of 2 mM for bare electrode
3[Fe (CN)
6] cyclic voltammetry curve in the solution shows a pair of tangible Fe (CN)
6 3-/4-Redox peak, oxidation peak current value are 11.1 μ A; When electrode on beautify chitosan solution (curve b), redox peak electric potential difference broadens, peak current descends to some extent simultaneously, and the oxidation peak current value is 9.931 μ A, may be because the shitosan that does not have electrochemical activity of electrode surface has hindered electron transport; After having modified Graphene/melamine/shitosan on the electrode (curve c), peak current obviously increases, and the oxidation peak current value is 21.71 μ A, and the electrochemical activity of Graphene excellence has strengthened the transmission of electronics greatly; Graphene/melamine/chitosan-modified electrode (immunosensor) is put into the Incubating Solution that contains melamine antibody hatch back (curve d), peak current descends, the oxidation peak current value is 10.31 μ A, this is because the antigen-reactive on melamine antibody and the electrode, be adsorbed on the electrode, hindered that the transmission of electronics causes, this illustrates that also melamine antigen has been modified on the electrode.
Embodiment 3 testing conditions optimizations
The influence of different testing conditions as immunoreactive time and antibody concentration, is determined by the DPV method.
1, the immune response time
The immunosensor that embodiment 1 makes uses the Incubating Solution that contains identical antibody concentration to hatch respectively after 0,10,20,40 and 60 minutes and cleans with PBS, at 2 mM K
3[Fe (CN)
6] carry out differential pulse voltammetry (DPV) scanning, result such as Fig. 5 in the solution.The DPV peak current of Graphene/melamine/chitosan-modified electrode (immunosensor) is increased in 40 minutes with incubation time and descends fast, descends slightly during by 60 minutes (Fig. 5).Incubation time increases not significantly influence of current-responsive again.Therefore, select 40 minutes as immunoreactive best incubation time.
2, hatch the influence of antibody concentration in the solution
Graphene/melamine/chitosan-modified electrode is immersed in 50 μ L and adds in the PBS solution of different antibodies concentration, hatches 40 min at 37 ℃, after PBS cleans, at 2 mM K
3[Fe (CN)
6] carry out differential pulse voltammetry (DPV) scanning, result such as Fig. 6 in the solution.As shown in Figure 6, when antibody concentration in solution increased to 4 μ g/mL by 0 μ g/mL, the DPV peak point current significantly descended.Along with further increase antibody concentration, in 4-10 μ g/mL scope, almost do not observe the reaction that reduces electric current.The result shows, in the hatching solution antibody concentration greater than 4 μ g/mL after the adsorbance of antibody no longer increase, show that binding site reaches capacity basically.For the immune response of competition, select one sufficiently high signal to be arranged and less than the concentration of saturation concentration.Therefore, select 4 μ g/mL to be best antibody concentration.
The drafting of embodiment 4 DPV scanning results and working curve
With K
3Fe (CN)
6Be probe,, utilize the ability of monoclonal antibody specific bond melamine, can realize melamine detection by the DPV detection method.
Preparing standard solution (Incubating Solution), standard solution are that one group of total amount is 50 μ L, contains the solution (pH=7.4) of PBS of the melamine antibody of melamine titer that concentration differs, 4 μ g/mL same concentrations.
Graphene/melamine/chitosan-modified electrode immersed hatch solution, the reaction of melamine antibody in free melamine and the melamine competition that is fixed on electrode surface and the solution in the solution.After 40 min are hatched down through 37 ℃, wash with PBS.Electrode after the flushing is a working electrode, at 2mM K
3[Fe (CN)
6] carry out differential pulse voltammetry (DPV) scanning in the solution, the recording responses electric current, as shown in Figure 7.
The result shows that along with the increase of melamine in Incubating Solution, the DPV peak current increases.Response current after definition Graphene/melamine/chitosan-modified electrode is hatched in the PBS buffer solution (blank) that only contains 4 μ g/mL antibody is I
0, the response current after the Incubating Solution that contains free melamine is hatched is I
x, the increase Δ I(Δ I=I of response current
x-I
0) in 5 to 1500ng/mL scopes, be to be directly proportional with melamine concentration.
Draw Δ I-C typical curve as shown in Figure 8.
With the concentration greater than the current signal correspondence of 3 times of noise signals is minimum detectability, and repeating more than 5 times experiment, to draw minimum detectability be 0.2 ng/mL.
The mensuration of content of melamine in embodiment 5 milk
The typical curve that the melamine of being set up according to embodiment 4 detects adopts method of the present invention to measure content of melamine in the milk sample, and quantivative approach is a standard addition method.
1) milk sample pre-treatment
Take by weighing 2 ± 0.0050 g milk (about 2 mL) in the sample hose of 10 mL, add the melamine titer, acetonitrile-0.05 M HCl extract (the V:V that newly joins with 7 mL, 1:1), potpourri sonic oscillation 30 min, centrifugal 10 min under 5000 r/m are transferred to supernatant in the volumetric flask that volume is 25 mL, residue repeats to extract 3 times with the identical extract of 6 mL, 6 mL and 6 mL, and supernatant is incorporated in the volumetric flask.
The supernatant of getting 1 mL is transferred in the sample hose of 5 mL at nitrogen and is blown under the condition concentration and evaporation under 55 ℃ of temperature, and the pH that concentrate adds 1 mL is used for electrochemical analysis after the dissolving of 7.4 phosphate buffer solutions.
2) four kinds of milk samples that add different amount melamine titers are carried out pre-treatment according to the described method of step 1), make the blank sample that does not add melamine, with 3 mark-on samples (standard addition method) that contain different melamine concentration, and then modified electrode is hatched and measured according to the method identical with embodiment 4, to each mark-on sample determination three times, obtain the actual concentrations of melamine in the sample according to typical curve, the recovery of melamine is between 90.8% to 117%, average out to 104.8%, statistics sees Table 1.
Table 1
| Melamine addition (ng/mL) | Melamine measured quantity (ng/mL) | The recovery (%) |
| 20 | 22.1，18.2，23.4 | 106.2 |
| 200 | 195.3，218.2，210.7 | 104 |
| 1200 | 1325.6，1113.2，1307.2 | 104.1 |
Claims (10)
1. an electrochemical immunosensor that detects melamine comprises basal electrode, it is characterized in that described basal electrode finishing Graphene-melamine-chitosan complexes, and seals non-specific avtive spot with bovine serum albumin.
2. electrochemical immunosensor according to claim 1 is characterized in that, described basal electrode is a glass-carbon electrode.
3. electrochemical immunosensor according to claim 1 and 2, it is characterized in that, described immunosensor makes by following method: the ultrasonic dispersion of the shitosan suspending liquid of Graphene back is mixed with melamine, to mix drop and be coated in electrode surface, utilize shitosan with Graphene and melamine bag by and be fixed in electrode surface, and electrode is immersed in the bovine serum albumen solution nonspecific avtive spot on enclosed-electrode surface.
4. electrochemical immunosensor according to claim 2 is characterized in that, described immunosensor makes by following step:
1) preparation of Graphene-shitosan suspending liquid: the HCl of 0.05M is heated to 80～90 ℃, adds load weighted shitosan, stirring and dissolving, it is the NaOH of 0.1M that cooling back adds concentration, regulates pH to 5, join the chitosan solution of 1mg/mL; Accurately take by weighing 5g Graphene sample in the 2mL chitosan solution, ultrasonic dispersion 1h makes Graphene-shitosan suspending liquid;
2) glass-carbon electrode is handled: diameter is the glass-carbon electrode Al that uses 1.0 and 0.3 μ m respectively of 3mm
2O
3Behind the powder emulsion sanding and polishing, each ultrasonic cleaning 3min in the second alcohol and water respectively;
3) electrode face finish: with 4 μ L Graphene-shitosan suspending liquid and 2 μ L concentration is the melamine aqueous solution of 0.2mg/mL, drips to be applied to step 2) the glass-carbon electrode surface that makes, 40 ℃ of oven dry 30min down;
4) nonspecific avtive spot sealing: the electrode after the finishing is immersed in 30min in 5% the BSA solution under 37 ℃, to seal remaining avtive spot, make described electrochemical immunosensor.
5. preparation method who detects the electrochemical immunosensor of melamine, it is characterized in that: the ultrasonic dispersion of the shitosan suspending liquid of Graphene back is mixed with melamine, to mix drop and be coated in electrode surface, utilize shitosan with Graphene and melamine bag by and be fixed in electrode surface, and electrode is immersed in the bovine serum albumen solution nonspecific avtive spot on enclosed-electrode surface.
6. the preparation method of electrochemical immunosensor according to claim 5 is characterized in that, described method may further comprise the steps:
1) preparation of Graphene-shitosan suspending liquid: the HCl of 0.05M is heated to 80～90 ℃, adds load weighted shitosan, stirring and dissolving, it is the NaOH of 0.1M that cooling back adds concentration, regulates pH to 5, join the chitosan solution of 1mg/mL; Accurately take by weighing 5g Graphene sample in the 2mL chitosan solution, ultrasonic dispersion 1h makes Graphene-shitosan suspending liquid;
2) glass-carbon electrode is handled: diameter is the glass-carbon electrode Al that uses 1.0 and 0.3 μ m respectively of 3mm
2O
3Behind the powder emulsion sanding and polishing, each ultrasonic cleaning 3min in the second alcohol and water respectively;
3) electrode face finish: with 4 μ L Graphene-shitosan suspending liquid and 2 μ L concentration is the melamine aqueous solution of 0.2mg/mL, drips to be applied to step 2) the glass-carbon electrode surface that makes, 40 ℃ of oven dry 30min down;
4) nonspecific avtive spot sealing: the electrode after the finishing is immersed in 30min in 5% the BSA solution under 37 ℃, to seal remaining avtive spot, make described electrochemical immunosensor.
7. one kind is detected the method for melamine based on the described electrochemical immunosensor of claim 1, may further comprise the steps:
1) standard solution preparation: preparing one group of pH that contains the free melamine of variable concentrations that comprises blank standard specimen and be 7.4 phosphate buffer solution is standard solution, wherein contains the melamine antibody of same concentrations;
2) set up working curve: immunosensor is immersed in the standard solution respectively hatch, hatch the back and wash immunosensor, at K with phosphate buffer solution
3[Fe (CN)
6] carry out differential pulse voltammetry (DPV) scanning, recording responses electric current in the solution; The response current of blank standard specimen is I
0, the response current that contains the standard specimen of free melamine is I
x, the increase Δ I(Δ I=I of response current
x-I
0) be directly proportional with melamine concentration C in the standard solution, draw Δ I-C typical curve, or adopt linear regression method to obtain Δ I-C equation of linear regression;
3) melamine is measured: testing sample is formulated as the phosphate buffer solution that contains with step 1) same concentrations melamine antibody, according to step 2) identical method hatches and differential pulse voltammetry scanning the recording responses electric current to immunosensor; Increase Δ I and typical curve according to response current obtain content of melamine.
8. the method for detection melamine according to claim 7 is characterized in that, described K
3[Fe (CN)
6] concentration of solution is 2 mM.
9. the method for detection melamine according to claim 7 is characterized in that, the immune response condition is for hatching 60 min under 37 ℃.
10. the method for detection melamine according to claim 7 is characterized in that, the concentration of described melamine antibody is 4 μ g/mL.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102636643A (en) * | 2012-04-06 | 2012-08-15 | 上海交通大学 | Immunosensor for detecting thiacloprid and preparation method of immunosensor |
| CN102914584A (en) * | 2012-09-29 | 2013-02-06 | 南昌大学 | Rapid detection system and detection method for lactogenesis mixing |
| CN103115954A (en) * | 2013-01-31 | 2013-05-22 | 中国人民共和国泰安出入境检验检疫局 | Method for fast measuring melamine in food by anthraquinone-2-sodium sulfonate sensor |
| CN103424445A (en) * | 2012-09-28 | 2013-12-04 | 上海出入境检验检疫局工业品与原材料检测技术中心 | Graphene/magnetic nanoparticle-modified electrode and its preparation method and use |
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| CN104267078A (en) * | 2014-10-09 | 2015-01-07 | 无锡百灵传感技术有限公司 | Electrochemical sensor for quickly detecting melamine |
| CN104280437A (en) * | 2013-12-17 | 2015-01-14 | 南京师范大学 | Immunosensor and method for detecting various beta-adrenergic receptor stimulant residues |
| CN104280540A (en) * | 2014-10-21 | 2015-01-14 | 南京师范大学 | Immunosensor and method for detecting various beta-adrenergic receptor stimulant residues |
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| CN107966484A (en) * | 2017-12-05 | 2018-04-27 | 南京师范大学淮安研究院 | Application of the electrochemical immunosensor in sulphonamides multi-relict context of detection |
| CN110243900A (en) * | 2019-02-25 | 2019-09-17 | 常州大学 | A kind of application of poly- melamine modified electrode |
| CN110501502A (en) * | 2019-09-02 | 2019-11-26 | 嘉兴学院 | Graphitized carbon-chitosan CA125 immunosensor method of preparation and use |
| CN111751546A (en) * | 2019-03-29 | 2020-10-09 | 中国科学院金属研究所 | Preparation method and application of calprotectin biosensor based on graphene |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102636643A (en) * | 2012-04-06 | 2012-08-15 | 上海交通大学 | Immunosensor for detecting thiacloprid and preparation method of immunosensor |
| CN103424445A (en) * | 2012-09-28 | 2013-12-04 | 上海出入境检验检疫局工业品与原材料检测技术中心 | Graphene/magnetic nanoparticle-modified electrode and its preparation method and use |
| CN103424445B (en) * | 2012-09-28 | 2015-10-21 | 上海出入境检验检疫局工业品与原材料检测技术中心 | A kind of Graphene/modified by magnetic nanoparticles, its electrode and its preparation method and application |
| CN102914584B (en) * | 2012-09-29 | 2015-04-15 | 南昌大学 | Rapid detection system and detection method for lactogenesis mixing |
| CN102914584A (en) * | 2012-09-29 | 2013-02-06 | 南昌大学 | Rapid detection system and detection method for lactogenesis mixing |
| CN103115954A (en) * | 2013-01-31 | 2013-05-22 | 中国人民共和国泰安出入境检验检疫局 | Method for fast measuring melamine in food by anthraquinone-2-sodium sulfonate sensor |
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| CN104267078A (en) * | 2014-10-09 | 2015-01-07 | 无锡百灵传感技术有限公司 | Electrochemical sensor for quickly detecting melamine |
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| CN104280540A (en) * | 2014-10-21 | 2015-01-14 | 南京师范大学 | Immunosensor and method for detecting various beta-adrenergic receptor stimulant residues |
| CN106525949A (en) * | 2016-12-13 | 2017-03-22 | 南京师范大学 | Electrochemical detection method of organophosphorus pesticide |
| CN107764887A (en) * | 2017-10-26 | 2018-03-06 | 天津科技大学 | A kind of 24 site microarray screen-printing electrochemical sensing devices and its application |
| CN107764887B (en) * | 2017-10-26 | 2024-05-10 | 天津科技大学 | 24-Site microarray screen printing electrochemical sensing device and application thereof |
| CN107966484A (en) * | 2017-12-05 | 2018-04-27 | 南京师范大学淮安研究院 | Application of the electrochemical immunosensor in sulphonamides multi-relict context of detection |
| CN110243900A (en) * | 2019-02-25 | 2019-09-17 | 常州大学 | A kind of application of poly- melamine modified electrode |
| CN110243900B (en) * | 2019-02-25 | 2021-04-27 | 常州大学 | Application of poly-melamine modified electrode |
| CN111751546A (en) * | 2019-03-29 | 2020-10-09 | 中国科学院金属研究所 | Preparation method and application of calprotectin biosensor based on graphene |
| CN111751546B (en) * | 2019-03-29 | 2023-11-10 | 中国科学院金属研究所 | Preparation method and application of calprotectin biosensor based on graphene |
| CN110501502A (en) * | 2019-09-02 | 2019-11-26 | 嘉兴学院 | Graphitized carbon-chitosan CA125 immunosensor method of preparation and use |
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