CN103558271A - Electrochemical biosensor for detecting penicillin and preparation method and application thereof - Google Patents
Electrochemical biosensor for detecting penicillin and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an electrochemical biosensor for detecting penicillin and a preparation method and application thereof. The electrochemical biosensor is characterized in that a working electrode is a magnetic glassy carbon electrode immobilized with MWCNTs-Fe3O4/Au-HRP-Ab. The preparation method comprises the following steps: carrying out carboxylation on a multi-walled carbon nanotube, preparing Fe3O4/Au composite nano particles, preparing a Fe3O4/Au magnetic nano particle-HRP-penicillin antibody, sequentially modifying the carbon nanotube subjected to carboxylation and Fe3O4/Au-HRP-Ab to the surface of the glassy carbon electrode of the carbon nanotube, forming the electrochemical biosensor by taking a platinum electrode as a counter electrode and taking a saturated calomel electrode as a reference electrode, immersing the electrochemical biosensor into a sample to be detected, and determining the concentration of the penicillin in the sample to be detected according to a quantitative relation between a corresponding current value and the concentration of the penicillin. The electrochemical biosensor has the advantages of high sensitivity, high selectivity, high accuracy and high detection speed.
Description
Technical field
The present invention relates to penicillin detection technique, especially relate to a kind of based on MWCNTs-Fe
3o
4electrochemica biological sensor for detection of penicillin of/Au and its preparation method and application.
Background technology
Penicillin is a kind of beta-lactam antibiotic; be widely used in treatment bovine mastitis and pre-bacteriological protection infection; yet due to the mankind's unreasonable use, often can cause the residual in a large number of penicillin in biological tissue, particularly in food, can cause potential harm to the mankind.First, Penicillin Content exceeds standard and understands the microorganisms resistance to the action of a drug to people and animals' enteron aisle, causes flora imbalance, thereby causes a series of diseases such as allergic reaction.Secondly, in the prevention of employing penicillin and treatment milk cow, after disease, its body is interior can the accumulation of generation microbiotic.The long-term edible milk that contains excessive penicillin, brings irreversible infringement will to people's internal organs, therefore, extremely important for the detection of penicillin in food.
At present, the detection means of relevant penicillin mainly concentrates on microbial method, high performance liquid chromatography, mass spectroscopy, immunofluorescence technique, conductance method, these methods can well be carried out quantitative and qualitative analysis to penicillin in sensitivity and selectivity, but also there is the operations such as derivatization, extraction and purifying, thereby make that it takes time and effort, complex steps is not suitable for practical application.Therefore, electrochemica biological sensor arises at the historic moment, and it had both combined the specificity of traditional antigen-antibody, combines again electrochemical low cost, high sensitivity and fast-response thereof, thereby has its huge using value at aspects such as food, medical science.Wherein, differential pulse voltammetry has higher sensitivity, is therefore commonly used for highly sensitive detection.
Carbon nano-tube, particularly multi-walled carbon nano-tubes (MWCNTs) have good chemical property and large specific surface area and stronger characterization of adsorption because of it, in the development of electrochemica biological sensor, are used widely.Carbon nano-tube is applied to overpotential, the transmission of promotion electronics, the increase response current that electrochemical sensor preparation can not only reduce substrate, to producing distinctive absorption and catalytic effect to Cucumber after its acidification.Fe
3o
4nano particle, except having the character of general nano particle, also has unique superparamagnetism, can play the effect of magnetic resolution and enrichment target biological molecules.Golden nanometer particle has the strong and synthetic operation of the stability point such as simply.Fe
3o
4/ Au nano particle can improve the charge capacity of horseradish peroxidase (HRP), and then increases catalytic current, the sensitivity that improves immune detection; While antibody and Fe
3o
4after the reaction of/Au composite nanoparticle, can the antibody free with it is separated by magnetic field, avoided centrifuging operation steps.But, also do not disclose at present any about utilizing carbon nano-tube and Fe both at home and abroad
3o
4/ Au nano composite material is reported for the preparation of the correlative study that detects the electrochemica biological sensor of penicillin as electrochemical sensing interface material.
Summary of the invention
Technical matters to be solved by this invention is to provide that a kind of detection speed is fast, the electrochemica biological sensor for detection of penicillin of accuracy and highly sensitive and easy operating and its preparation method and application.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of electrochemica biological sensor for detection of penicillin, comprise electrode, contrast electrode and working electrode, described is platinum electrode to electrode, described contrast electrode is saturated calomel electrode, the described working electrode MWCNTs-Fe that has been immobilized
3o
4the magnetic glassy carbon electrode of/Au-HRP-Ab.
A preparation method for the electrochemica biological sensor of penicillin, concrete steps are as follows:
(1) carbon nano-tube is carboxylated
Ultrasonic 7-9h after multi-walled carbon nano-tubes is mixed in the ratio of mass volume ratio 15mg:20ml with the mixed liquor being comprised of with nitric acid sulfuric acid, then by aperture, be the Kynoar micropore filtering film vacuum filtration of 0.22 μ m, get that filtrate is repeatedly washed with ultrapure water until filtrate is neutral, obtain carboxylated carbon nano-tube aqueous solutions;
(2) Fe
3o
4the preparation of/Au composite nanoparticle
By Fe
3o
4after magnetic nano-particle repeatedly washs with ultrapure water, adopt tetramethyl azanol as spreading agent constant volume, obtain the Fe that concentration is 1 mg/mL
3o
4nano suspending liquid; Then, by Fe
3o
4the sodium citrate aqueous solution of nano suspending liquid and 0.01wt% after the ratio of 1:20 is mixed by volume, is heated to boil under stirring condition, then adds rapidly 3/5Fe
3o
4the concentration of nano suspending liquid volume is the chlorauric acid solution of 1.0mg/mL, and sustained response 20-30min, until solution colour becomes claret, obtains Fe
3o
4/ Au composite nanoparticle, abandoning supernatant after magnetic resolution, again with ultrapure water washing, is mixed with the Fe of 1mg/mL
3o
4/ Au composite nanoparticle solution, in 4 ℃ of stored refrigerated;
(3) Fe
3o
4/ Au-HRP-Ab preparation
By Fe
3o
4/ Au nano suspending liquid Na
2cO
3adjust pH to 8.0, add volume to be respectively Fe
3o
4the 1.0mg/mL horseradish peroxidase of/Au nano suspending liquid 1/5 and 10 μ g/mL penicillin antibody (Pen-Ab) stir and spend the night at 4 ℃, after magnetic resolution, potpourri are dispersed in and Fe
3o
4the isopyknic pH7.4 of/Au nano suspending liquid seals non-specific avtive spot containing in the phosphate buffer of 1wt% bovine serum albumin, then after cleaning up with PBS phosphate buffer, ultrasonic being dispersed in and Fe
3o
4in the isopyknic phosphate buffer of/Au nano suspending liquid, obtain Fe
3o
4/ Au-HRP-Ab solution;
(4) electrode pre-service
Bare electrode is used to deionized water, absolute ethyl alcohol and deionized water ultrasonic 5min respectively successively, with the alumina powder that particle diameter is 1 μ m, 0.3 μ m, 0.05 μ m, repeatedly polish to minute surface successively again, then be placed in respectively ethanol, deionized water for ultrasonic cleaning 5min, finally with nitrogen, dry up standby;
(5) working electrode is modified and sensor preparation
The carboxylated carbon nano-tube solution 5 μ L of preparation in step (1) are dripped in glass-carbon electrode surface, dry under room temperature, obtained carbon nano tube modified glass-carbon electrode (MWCNTs/GCE); By the Fe of preparation in step (3)
3o
4/ Au-HRP-Ab solution 5 μ L drip in carbon nano tube modified glass-carbon electrode surface, dry under room temperature, obtain Fe
3o
4the glass-carbon electrode that/Au-HRP-Ab-MWCNTs modifies; To fix Fe
3o
4/ Au-HRP-Ab-MWCNTs glass-carbon electrode is as working electrode, adopts platinum electrode as to electrode, and saturated calomel electrode forms the electrochemica biological sensor for detection of penicillin of three-electrode system as contrast electrode.
In step (1), the volume ratio of sulfuric acid and nitric acid is 3:1.
Apply the method that above-mentioned electrochemica biological sensor detects penicillin concn, by Fe
3o
4/ Au-HRP-Ab-MWCNTs modifies naked glass-carbon electrode surface as working electrode, adopt platinum electrode as to electrode, saturated calomel electrode forms the electrochemica biological sensor of three-electrode system as contrast electrode, electrochemica biological sensor is immersed in testing sample, according to the quantitative relationship of respective electrical flow valuve and penicillin concn, determine the concentration of penicillin in testing sample.
Compared with prior art, the invention has the advantages that: a kind of electrochemica biological sensor for detection of penicillin of the present invention and its preparation method and application, based on MWCNTs-Fe
3o
4the detection penicillin of/Au-HRP-Ab immunosensor, first, by carbon nano tube modified surperficial to naked glass-carbon electrode, secondly passes through strong suction-operated of magnet by Fe
3o
4/ Au-HRP-Ab compound is fixed to electrode surface, forms electroactive immune magnetic Nano probe.While there is target analytes penicillin in detection system, object and the antibody generation specific immune response that is fixed on electrode surface, increase along with object concentration, more immune complexs will hinder the oxidation of p-dihydroxy-benzene in chem workstation, the transmission effects of impeded electrode surface electronic, reduces redox electric current, thereby realize, penicillin standard items is detected, Criterion curve, carries out to containing penicillin sample the object quantitatively detecting to reach.
The present invention passes through at many walls of glass-carbon electrode finishing nanotube (MWCNTs), Fe
3o
4/ Au, HRP (horseradish peroxidase) and penicillin antibody (Pen-Ab), built the high-sensitive biosensor that is applicable to penicillin fast detecting.This Fe
3o
4the interfacial energy that/Au/MWCNTs composite materials is modified provides larger active area and less interface resistance, and Fe
3o
4in/Au, nanogold particle has very strong catalytic, catalytic efficiency has been strengthened in the dual catalytic effect of nanotube and golden nanometer particle, than directly modifying the sensor (detectability 1.05 ng/mL) of hydroxylation carbon nano-tube assembling or directly modify Fe at glass-carbon electrode at glass-carbon electrode separately
3o
4the immunosensor of/Au-HRP-Ab (detectability 1.00 ng/mL) detectability has improved 10 times of left and right, thereby has greatly strengthened sensitivity (detectability 0.1050 ng/mL), stability and the reappearance of prepared modified electrode; By current time, test, record this sensor the response time of penicillin is less than to 20s, than the response time 30min of traditional high effective liquid chromatography for measuring, the minute 2h of enzyme immunization (ELISA) wants much less, greatly shortened the current-responsive time, greatly improve detection speed, again demonstrated the huge applications of this sensor when measuring micro-penicillin.
In sum, the present invention is for detection of electrochemica biological sensor of penicillin and its preparation method and application, for being fixed on Fe
3o
4penicillin antibody capable on/Au is successfully realized its Direct Electrochemistry response, has reached object quick to penicillin, Sensitive Detection, thereby for supervising food security and guaranteeing that people's interests provide the support of trace residue detection technique.
Accompanying drawing explanation
Fig. 1 is based on Fe
3o
4/ Au-HRP-Ab-CNTs/GCE electrochemical sensor detects the schematic diagram of penicillin;
Fig. 2 is the Fe of preparation
3o
4transmission electron microscope picture;
Fig. 3 is the Fe of preparation
3o
4the transmission electron microscope picture of/Au compound;
Fig. 4 is the Fe of preparation
3o
4the particle diameter distribution plan of/Au compound;
Fig. 5 is preparation Fe
3o
4the phenogram of/Au-HRP-Ab compound; A: the Fe of preparation
3o
4ultraviolet-visible absorption spectroscopy; B: the Fe of preparation
3o
4the ultraviolet-visible absorption spectroscopy of/Au compound; C: the Fe of preparation
3o
4the ultraviolet-visible absorption spectroscopy of/Au-HRP-Ab compound;
Fig. 6 is the ac impedance spectroscopy of different modifying electrode;
Fig. 7 is the canonical plotting that object penicillin current signal and penicillin concn are set up.
Embodiment
Below in conjunction with accompanying drawing, embodiment is described in further detail the present invention.
Specific embodiment one
A kind of electrochemica biological sensor for detection of penicillin, this electrochemical sensor is three-electrode system, comprise electrode, contrast electrode and working electrode, to electrode, be wherein platinum electrode, contrast electrode is saturated calomel electrode, working electrode has been immobilized multi-walled carbon nano-tubes (MWCNTs)-Fe
3o
4the glass-carbon electrode of/Au magnetic nano-particle-HRP-penicillin antibody (Pen-Ab).
Specific embodiment two
A preparation method for the electrochemica biological sensor of penicillin, as shown in Figure 1, concrete steps are as follows for the principle of electrochemical sensor detection penicillin:
(1) carbon nano-tube is carboxylated
By multi-walled carbon nano-tubes (MWCNTs) and by sulfuric acid and nitric acid (3:1, v/v) ultrasonic 7-9h after the mixed liquor forming mixes in the ratio of mass volume ratio 15mg:20ml, then by Kynoar micropore filtering film (aperture is 0.22 μ m) vacuum filtration, get that filtrate is repeatedly washed with ultrapure water until filtrate is neutral, obtain carboxylated carbon nano-tube aqueous solutions;
(2) Fe
3o
4the preparation of/Au composite nanoparticle
By Fe
3o
4magnetic nano-particle (can or directly be bought the Fe that the present invention adopts by coprecipitation and thermal decomposition method preparation in Xi'an Jin Ci nanometer biotechnology company limited
3o
4transmission electron microscope picture as shown in Figure 2, visible Fe
3o
4particle is uniformly dispersed, and particle diameter is roughly 20nm) with ultrapure water, repeatedly wash after, adopt tetramethyl azanol as spreading agent constant volume, obtain the Fe that concentration is 1 mg/mL
3o
4nano suspending liquid; Then, by Fe
3o
4the sodium citrate aqueous solution of nano suspending liquid and 0.01wt% after the ratio of 1:20 is mixed by volume, is heated to boil under stirring condition, then adds rapidly 3/5Fe
3o
4the concentration of nano suspending liquid volume is the chlorauric acid solution of 1.0mg/mL, and sustained response 20-30min, until solution colour becomes claret, obtains Fe
3o
4/ Au composite nanoparticle is (by the Fe of Fig. 3
3o
4the transmission electron microscope picture explanation Fe of/Au compound
3o
4the particle diameter of/Au is roughly 35nm left and right, and this is than simple Fe
3o
4the large 15nm of particle diameter left and right, has also illustrated that Au nano particle is successfully coated on Fe
3o
4surface.The Fe of Fig. 4 particle diameter distribution plan explanation preparation
3o
4/ Au particle is uniformly dispersed, and agglomeration does not occur), abandoning supernatant after magnetic resolution, again with ultrapure water washing, is mixed with the Fe of 1mg/mL
3o
4/ Au composite nanoparticle solution, in 4 ℃ of stored refrigerated; (Fig. 5 is preparation Fe
3o
4the phenogram of/Au-HRP-Ab compound; A: the Fe of preparation
3o
4ultraviolet-visible absorption spectroscopy; B: the Fe of preparation
3o
4the ultraviolet-visible absorption spectroscopy of/Au compound; C: the Fe of preparation
3o
4the ultraviolet-visible absorption spectroscopy of/Au-HRP-Ab compound, visible simple Fe
3o
4(a) there is no characteristic absorption peak, and Fe
3o
4there is obvious characteristic absorption peak at 540nm in/Au compound (b), this is the characteristic absorption peak of Au, and Fe
3o
4also having there is the characteristic absorption peak of horseradish peroxidase in/Au-HRP-Ab compound (c), illustrates that compound successfully synthesizes at 280nm)
(4) electrode pre-service
Bare electrode is used to deionized water, absolute ethyl alcohol and deionized water ultrasonic 5min respectively successively, with the alumina powder that particle diameter is 1 μ m, 0.3 μ m, 0.05 μ m, repeatedly polish to minute surface successively again, then be placed in respectively ethanol, deionized water for ultrasonic cleaning 5min, finally with nitrogen, dry up standby;
(5) working electrode is modified and sensor preparation
The carboxylated carbon nano-tube solution 5 μ L of preparation in step (1) are dripped in glass-carbon electrode surface, dry under room temperature, obtained carbon nano tube modified glass-carbon electrode (MWCNTs/GCE); By the Fe of preparation in step (3)
3o
4/ Au-HRP-Ab solution 5 μ L drip in carbon nano tube modified glass-carbon electrode surface, dry under room temperature, obtain Fe
3o
4the glass-carbon electrode that/Au-HRP-Ab-MWCNTs modifies; To fix Fe
3o
4/ Au-HRP-Ab-MWCNTs glass-carbon electrode is as working electrode, adopts platinum electrode as to electrode, and saturated calomel electrode forms the electrochemica biological sensor for detection of penicillin of three-electrode system as contrast electrode.
The glass-carbon electrode of processing in step (4) is measured containing 2 mM p-dihydroxy-benzene and 5 mM H on CHI660B electrochemical workstation
2 o
2 solution in AC impedance signal.Experimental result, as Fig. 6, is a straight line under naked GCE low frequency substantially, shows that naked GCE has lower AC impedance value, is convenient to the transmission of electrochemical sensing interface electronics; It is obviously less that MWCNTs/GCE compares current impedance than bare electrode (naked GCE), is more conducive to electronics and shifts, and current-responsive value may be larger.And Fe
3o
4/ Au-HRP-Ab-MWCNTs/GCE+2 ppm penicillin antigen is compared Fe
3o
4/ Au-HRP-Ab-MWCNTs/GCE impedance is larger, thereby this may be at electrode surface, to have formed the transmission that immune complex has stoped electronics because specific immunity recognition reaction has occurred antigen-antibody.
Specific embodiment three
A kind of based on multi-walled carbon nano-tubes (MWCNTs)-Fe
3o
4the electrochemica biological sensor of the glass-carbon electrode of/Au magnetic nano-particle-HRP-penicillin antibody (Pen-Ab) detects the method for penicillin concn
By carboxylated carbon nano-tube, the Fe of preparation in above-mentioned specific embodiment two
3o
4/ Au-HRP-Ab modifies naked glass-carbon electrode surface successively as working electrode, adopts platinum electrode as to electrode, and saturated calomel electrode forms the electrochemica biological sensor of three-electrode system as contrast electrode.While there is target analytes penicillin in detection system, object and the antibody generation specific immune response that is fixed on electrode surface, increase along with object concentration, more immune complexs will hinder the oxidation of p-dihydroxy-benzene, the transmission effects of impeded electrode surface electronic, reduce redox electric current, thereby realize, penicillin standard items are detected, Criterion curve, electrochemica biological sensor is immersed in testing sample, according to the quantitative relationship of respective electrical flow valuve and penicillin concn, determine the concentration of penicillin in testing sample, thereby reach, to containing penicillin sample, carry out the object (as shown in Figure 1) quantitatively detecting.
Specific embodiment four
Fe of the present invention
3o
4the electrochemica biological sensor of/Au-HRP-Ab-MWCNTs is to the sensitivity of penicillin and the range of linearity
By the Fe that utilizes specific embodiment two methods to prepare
3o
4/ Au-HRP-Ab-MWCNTs sensor is placed in penicillin standard solution and reacts, standard solution concentration is followed successively by: 0 ng/mL, 0.005 ng/mL, 0.02 ng/mL, 0.08 ng/mL, 0.2 ng/mL, 0 .6 ng/mL,, 2 ng/mL, 5 ng/mL,, 10 ng/mL, 50 ng/mL(standard items solvents are PBS buffer solution), operating voltage 0～1V, working buffer system is NaH
2pO
4-Na
2hPO
4(pH=7.4), record differential pulse method (DPV) gained response current, Criterion curve: y=4.5716LogC+18.244, R
2=0.9985 (as shown in Figure 7), obtaining this electrochemica biological sensor is 0.005-50 ng/mL to the range of linearity of penicillin, detects and is limited to 0.1050 ng/mL; This electrode favorable reproducibility, stable performance, makes simply and easily upgrades.
From measuring, on glass-carbon electrode, directly modify the sensor of hydroxylation carbon nano-tube assembling, the detection of penicillin is limited to 1.05 ng/mL, at glass-carbon electrode, directly modify Fe
3o
4the immunosensor of/Au-HRP-Ab is 1.00 ng/mL to the detectability of penicillin.But this method is by multi-walled carbon nano-tubes and Fe
3o
4the detectability of the co-modified galvanochemistry magnetic immuno sensor in glass-carbon electrode surface composition of/Au nano material is 0.1050 ng/mL, than modifying single material tests limit, 10 times of left and right have been improved, absolutely proved that this sensor has than the stronger advantage of independent material modification, has larger using value.
Specific embodiment five
High selectivity confirmatory experiment
Other microbiotic that add respectively same concentrations in PBS solution, be respectively ampicillin, streptomysin, Roxithromycin, neomycin, kanamycins, gentamicin, contrast with penicillin, its impact is respectively 9%, 5.0%, 6.0%, 3.5%, 4.5% and 2.5% of penicillin current-responsive, result shows that other materials of above-mentioned interpolation are smaller on the detection impact of penicillin, illustrates that thus sensor has high selectivity.
Specific embodiment six
Get commercially available milk sample some, in milk sample, add the penicillin of known quantity, then add appropriate ammonium sulfate (6g/20 mL milk), in 4 ℃ with centrifugal 30 min of 8000 r/min.The PBS buffer solution of its supernatant preparation variable concentrations of leaching.Voltage 0～1V, working buffer system is NaH
2pO
4-Na
2hPO
4(pH=7.4), by the Fe of preparation in above-mentioned specific embodiment two
3o
4the electrochemica biological sensor that/Au-HRP-Ab-CNTs/GCE modifies is placed in the PBS buffer solution that contains appropriate extract, and differential pulse method (DPV) records the electrochemical properties of sample, and result is as shown in table 1.
The testing result of table 1 Penicillin in Milk
| Milk sample | Mark-on concentration/ng/mL | Measure concentration/ng/mL | The recovery/% |
| 1 | 3 | 3.40 | 113.3 |
| 2 | 5 | 4.79 | 98.6 |
From table 1 testing result, Fe
3o
4the average recovery rate that the electrochemica biological sensor that/Au-HRP-Ab-MWCNTs modifies detects the method for penicillin is 98.6～113.3%, shows prepared by the present invention, Fe
3o
4/ Au-HRP-Ab-MWCNTs electrochemica biological sensor is high for the detection precision of penicillin, and result accurately and reliably.
Specific embodiment seven
Accurately weigh chicken and the pork sample of some equivalent, pack centrifuge tube into, add respectively appropriate penicillin, after ultrasonic 3min, 1:5 is to the phosphate buffer solution that adds 0.2 mol/L pH=7.4 in test tube by volume, sonic oscillation 15min, then centrifugal 5min at 4 ℃, get supernatant, the chicken by volume ratio of 1:2 adds saturated ammonium sulfate, pork adds trichloroacetic acid for the ratio of 1:0.5 by volume, after mixing, pack the centrifugal 25min of centrifuge tube into, now solution presents 3 layers, get middle layer clear liquid, 1:1 adds saturated ammonium sulfate (it is 1:1 trichloroacetic acid that pork adds volume ratio) by volume, secondary centrifuging in an identical manner.Chicken and pork get respectively supernatant by volume 1:1 add normal hexane extraction 1 time, standing 5min, obtains final processing sample.Voltage 0～1V, working buffer system is NaH
2pO
4-Na
2hPO
4(pH=7.4), by the Fe of preparation in above-mentioned specific embodiment two
3o
4the electrochemica biological sensor that/Au-HRP-Ab-CNTs/GCE modifies is placed in the PBS buffer solution that contains appropriate extract, and differential pulse method (DPV) records the electrochemical properties of sample, and result is as shown in table 2.
The testing result of penicillin in table 2 chicken and pork
From table 2 testing result, Fe
3o
4the average recovery rate that the electrochemica biological sensor that/Au-HRP-Ab-MWCNTs modifies detects the method for penicillin is 92.8～109%, shows Fe prepared by the present invention
3o
4the electrochemica biological sensor that/Au-HRP-Ab-MWCNTs modifies is high for the detection precision of penicillin, and result accurately and reliably.
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned giving an example.Those skilled in the art are in essential scope of the present invention, and the variation of making, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.
Claims (4)
1. the electrochemica biological sensor for detection of penicillin, comprise electrode, contrast electrode and working electrode, described is platinum electrode to electrode, and described contrast electrode is saturated calomel electrode, it is characterized in that: the described working electrode MWCNTs-Fe that has been immobilized
3o
4the magnetic glassy carbon electrode of/Au-HRP-Ab.
2. a preparation method for the electrochemica biological sensor for detection of penicillin according to claim 1, is characterized in that concrete steps are as follows:
(1) carbon nano-tube is carboxylated
Ultrasonic 7-9h after multi-walled carbon nano-tubes is mixed in the ratio of mass volume ratio 15mg:20ml with the mixed liquor being comprised of with nitric acid sulfuric acid, then by aperture, be the Kynoar micropore filtering film vacuum filtration of 0.22 μ m, get that filtrate is repeatedly washed with ultrapure water until filtrate is neutral, obtain carboxylated carbon nano-tube aqueous solutions;
(2) Fe
3o
4the preparation of/Au composite nanoparticle
By Fe
3o
4after magnetic nano-particle repeatedly washs with ultrapure water, adopt tetramethyl azanol as spreading agent constant volume, obtain the Fe that concentration is 1 mg/mL
3o
4nano suspending liquid; Then, by Fe
3o
4the sodium citrate aqueous solution of nano suspending liquid and 0.01wt% after the ratio of 1:20 is mixed by volume, is heated to boil under stirring condition, then adds rapidly 3/5Fe
3o
4the concentration of nano suspending liquid volume is the chlorauric acid solution of 1.0mg/mL, and sustained response 20-30min, until solution colour becomes claret, obtains Fe
3o
4/ Au composite nanoparticle, abandoning supernatant after magnetic resolution, again with ultrapure water washing, is mixed with the Fe of 1mg/mL
3o
4/ Au composite nanoparticle solution, in 4 ℃ of stored refrigerated;
(3) Fe
3o
4/ Au-HRP-Ab preparation
By Fe
3o
4/ Au nano suspending liquid Na
2cO
3adjust pH to 8.0, add volume to be respectively Fe
3o
4the 1.0mg/mL horseradish peroxidase of/Au nano suspending liquid 1/5 and 10 μ g/mL penicillin antibody (Pen-Ab) stir and spend the night at 4 ℃, after magnetic resolution, potpourri are dispersed in and Fe
3o
4the isopyknic pH7.4 of/Au nano suspending liquid seals non-specific avtive spot containing in the phosphate buffer of 1wt% bovine serum albumin, then after cleaning up with PBS phosphate buffer, ultrasonic being dispersed in and Fe
3o
4in the isopyknic phosphate buffer of/Au nano suspending liquid, obtain Fe
3o
4/ Au-HRP-Ab solution;
(4) electrode pre-service
Bare electrode is used to deionized water, absolute ethyl alcohol and deionized water ultrasonic 5min respectively successively, with the alumina powder that particle diameter is 1 μ m, 0.3 μ m, 0.05 μ m, repeatedly polish to minute surface successively again, then be placed in respectively ethanol, deionized water for ultrasonic cleaning 5min, finally with nitrogen, dry up standby;
(5) working electrode is modified and sensor preparation
The carboxylated carbon nano-tube solution 5 μ L of preparation in step (1) are dripped in glass-carbon electrode surface, dry under room temperature, obtained carbon nano tube modified glass-carbon electrode (MWCNTs/GCE); By the Fe of preparation in step (3)
3o
4/ Au-HRP-Ab solution 5 μ L drip in carbon nano tube modified glass-carbon electrode surface, dry under room temperature, obtain Fe
3o
4the glass-carbon electrode that/Au-HRP-Ab-MWCNTs modifies; To fix Fe
3o
4/ Au-HRP-Ab-MWCNTs glass-carbon electrode is as working electrode, adopts platinum electrode as to electrode, and saturated calomel electrode forms the electrochemica biological sensor for detection of penicillin of three-electrode system as contrast electrode.
3. the preparation method of the electrochemica biological sensor for detection of penicillin according to claim 2, is characterized in that: in step (1), the volume ratio of sulfuric acid and nitric acid is 3:1.
4. application rights requires a method for the electrochemica biological sensor detection penicillin concn described in 1 or 2, it is characterized in that comprising the following steps: by Fe
3o
4/ Au-HRP-Ab-MWCNTs modifies naked glass-carbon electrode surface as working electrode, adopt platinum electrode as to electrode, saturated calomel electrode forms the electrochemica biological sensor of three-electrode system as contrast electrode, electrochemica biological sensor is immersed in testing sample, according to the quantitative relationship of respective electrical flow valuve and penicillin concn, determine the concentration of penicillin in testing sample.
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| CN104931693A (en) * | 2015-06-11 | 2015-09-23 | 上海海洋大学 | Electrochemical immunosensor and preparation method thereof |
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| CN115096971A (en) * | 2022-06-29 | 2022-09-23 | 北部湾大学 | Glassy carbon electrode for detecting mercury ions and preparation method and application thereof |
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