CN103278541A - Electrochemical biosensor for detecting bisphenol-A, and preparation method and application thereof - Google Patents
Electrochemical biosensor for detecting bisphenol-A, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an electrochemical biosensor for detecting bisphenol-A, and a preparation method and an application thereof. The electrochemical biosensor comprises a counter electrode, a reference electrode and a working electrode. The electrochemical biosensor is characterized in that the working electrode is a glassy carbon electrode surface-modified by a graphene/gold complex, tyrosinase and chitosan. The preparation method comprises a step of preparing the graphene oxide; a step of preparing the graphene/gold complex; and a step of polishing and cleaning a bare glassy carbon electrode. A three electrode system electrochemical enzyme sensor is formed in the way that the graphene/gold complex, the tyrosinase and the chitosan are modified on the glassy carbon electrode to be used as the working electrode, a platinum electrode is used as the counter electrode and a saturated calomel electrode is used as the reference electrode. The concentration of bisphenol-A in a to-be-detected sample is determined by immersing the electrochemical biosensor in the to-be-detected sample and according to a quantitative relationship between corresponding current value and the concentration of bisphenol-A. The electrochemical biosensor has high sensitivity, strong selectivity, high accuracy and fast detection speed. The preparation method is simple and practical, and convenient for operations in actual detection.
Description
Technical field
The present invention relates to the detection method of bisphenol-A, especially relate to for detection of bisphenol-A based on electrochemica biological sensor of Graphene/golden composite nano materials and preparation method thereof.
Background technology
Bisphenol-A, namely 4,4-dihydroxy-diphenyl propane (being called for short BPA) is that (endorsing disrupting chemicals EDCs), causes serious pollution and harm to ecologic environment to a kind of environment incretion interferent matter with estrogen active.Bisphenol-A is piled up in human body by food chain, causes human, animal various diseases, can cause the distortion of the mankind, animal reproduction cell and the decline of breeding potential, and can cause feminizing of gastropod male.At present, the detection method of relevant bisphenol-A mainly concentrates on gas chromatography, gas phase-mass spectrometry, liquid chromatography, liquid phase-mass spectrometry etc., although these method sensitivity are higher, accuracy good, but need carry out loaded down with trivial details pre-service to testing sample, the mostly expensive and huge heaviness of required instrument, need the professional and technical personnel to carry out daily servicing, have length consuming time, cost height, be not suitable for many shortcomings such as on-the-spot detection.In view of bisphenol-A to the havoc of the mankind and ecologic environment and the limitation of current detection technology, the technology that development detects bisphenol-A is very necessary.Demonstrate good redox property owing to have the bisphenol-A of phenolic hydroxyl group at electrode surface, the preparation of electrochemica biological sensor is expected to success and overcomes the above problems.
Because the Graphene material with carbon element can provide more reactivity site, can be good at keeping the activity of enzyme, simultaneously Graphene also has good adsorption ability, bigger surface area, more catalytic site, stronger electric conductivity and very high advantages such as physical strength, so Graphene is applied to the electrochemica biological sensor field and has special advantages.But there is bigger Van der Waals force (5.9kJmol-1) in the graphene film interlayer, and accumulation and gathering easily take place, thereby has limited the development of high-sensitivity detection technology to a certain extent.At present, also not disclose both at home and abroad and anyly report about utilizing Graphene to prepare for detection of the correlative study of the electrochemica biological sensor of bisphenol A concentration.
Summary of the invention
Technical matters to be solved by this invention provide a kind of have the fast and easy operating of highly sensitive, detection speed for detection of electrochemica biological sensor of bisphenol-A and preparation method thereof.
The present invention solves the problems of the technologies described above the technical scheme that adopts: a kind of electrochemica biological sensor for detection of bisphenol-A, comprise to electrode, contrast electrode and working electrode described working electrode the has been finishing glass-carbon electrode of Graphene/Au composite, tyrosinase and shitosan.
Described is platinum electrode to electrode, and described contrast electrode is saturated calomel electrode.
A kind of preparation method of the electrochemica biological sensor for detection of bisphenol-A, concrete steps are as follows:
(1) preparation of graphene oxide: potassium permanganate and crystalline flake graphite mixed by mass ratio 6:1 obtain mixed-powder, to obtain mixed liquor after the concentrated sulphuric acid and the phosphoric acid 9:1 mixing by volume, mixed-powder is mixed by mass volume ratio 21g:400ml with mixed liquor, be cooled to room temperature again after 45-55 ℃ of constant temperature stirs 10-14h, the massfraction that adds 3/400 mixeding liquid volume under the condition of ice bath is 30% H
2O
2Solution, fully the reaction back is to room temperature, use the nonwoven fabrics suction filtration, with the filtrate centrifugal 4h under 4000rpm that obtains behind the suction filtration, remove floating layer, the centrifugal solid matter that obtains is used the deionized water of equivalent successively, massfraction is 30% HCl solution, and ethanol cleans, after having cleaned at every turn, solid matter nonwoven fabrics suction filtration, the solid matter that obtains behind the suction filtration is centrifugal 4h under 4000rpm, discards floating layer, after repeated washing repeatedly, place-80 ℃ of vacuum freezing drying ovens dry the remaining solid material, the gained material is graphene oxide;
(2) preparation of Graphene/Au composite: get the 100mg graphene oxide and be dissolved in the 100ml deionized water, ultrasonicly be uniformly dispersed to graphene oxide, obtain stable dispersion liquid, the chlorauric acid solution that adds 50ml 2mM again in the dispersion liquid, behind the ultrasonic 15min, be transferred to and heat 2min in the micro-wave oven, again dispersion liquid is transferred in the three-necked bottle, dropping 6ml massfraction is 80% hydrazine hydrate solution, 80 ℃ of constant temperature magnetic agitation 30min, be cooled to room temperature, product discards floating layer in the centrifugal 20min of 8000rpm, and the centrifugal solid matter that obtains is used a large amount of deionized waters respectively, centrifuge washing is repeatedly under 8000rpm for absolute ethyl alcohol, place-80 ℃ of vacuum freezing drying ovens dry the gained solid matter, the gained material is Graphene/Au composite;
(3) polishing of naked glass-carbon electrode and cleaning: naked glass-carbon electrode is used deionized water, absolute ethyl alcohol, deionized water ultrasonic 5min respectively successively, be the alumina powder of 1 μ m, 0.3 μ m, the 0.05 μ m 5min that respectively polishes with particle diameter successively again, before each replacing different-grain diameter alumina powder, powder slurry remaining on polishing cloth and the glass-carbon electrode is rinsed well with deionized water, glass-carbon electrode is dried up standby with nitrogen at last;
(4) electrode modification: Graphene/Au composite suspending liquid and the concentration that with concentration is 3mg/ml be 4.5mg/ml tyrosinase solution by equal-volume than after mixing, concussion mixes 1-2h, adding with the isopyknic concentration of tyrosinase solution again is the chitosan solution of 1.5mg/mL, concussion mixes 15-20min, obtains Graphene/gold-tyrosinase-shitosan (GR/Au-Tyr-CS) mixed solution; Graphene/gold-tyrosinase-shitosan mixed solution dripped be applied to the naked glass-carbon electrode surface that step (3) obtains, cover with beaker, be dried to solvent evaporates under the room temperature condition, be fixed the Graphene/gold-tyrosinase-shitosan glass-carbon electrode of tyrosinase as working electrode, adopt the platinum electrode conduct to electrode, saturated calomel electrode constitutes the electrochemical enzymatic sensor of three-electrode system as contrast electrode.
In the step (4) Graphene/Au composite is dissolved in the Graphene/Au composite suspending liquid that is mixed with 3mg/ml in the deionized water, ultrasonic standby to being uniformly dispersed; Be the tyrosinase solution for standby that 7.0 PBS buffer preparation becomes 4.5mg/ml with tyrosinase pH; Shitosan is dissolved in the chitosan solution that is mixed with 1.5mg/mL in the 2M acetum, and it is standby to regulate pH to 6.0 with NaOH.
A kind of Applied Electrochemistry biology sensor detects the method for bisphenol A concentration, may further comprise the steps: Graphene/gold-tyrosinase-shitosan mixed liquor is modified naked glass-carbon electrode surface as working electrode, by suction-operated the enzyme loading is fixed to electrode surface, adopt the platinum electrode conduct to electrode, saturated calomel electrode constitutes the electrochemical enzymatic sensor of three-electrode system as contrast electrode, electrochemical sensor is immersed in the testing sample, according to the quantitative relationship of respective electrical flow valuve and bisphenol A concentration, determine the concentration of bisphenol-A in the testing sample.
Compared with prior art, the invention has the advantages that: a kind of electrochemica biological sensor for detection of bisphenol-A of the present invention and its preparation method and application, by at glass-carbon electrode finishing Graphene/metal/composite material, shitosan and tyrosinase, made up and be applicable to that bisphenol-A detects the high-sensitive biosensor of analyzing.When having the target analytes bisphenol-A in the testing environment, irreversible redox reaction takes place with the tyrosinase that is fixed on electrode surface in object, increase along with object concentration, response current also strengthens thereupon, with this phenomenon the bisphenol-A standard items are detected, set up typical curve, to reach containing the purpose that the bisphenol-A sample quantitatively detects.
The present invention prepares Graphene/Au composite by graphene oxide is mixed the back electronation with chlorauric acid solution, the introducing of nanogold particle, not only avoid the Graphene lamella to come back to crystal structure of graphite because of gathering effectively, and improved electronic conductivity, adsorbability and the catalytic activity of grapheme material greatly.The interfacial energy that this GR/Au-Tyr-CS compound substance is modified provides bigger active area and littler interface resistance, and nanogold particle has very strong catalytic in Graphene/Au composite, the synergy of nanogold particle and tyrosinase has been strengthened catalytic efficiency in Graphene/Au composite, thereby strengthened the sensitivity (detection is limited to 8.9nmol/L) of tested electrode greatly, stability, (electrode is made favorable reproducibility to reappearance, be 20h serviceable life) and accuracy (recovery reaches 92.8%～110.3%), greatly shortened the current-responsive time, the tyrosinase that is fixed on Graphene/gold-shitosan can successfully be realized its Direct Electrochemistry response, reached quick to bisphenol-A, the purpose of Sensitive Detection, its preparation method is simple, and actual detected is easy and simple to handle.
Description of drawings
Fig. 1 is the schematic diagram that detects bisphenol-A based on Graphene/gold-tyrosinase-shitosan electrochemica biological sensor;
Fig. 2 is the electron diffraction diagram of the graphene oxide of preparation;
Fig. 3 is the transmission electron microscope picture of the Graphene/Au composite of preparation;
Fig. 4 is the electrochemical impedance collection of illustrative plates of different electrode modifications;
Fig. 5 is the canonical plotting that object bisphenol-A current signal and bisphenol A concentration are set up.
Embodiment
Describe in further detail below in conjunction with the present invention of accompanying drawing embodiment.
Specific embodiment one
A kind of electrochemica biological sensor for detection of bisphenol A concentration, this electrochemical sensor is three-electrode system, be platinum electrode to electrode wherein, contrast electrode is saturated calomel electrode, working electrode has been immobilized Graphene/gold-tyrosinase-chitosan-modified glass-carbon electrode (GR/Au-Tyr-CS/GCE).
Specific embodiment two
A kind of preparation method of the electrochemica biological sensor for detection of bisphenol-A, concrete steps are as follows:
(1) preparation of graphene oxide
Potassium permanganate and crystalline flake graphite mixed by mass ratio 6:1 obtain mixed-powder, to obtain mixed liquor after the concentrated sulphuric acid and the phosphoric acid 9:1 mixing by volume, mixed-powder is mixed by mass volume ratio 21g:400ml with mixed liquor, be cooled to room temperature again after 45-55 ℃ of constant temperature stirs 10-14h, the massfraction that adds 3/400 mixeding liquid volume under the condition of ice bath is 30% H
2O
2Solution, fully the reaction back is to room temperature, use the nonwoven fabrics suction filtration, with the filtrate centrifugal 4h under 4000rpm that obtains behind the suction filtration, remove floating layer, the centrifugal solid matter that obtains is used the deionized water of equivalent successively, massfraction is 30% HCl solution, and ethanol cleans, after each the cleaning, solid matter nonwoven fabrics suction filtration, the solid matter that obtains behind the suction filtration is centrifugal 4h under 4000rpm, discards floating layer, after repeated washing repeatedly, place-80 ℃ of vacuum freezing drying ovens dry the remaining solid material, the gained material is graphene oxide; The electron diffraction diagram of graphene oxide as shown in Figure 2, the prepared graphene oxide of this method meets Graphene symmetry six-membered ring structure, shows that graphene oxide prepares successfully;
(2) preparation of Graphene/Au composite
Getting the 100mg graphene oxide is dissolved in the 100ml deionized water, ultrasonicly be uniformly dispersed to graphene oxide, obtain stable dispersion liquid, the chlorauric acid solution that adds 50ml 2mM again in the dispersion liquid, behind the ultrasonic 15min, be transferred to and heat 2min in the micro-wave oven, again dispersion liquid is transferred in the three-necked bottle, dropping 6ml massfraction is 80% hydrazine hydrate solution, 80 ℃ of constant temperature magnetic agitation 30min, be cooled to room temperature, product discards floating layer in the centrifugal 20min of 8000rpm, and the centrifugal solid matter that obtains is used a large amount of deionized waters respectively, centrifuge washing is repeatedly under 8000rpm for absolute ethyl alcohol, place-80 ℃ of vacuum freezing drying ovens dry the gained solid matter, the gained material is Graphene/Au composite; The transmission electron microscope picture of Graphene/Au composite has finely dispersed nanogold particle on the Graphene fold bottom as shown in Figure 3, shows that the method has successfully prepared Graphene/gold nano compound substance;
(3) polishing of naked glass-carbon electrode and cleaning
Naked glass-carbon electrode is used deionized water, absolute ethyl alcohol, deionized water ultrasonic 5min respectively successively, be the alumina powder of 1 μ m, 0.3 μ m, the 0.05 μ m 5min that respectively polishes with particle diameter successively again, before each replacing different-grain diameter alumina powder, powder slurry remaining on polishing cloth and the electrode is rinsed well with deionized water, dried up naked glass-carbon electrode standby with nitrogen at last;
(4) electrode modification
Graphene/Au composite the suspending liquid that with concentration is 3mg/ml is after 4.5mg/ml tyrosinase solution mixes by equal-volume with concentration, concussion mixes 1-2h, adding with the isopyknic concentration of tyrosinase solution again is the chitosan solution of 1.5mg/mL, concussion mixes 15-20min, obtains Graphene/gold-tyrosinase-shitosan (GR/Au-Tyr-CS) mixed solution; Graphene/gold-tyrosinase-shitosan mixed solution dripped be applied to the naked glass-carbon electrode surface that step (3) obtains, cover with beaker, be dried to solvent evaporates under the room temperature condition, be fixed the Graphene/gold-tyrosinase-shitosan glass-carbon electrode of tyrosinase as working electrode, adopt the platinum electrode conduct to electrode, saturated calomel electrode constitutes the electrochemical enzymatic sensor of three-electrode system as contrast electrode.
Wherein in the step (4) Graphene/Au composite is dissolved in the Graphene/Au composite suspending liquid that is mixed with 3mg/ml in the deionized water, ultrasonic standby to being uniformly dispersed; Be the tyrosinase solution for standby that 7.0 PBS buffer preparation becomes 4.5mg/ml with tyrosinase pH; Shitosan is dissolved in the chitosan solution that is mixed with 1.5mg/mL in the 2M acetum, and it is standby to regulate pH to 6.0 with NaOH.
Specific embodiment three
A kind of method that detects bisphenol-A based on the electrochemical enzymatic sensor of Graphene/Au composite-tyrosinase-shitosan (GR/Au-Tyr-CS)
Graphene/gold-tyrosinase-shitosan (GR/Au-Tyr-CS) mixed liquor of preparation in the above-mentioned specific embodiment two is modified naked glass-carbon electrode surface, by suction-operated the enzyme loading is fixed to electrode surface, adopt the platinum electrode conduct to electrode, saturated calomel electrode is as the electrochemical enzymatic sensor of contrast electrode formation three-electrode system, constitutive enzyme sensor.When having the target analytes bisphenol-A in the testing environment, irreversible redox reaction takes place with the tyrosinase that is fixed on electrode surface in object, increase along with object concentration, response current also strengthens thereupon, with this phenomenon the bisphenol-A standard items are detected, set up typical curve, electrochemical sensor is immersed in the testing sample, quantitative relationship according to electrochemical source of current response and bisphenol A concentration, determine the concentration of bisphenol-A in the testing sample, thereby reach containing the purpose (as shown in Figure 1) that the bisphenol-A sample quantitatively detects.
Specific embodiment four
The performance measurement of a kind of electrochemical enzymatic sensor based on Graphene/Au composite-tyrosinase-shitosan (GR/Au-Tyr-CS) of the embodiment of the invention two preparation, specific as follows:
(1) preparation of Graphene/Au composite: with above-mentioned specific embodiment two;
(2) polishing of naked glass-carbon electrode and cleaning: with above-mentioned specific embodiment two;
(3) test of naked glass-carbon electrode: take by weighing 3.73g KCl and 0.082g K
3Fe (CN)
6Be dissolved in the 50ml volumetric flask, be made into 5mM K
3Fe (CN)
6With 1 M KCl solution, get this solution of 15ml and place electrolytic cell; Be working electrode with the electrode of polishing, mercurous chloride electrode is contrast electrode, platinum electrode is tested for electrode is formed three-electrode system, operating voltage is-0.3～0.6V, sweep speed and be 100mV/s, peak shape is stable, shape good until obtaining, the redox peak to peak value is less than the cyclic voltammogram of 65mV, to guarantee glass-carbon electrode any surface finish contamination-free, reduces experimental error;
(4) electrode modification has been fixed the GR/Au-Tyr-CS/GCE electrode of tyrosinase: with above-mentioned specific embodiment two;
(5) be that the graphene suspension of 3mg/ml is after 4.5mg/ml tyrosinase solution mixes by equal-volume with concentration with concentration, concussion mixes 1-2h, adding with the isopyknic concentration of tyrosinase solution again is the chitosan solution of 1.5mg/mL, concussion mixes 15-20min, obtains Graphene-tyrosinase-shitosan (GRu-Tyr-CS) mixed solution; Graphene-tyrosinase-shitosan mixed solution dripped be applied to the naked glass-carbon electrode surface that step (3) obtains, cover with beaker, be dried to solvent evaporates under the room temperature condition, obtain Graphene-tyrosinase-shitosan electrode (GR-Tyr-CS/GCE);
(6) be that 4.5mg/ml tyrosinase solution and concentration are the chitosan solution of 1.5mg/mL with concentration, concussion mixes 15-20min, obtains tyrosinase-shitosan (Tyr-CS) mixed solution; Tyrosinase-shitosan mixed solution dripped be applied to the naked glass-carbon electrode surface that step 3 obtains, cover with beaker, be dried to solvent evaporates under the room temperature condition, obtain tyrosinase-shitosan electrode (Tyr-CS/GCE);
(7) GR/Au-Tyr-CS/GCE and GR-Tyr-CS/GCE, Tyr-CS/GCE and bare electrode (Bare GCE) are measured at the CHI660B electrochemical workstation respectively contained 5mM K
3[Fe (CN)
6]/K
4[Fe (CN)
6] AC impedance signal in the electrolytic solution of probe.Experimental result as shown in Figure 4, it is obviously less that GR/Au-Tyr-CS/GCE and GR-Tyr-CS/GCE, Tyr-CS/GCE and bare electrode (Bare GCE) are compared current impedance, more is conducive to electronics and shifts, the current-responsive value may be bigger;
(8) above-mentioned adorned GR/Au-Tyr-CS/GCE and GR-Tyr-CS/GCE, Tyr-CS/GCE and Bare GCE electrode are measured at CHI660B respectively contained 5mMK
3[Fe (CN)
6] cyclic voltammetry curve in (0.1moL/LKCl) the electrolytic solution, experiment shows that three kinds of electrodes of GR/Au-Tyr-CS/GCE and other compare, and background current is bigger, and chemical property is higher.Verified the superiority of GR/Au-Tyr-CS/GCE electrochemical characteristic;
(9) adopt difference volt-ampere impulse method, GR/Au-Tyr-CS/GCE and GR-Tyr-CS/GCE, Tyr-CS/GCE and the immersion of Bare GCE electrode of method for preparing contained the working buffer system KH of same concentrations bisphenol-A
2PO
4-K
2HPO
4(pH=7.0) in, experiment shows that GR/Au-Tyr-CS/GCE is than other three kinds of electrodes, current-responsive value to bisphenol-A is bigger, further shows the superiority of GR/Au-Tyr-CS/GCE in bisphenol-A detects, for exploitation GR/Au-Tyr-CS/GCE sensor is established theoretical foundation.
Specific embodiment five
The electrochemical enzymatic sensor of Graphene/Au composite of the present invention-tyrosinase-shitosan (GR/Au-Tyr-CS) is to bisphenol-A sensitivity and the range of linearity
To utilize the GR/Au-Tyr-CS/GCE sensor of specific embodiment two methods preparation to place the bisphenol-A standard solution to react, standard solution concentration is followed successively by: in the PBS buffer solution of 0.25 μ mol/L, 0.5 μ mol/L, 1 μ mol/L, 1.5 μ mol/L, 2 μ mol/L, voltage 0～1V, the working buffer system is KH
2PO
4-K
2HPO
4(pH=7.0), record differential pulse method (DPV) gained response current, set up typical curve: y=3.37244C+0.40494, R
2=0.99542 (as shown in Figure 5), obtaining this enzyme sensor is 2s to the bisphenol-A response time, and the range of linearity of measuring bisphenol-A is 250-2000nmol/L, detects and is limited to 8.9nmol/L; Electrode is made favorable reproducibility, and be 20h serviceable life.
Specific embodiment six
The high selectivity of Graphene/gold-tyrosinase-shitosan (GR/Au-Tyr-CS) electrochemical enzymatic sensor that the present invention makes up
To utilize the GR/Au-Tyr-CS/GCE sensor of specific embodiment two methods preparation to place the PBS buffer solution that contains same concentrations bisphenol-A, estradiol, oestrone respectively, voltage 0～1V, the working buffer system is KH
2PO
4-K
2HPO
4(pH=7.0), record differential pulse method (DPV) gained response current, draw bisphenol-A working buffer system gained response current maximum, and other two kinds of corresponding electric currents of hormone gained all a little less than, account for 14%, 37% of bisphenol-A response current respectively, show the good selectivity of GR/Au-Tyr-CS/GCE sensor.
Specific embodiment seven
After disposal plastic cup and milk box shredded, pulverize with comminutor, take by weighing the 1.0g sample respectively, place 250ml tool plug conical flask, add the 50ml absolute methanol, leave standstill 24h; Rotary evaporation refilters then, filtrate with methanol constant volume to 2ml as blank sample, carry out mark-on and reclaim to detect.Voltage 0～1V, the working buffer system is KH
2PO
4-K
2HPO
4(pH=7.0), the GR/Au-Tyr-CS/GCE that above-mentioned specific embodiment two methods are prepared places the PBS buffer solution that contains an amount of sample liquid that bisphenol-A is detected, and testing result is as shown in table 1,
The testing result of bisphenol-A in table 1 plastic cup and the milk box
By table 1 testing result as can be known, the average recovery rate that the electrochemical enzymatic sensor of Graphene/Au composite-tyrosinase-shitosan (GR/Au-Tyr-CS) detects the method for bisphenol-A is 98～110.3%, show the chemical biosensor of Graphene/Au composite-tyrosinase-shitosan that the present invention prepares for the detection precision height of bisphenol-A, the result accurately and reliably.
Specific embodiment eight
Accurately take by weighing and rub uniform 1g ham sausage sample, 1g bullamacow product sample and 2.0g florisil silica (Florisil) filler.Filler is put into mortar, gently grind to mixing.A slice filter paper is put in 10ml syringe tube bottom, will grind uniform ham sausage sample and bullamacow product sample and be seated in the corresponding syringe tube with filler mixture respectively, covers with a slice filter paper, with the compacting of injection plug again.4ml normal hexane wash away grease is used the elution of 4ml methylene chloride again, and eluent is concentrated into 0.lml with little nitrogen stream in 30 ℃ of water-baths, and methanol constant volume, is carried out mark-on and reclaimed detection as blank sample to 2ml.Voltage is under 0～1V condition, and the working buffer system is KH
2PO
4-K
2HPO
4(pH=7.0), the GR/Au-Tyr-CS/GCE that above-mentioned specific embodiment two methods are prepared places the PBS buffer solution that contains an amount of sample liquid that bisphenol-A is detected, and testing result is as shown in table 2,
The testing result of bisphenol-A in table 2 ham sausage and the bullamacow goods
By table 2 testing result as can be known, the average recovery rate that the electrochemical enzymatic sensor of Graphene/Au composite-tyrosinase-shitosan (GR/Au-Tyr-CS) detects the method for bisphenol-A is 92.8～109%, show the chemical biosensor of Graphene/Au composite-tyrosinase-shitosan that the present invention prepares for the detection precision height of bisphenol-A, the result accurately and reliably.
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention also is 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 (5)
1. the electrochemica biological sensor for detection of bisphenol-A comprises electrode, contrast electrode and working electrode, it is characterized in that: described working electrode the has been finishing glass-carbon electrode of Graphene/Au composite, tyrosinase and shitosan.
2. the electrochemica biological sensor for detection of bisphenol-A according to claim 1, it is characterized in that: described is platinum electrode to electrode, and described contrast electrode is saturated calomel electrode, and described contrast electrode is saturated calomel electrode.
3. method for preparing the described electrochemica biological sensor for detection of bisphenol-A of claim 1 is characterized in that concrete steps are as follows:
(1) preparation of graphene oxide: potassium permanganate and crystalline flake graphite mixed by mass ratio 6:1 obtain mixed-powder, to obtain mixed liquor after the concentrated sulphuric acid and the phosphoric acid 9:1 mixing by volume, mixed-powder is mixed by mass volume ratio 21g:400ml with mixed liquor, be cooled to room temperature again after 45-55 ℃ of constant temperature stirs 10-14h, the massfraction that adds 3/400 mixeding liquid volume under the condition of ice bath is 30% H
2O
2Solution, fully the reaction back is to room temperature, use the nonwoven fabrics suction filtration, with the filtrate centrifugal 4h under 4000rpm that obtains behind the suction filtration, remove floating layer, the centrifugal solid matter that obtains is used the deionized water of equivalent successively, massfraction is 30% HCl solution, and ethanol cleans, after each the cleaning, solid matter nonwoven fabrics suction filtration, the solid matter that obtains behind the suction filtration is centrifugal 4h under 4000rpm, discards floating layer, after repeated washing repeatedly, place-80 ℃ of vacuum freezing drying ovens dry the remaining solid material, the gained material is graphene oxide;
(2) preparation of Graphene/Au composite: get the 100mg graphene oxide and be dissolved in the 100ml deionized water, ultrasonicly be uniformly dispersed to graphene oxide, obtain stable dispersion liquid, the chlorauric acid solution that adds 50ml 2mM again in the dispersion liquid, behind the ultrasonic 15min, be transferred to and heat 2min in the micro-wave oven, again dispersion liquid is transferred in the three-necked bottle, dropping 6ml massfraction is 80% hydrazine hydrate solution, 80 ℃ of constant temperature magnetic agitation 30min, be cooled to room temperature, product discards floating layer in the centrifugal 20min of 8000rpm, and the centrifugal solid matter that obtains is used a large amount of deionized waters respectively, centrifuge washing is repeatedly under 8000rpm for absolute ethyl alcohol, place-80 ℃ of vacuum freezing drying ovens dry the gained solid matter, the gained material is Graphene/Au composite;
(3) polishing of naked glass-carbon electrode and cleaning: naked glass-carbon electrode is used deionized water, absolute ethyl alcohol, deionized water ultrasonic 5min respectively successively, be the alumina powder of 1 μ m, 0.3 μ m, the 0.05 μ m 5min that respectively polishes with particle diameter successively again, before each replacing different-grain diameter alumina powder, powder slurry remaining on polishing cloth and the glass-carbon electrode is rinsed well with deionized water, glass-carbon electrode is dried up standby with nitrogen at last;
(4) electrode modification: Graphene/Au composite suspending liquid and the concentration that with concentration is 3mg/ml be 4.5mg/ml tyrosinase solution by equal-volume than after mixing, concussion mixes 1-2h, adding with the isopyknic concentration of tyrosinase solution again is the chitosan solution of 1.5mg/mL, concussion mixes 15-20min, obtains Graphene/gold-tyrosinase-shitosan mixed solution; Graphene/gold-tyrosinase-shitosan mixed solution dripped be applied to the naked glass-carbon electrode surface that step (3) obtains, cover with beaker, be dried to solvent evaporates under the room temperature condition, be fixed the Graphene/gold-tyrosinase-shitosan glass-carbon electrode of tyrosinase as working electrode, adopt the platinum electrode conduct to electrode, saturated calomel electrode constitutes the electrochemical enzymatic sensor of three-electrode system as contrast electrode.
4. the preparation method of the electrochemica biological sensor for detection of bisphenol-A according to claim 2, it is characterized in that: in the step (4) Graphene/Au composite is dissolved in the Graphene/Au composite suspending liquid that is mixed with 3mg/ml in the deionized water, ultrasonic standby to being uniformly dispersed; Be the tyrosinase solution for standby that 7.0 PBS buffer preparation becomes 4.5mg/ml with tyrosinase pH; Shitosan is dissolved in the chitosan solution that is mixed with 1.5mg/mL in the 2M acetum, and it is standby to regulate pH to 6.0 with NaOH.
5. an application rights requires the described electrochemica biological sensor of 1-4 to detect the method for bisphenol A concentration, it is characterized in that may further comprise the steps: Graphene/gold-tyrosinase-shitosan mixed liquor is modified naked glass-carbon electrode surface as working electrode, by suction-operated the enzyme loading is fixed to electrode surface, adopt the platinum electrode conduct to electrode, saturated calomel electrode constitutes the electrochemical enzymatic sensor of three-electrode system as contrast electrode, electrochemical sensor is immersed in the testing sample, according to the quantitative relationship of respective electrical flow valuve and bisphenol A concentration, determine the concentration of bisphenol-A in the testing sample.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310138092.5A CN103278541B (en) | 2013-04-19 | 2013-04-19 | Electrochemical biosensor for detecting bisphenol-A, and preparation method and application thereof |
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| CN103472118A (en) * | 2013-09-26 | 2013-12-25 | 南京师范大学 | Electrochemical immunodetection method for detecting estrols |
| CN103487492A (en) * | 2013-09-17 | 2014-01-01 | 常州大学 | Electrochemical sensor for detecting trace amount of bisphenol A, manufacturing method thereof and application |
| CN103630591A (en) * | 2013-11-13 | 2014-03-12 | 江苏省洪泽湖水利工程管理处 | Electrochemical method for determining phenol in environmental water sample based on graphene-chitosan modified electrode |
| CN103760343A (en) * | 2014-01-16 | 2014-04-30 | 山东省城市供排水水质监测中心 | Method for rapidly detecting microcystic toxins in water based on portable blood glucose meter |
| CN103926280A (en) * | 2014-04-29 | 2014-07-16 | 扬州大学 | Preparation method for electrochemical enzyme sensor |
| CN103979531A (en) * | 2014-05-22 | 2014-08-13 | 天津大学 | Application of electrochemical sensor prepared from graphene material in bisphenol A detection |
| CN105203612A (en) * | 2014-06-10 | 2015-12-30 | 中国科学院大连化学物理研究所 | Biosensor based on metal organic skeleton material and application thereof |
| CN108051490A (en) * | 2017-11-08 | 2018-05-18 | 韶关学院 | L-lysine electrochemical sensor based on grapheme material and preparation method thereof |
| CN110646479A (en) * | 2019-06-27 | 2020-01-03 | 吉林化工学院 | Ratio electrochemical sensor for detecting paracetamol |
| CN112924507A (en) * | 2019-12-06 | 2021-06-08 | 中国科学院大连化学物理研究所 | Preparation method and application of electrochemical sensor based on tyrosinase nanocapsule |
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| CN108918613A (en) * | 2018-06-22 | 2018-11-30 | 江苏大学 | Based on gold nanoparticle/graphite alkene/chitosan trace cadmium ion electrochemical sensor, preparation method and its usage |
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| CN103487492B (en) * | 2013-09-17 | 2015-08-12 | 常州大学 | A kind of electrochemical sensor detecting trace amount of bisphenol A and preparation method thereof and application |
| CN103487492A (en) * | 2013-09-17 | 2014-01-01 | 常州大学 | Electrochemical sensor for detecting trace amount of bisphenol A, manufacturing method thereof and application |
| CN103472118A (en) * | 2013-09-26 | 2013-12-25 | 南京师范大学 | Electrochemical immunodetection method for detecting estrols |
| CN103472118B (en) * | 2013-09-26 | 2016-03-30 | 南京师范大学 | A kind of electrochemical immunoassay method detecting female phenol |
| CN103630591A (en) * | 2013-11-13 | 2014-03-12 | 江苏省洪泽湖水利工程管理处 | Electrochemical method for determining phenol in environmental water sample based on graphene-chitosan modified electrode |
| CN103760343A (en) * | 2014-01-16 | 2014-04-30 | 山东省城市供排水水质监测中心 | Method for rapidly detecting microcystic toxins in water based on portable blood glucose meter |
| CN103926280A (en) * | 2014-04-29 | 2014-07-16 | 扬州大学 | Preparation method for electrochemical enzyme sensor |
| CN103979531A (en) * | 2014-05-22 | 2014-08-13 | 天津大学 | Application of electrochemical sensor prepared from graphene material in bisphenol A detection |
| CN105203612A (en) * | 2014-06-10 | 2015-12-30 | 中国科学院大连化学物理研究所 | Biosensor based on metal organic skeleton material and application thereof |
| CN108051490A (en) * | 2017-11-08 | 2018-05-18 | 韶关学院 | L-lysine electrochemical sensor based on grapheme material and preparation method thereof |
| CN110646479A (en) * | 2019-06-27 | 2020-01-03 | 吉林化工学院 | Ratio electrochemical sensor for detecting paracetamol |
| CN112924507A (en) * | 2019-12-06 | 2021-06-08 | 中国科学院大连化学物理研究所 | Preparation method and application of electrochemical sensor based on tyrosinase nanocapsule |
| CN112924507B (en) * | 2019-12-06 | 2022-05-17 | 中国科学院大连化学物理研究所 | Preparation method and application of electrochemical sensor based on tyrosinase nanocapsule |
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