CN101825602A - Alcohol dehydrogenase sensor based on poly-brilliant cresyl blue and carbon nanotube combination electrode - Google Patents
Alcohol dehydrogenase sensor based on poly-brilliant cresyl blue and carbon nanotube combination electrode Download PDFInfo
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- CN101825602A CN101825602A CN200910063973A CN200910063973A CN101825602A CN 101825602 A CN101825602 A CN 101825602A CN 200910063973 A CN200910063973 A CN 200910063973A CN 200910063973 A CN200910063973 A CN 200910063973A CN 101825602 A CN101825602 A CN 101825602A
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Abstract
The invention provides an alcohol dehydrogenase sensor based on a poly-brilliant cresyl blue and carbon nanotube combination electrode, which is expressed as alcohol dehydrogenase/poly-brilliant cresyl blue/single-walled carbon nanotube/galssy carbon electrode (ADH/PBCB/SWCNT/GCE), wherein a poly-brilliant cresyl blue/single-walled carbon nanotube modified electrode is prepared through a one-step constant potential method with the potential controlled to be +0.9V within 60-90 seconds, then a poly-brilliant cresyl blue/carbon nanotube nano-composite modified glassy carbon electrode is prepared by the constant potential method through fixing the alcohol dehydrogenase on an electrode surface by using 0.2-0.4 percent by weight of polysaccharide hydrogel, and the nano-composite modified glassy carbon electrode is used for detecting nicotinamide adenine dinucletide (NADH) with excellent properties of simple and convenient method, wide linearity, fast response, high sensitivity, low detection limit, good repeatability, stability and anti-interference. The dehydrogenase sensor is prepared by fixing the alcohol dehydrogenase on the surface of the poly-brilliant cresyl blue/carbon nanotube nano-composite modified glassy carbon electrode by using the polysaccharide hydrogel and has the maintained enzymatic activity, high stability and good repeatability.
Description
Technical field
The present invention relates to a kind of alcohol dehydrogenase sensor, especially relate to poly-brilliant cresyl blue and carbon nano-tube combination electrode, and the immobilized preparation of alcohol dehydrogenase.
Technical background
Most redox reactions all are to carry out under dehydrogenasa or oxidasic catalysis in the biosome, and wherein dehydrogenasa is particularly important.Dehydrogenasa is the main primitive that constitutes life as the big molecule in the biosome, the many physiology courses such as metabolism in the life entity are finished in participation, in these life processes, dehydrogenasa all will experience electronic transfer process, transforms mutually between its oxidized form and reduced form.Electron transfer process in postgraduate's object can disclose the secret of life process.The electrochemical research of dehydrogenasa is studying a question of paying special attention to of bioelectrochemistry circle and biological educational circles.Its research has important theory and application directs meaning for deeply being familiar with the physiological action and the bioelectronics device such as electron transfer reaction pass through mechanism and development of new biology sensor, novel biological fuel battery of enzyme in life entity.
The electrochemica biological sensor that the amplification that detects based on the height selectivity of bio-identification and electrochemical signals combines, have highly sensitive, selectivity good, be easy to advantages such as microminiaturization and robotization, be widely used in fields such as bioanalysis and environment measurings.As far back as the sixties in 20th century, biocatalytic Activity just has been used to the development of electrochemica biological sensor.But mainly use oxidase as biological sensitive materials, as glucose oxidase, peroxidase etc., by detecting the oxygen that consumes in the oxidase catalyzed biological respinse or produce hydrogen peroxide, content (first generation electrochemica biological sensor) that can the indirect determination substrate; Perhaps use electron mediator, change the path of catalytic reaction, reach the purpose (second generation electrochemical sensor) of measuring substrate, be exactly to adopt the electric current that produces in the electrochemical gaging glucose oxidase course of reaction as most of blood glucose meter in the market, and draw blood glucose value.
The blood glucose meter that adopts glucose oxidase is when measuring blood glucose value, and the influence of oxygen is inevitably, because in electronics oxygen and medium competition when electrode shifts, high concentration oxygen can cause the blood glucose value signal on the low side, low concentration oxygen then can cause the blood glucose value signal higher.Erect image galvanochemistry disturbing factor is the same, and the influence of oxygen is the most obvious when low sugar, and this influence is too obvious usually, so that these test paper can not be used for the mensuration of the blood sample (vein, capillary and arterial blood) in all sources.Adopt the enzyme of glucose dehydrogenase, can avoid this problem as glucose response.The participation different with glucose oxidase, that the glucose dehydrogenase course of reaction is not aerobic, the influence of oxygen reduces greatly, and stability is higher, and test result is more accurate.Therefore, the redox reaction with electrochemical method research dehydrogenasa provides theory and experiment basis for constructing the dehydrogenasa biology sensor, has wide application value and prospect.
Though the participation that the dehydrogenase reaction process is not aerobic needs NAD (P) to participate in the transmission of electronics as prothetic group.The redox behavior of NAD-NADH on electrode is irreversible regrettably, and NAD carries out electrochemical reduction can not obtain NADH, but produces a kind of dipolymer.NADH generates NAD through electrochemical oxidation, but overpotential is bigger, and NADH is in recycling under the current potential of+0.75V on the glass-carbon electrode, and current potential is apparently higher than its normal potential-0.32V.Therefore reducing the oxidation overpotential of NADH, to NADH catalytic oxidation efficiently, is the condition precedent that makes up galvanochemistry dehydrogenasa biology sensor.Dyestuff such as methylenum careuleum, thionine, methylene green and brilliant cresyl blue etc. are fixed on electrode surface, are developed into the bionical interface that NADH is had good catalytic activity, reduce NADH oxidation overpotential and can reach 500-600mV.Fixing method has: absorption method, field electrochemical polymerization and self assembly embrane method etc.The disadvantage at the bionical interface of absorbent-type is unstable; The loss of enzyme is more in the field electrochemical polymerization; The self assembly embrane method expends time in longer.Therefore the fixing means of studying enzyme and mediator is the key that solves sensor stability.
Nano particle has unique chemical character and physical property, as surface effect, micro-dimension effect, quantum effect and macro quanta tunnel effect etc., nanometer technology has improved the detection performance of biology sensor, and has inspired novel biology sensor after introducing field of biosensors.Because had size, transducer, probe or the nanometer micro-system of sub-micron, the chemistry of biology sensor and physical property and its detection sensitivity to biomolecule or cell significantly improve, the reaction time of detecting is also shortened, and can realize high-throughout real-time check and analysis.
Nanometer technology is constructed the dehydrogenasa biology sensor, more existing reports, and the seminar of teaching as Sweden Gorton has carried out systematic research.Some seminars that China Chinese Academy of Sciences should change in Changchun institute, Nanjing University, Nanjing Normal University have also carried out effective exploration.Its method mainly is to utilize carbon nano-tube or gold nano colloidal sol modified electrode, as the carrier of appendix enzyme or mediator, with absorption method and investment fixed mediums body and enzyme, constructs dehydrogenase sensor.These researchs provide new thinking and method for constructing the dehydrogenasa biology sensor.Construct the dehydrogenasa electrochemical sensor, need the problem of solution also a lot, as select suitable nano material and suitable nanometer technology to construct the nano bionic interface, the effective cycling and reutilization of NADH, select suitable mediator, select suitable immobilization material and method with activity of keeping enzyme or the like.
Utilize poly-brilliant cresyl blue catalytic oxidation NADH, some reports are arranged, but adopt cyclic voltammetry mostly at the poly-brilliant cresyl blue of glass-carbon electrode surface preparation.Prepare poly-brilliant cresyl blue/carbon nano tube modified electrode and construct alcohol dehydrogenase sensor and yet there are no report.
Summary of the invention
The present invention relates to a kind of alcohol dehydrogenase sensor, especially relate to poly-brilliant cresyl blue and carbon nano-tube combination electrode, preparation methods such as immobilization alcohol dehydrogenase.
The alcohol dehydrogenase electrochemical sensor is characterized in that being expressed as alcohol dehydrogenase/poly-brilliant cresyl blue/Single Walled Carbon Nanotube/glass-carbon electrode (ADH/PBCB/SWCNT/GCE)..
The preparation process of alcohol dehydrogenase electrochemical sensor is as follows:
A) commercially available Single Walled Carbon Nanotube is dispersed into uniform black colloidal sol through 40 ℃ of nitration mixture constant temperature are ultrasonic, filters purifying, clean, vacuum drying makes the Single Walled Carbon Nanotube aqueous solution.Drip and to be coated with 2 μ L～10 μ L Single Walled Carbon Nanotube aqueous solution to clean glass-carbon electrode surface, drying at room temperature makes the Single Walled Carbon Nanotube modified electrode.
B) this electrode is put into brilliant cresyl blue solution, adopt three-electrode system, potassium nitrate is supporting electrolyte, and phosphate buffer solution is regulated the pH value, and electrode is taken out in the CONTROLLED POTENTIAL polarization, cleans, and makes poly-brilliant cresyl blue/Single Walled Carbon Nanotube modified electrode.
C) get the polysaccharide hydrogel solution, add a little organic molecule, add alcohol dehydrogenase again, make enzyme colloidal sol.Get 10 μ L enzyme colloidal sols and drip and be applied to brilliant cresyl blue/Single Walled Carbon Nanotube modified electrode surface, 4 ℃ dry down, makes alcohol dehydrogenase sensor.
In the step (2), described pH value of solution value is 8.0, and CONTROLLED POTENTIAL is+0.9V that the polarization time is 60s～90s.
In the step (3), the polysaccharide hydrogel solution uses a kind of in carragheen, agarose, sodium alginate, the methylcellulose etc., and concentration is 0.2%～0.8% (W/W).
In the step (3), it is dimethyl sulfoxide that micromolecule is arranged, dimethyl formamide, and a kind of in the glycerine etc., addition is the polysaccharide hydrogel: organic not molecule=3: 1 (V/V).
Beneficial effect of the present invention is embodied in: prepared poly-brilliant cresyl blue/carbon nanotube nanocomposite modified glassy carbon electrode with potentiostatic method, that the method has is easy, characteristics fast and efficiently.In electrode surface, the polysaccharide hydrogel has stronger elasticity, high stability and good bioaffinity with the polysaccharide aquogel fixed enzyme, is the desirable XC polymer of immobilized enzyme; The polysaccharide hydrogel is very stable in various organic solvents; The polysaccharide hydrogel has certain aperture, and substrate molecule can freely diffuse in the Ago-Gel, with the effect of immobilized protein enzyme; The polysaccharide hydrogel is reticulate texture, wherein forms many water cavities, for enzyme provides suitable little water environment; The original conformation and the biologically active that keep enzyme.When preparation polysaccharide hydrogel, notice that the concentration of polysaccharide is suitable, the control heating-up temperature.In hydrogel, add organic molecule, quickened the gelation process of polysaccharide hydrogel, form all even stable hydrogel, protein can be securely fixed in electrode surface, increase the serviceable life of enzyme sensor.Poly-brilliant cresyl blue/Single Walled Carbon Nanotube the modified electrode that makes is used to detect nicotinamide adenine dinucleotide, it is easy to have method, and linear wide (3.0～104.2 μ M), response fast (＜5s), highly sensitive (9.884nA/ μ M), low (the 1.0 μ M of detection limit, S/N=3), favorable reproducibility, excellent properties such as stable and anti-interference.
The alcohol dehydrogenase sensor that makes is used for the detection of ethanol, in concentration of alcohol scope 0.4～2.4mM, linear relationship is arranged, and detects and is limited to 0.1mM (S/N=3), has the high and good reappearance of high activity (Michaelis constant is 2.3mM), stability of enzyme.
Description of drawings
The invention will be further described below in conjunction with drawings and Examples.
Accompanying drawing is a manufacture craft schematic flow sheet of the present invention.
Among the figure: GC: glass-carbon electrode, SWNT: Single Walled Carbon Nanotube, BCB: brilliant cresyl blue, PBCB: poly-brilliant cresyl blue.
Embodiment
Be expressed as alcohol dehydrogenase/poly-brilliant cresyl blue/Single Walled Carbon Nanotube/glass-carbon electrode (ADH/PBCB/SWCNT/GCE), promptly with the poly-brilliant cresyl blue of constant potential single stage method preparation/Single Walled Carbon Nanotube modified electrode, control of Electric potentials is at+0.9V, time 60s～90s uses 0.2%～0.4% (W%) polysaccharide hydrogel at the fixing alcohol dehydrogenase of electrode surface again.Its step is as follows: (1) is dispersed into uniform black colloidal sol with commercially available Single Walled Carbon Nanotube through 40 ℃ of nitration mixture constant temperature are ultrasonic, filter purifying, clean, vacuum drying, make the Single Walled Carbon Nanotube aqueous solution, drip and to be coated with 2 μ L～10 μ L Single Walled Carbon Nanotube aqueous solution to clean glass-carbon electrode surface, drying at room temperature makes the Single Walled Carbon Nanotube modified electrode; (2) this electrode is put into brilliant cresyl blue solution, adopt three-electrode system, potassium nitrate is supporting electrolyte, and phosphate buffer solution is regulated the pH value, and electrode is taken out in the CONTROLLED POTENTIAL polarization, cleans, and makes poly-brilliant cresyl blue/Single Walled Carbon Nanotube modified electrode; (3) get the polysaccharide hydrogel solution, add a little organic molecule, add alcohol dehydrogenase again, make enzyme colloidal sol.Get 10 μ L enzyme colloidal sols and drip and be applied to brilliant cresyl blue/Single Walled Carbon Nanotube modified electrode surface, 4 ℃ dry down, makes alcohol dehydrogenase sensor.In the step (2), described pH value of solution value is 8.0, and CONTROLLED POTENTIAL is+0.9V that the polarization time is 60s～90s.In the step (3), the polysaccharide hydrogel solution uses a kind of in carragheen, agarose, sodium alginate, the methylcellulose etc., and concentration is 0.2%～0.8% (W/W).In the step (3), it is dimethyl sulfoxide that micromolecule is arranged, dimethyl formamide, and a kind of in the glycerine etc., addition is the polysaccharide hydrogel: organic not molecule=3: 1 (V/V).
(1) preparation of Single Walled Carbon Nanotube modified glassy carbon electrode
Glass-carbon electrode is polished into minute surface with abrasive paper for metallograph (granularity 10000 orders), with 1.0,0.3,0.05 micrometer alumina particle it is polished respectively again, place water, absolute ethyl alcohol, water thoroughly ultrasonic clean then respectively.Clean glass-carbon electrode is put into 0.5M H
2SO
4In the solution ,-1.0～+ activation of 1.0V (reference SCE) sweep limit cyclic voltammetry, scanning is till reaching stable cyclic voltammogram.Take out electrode, after washing was clean, nitrogen dries up, and drips to be coated with 3 μ LSWNT aqueous solution in the surface, and was standby behind the room temperature slow curing.
(2) preparation of poly-brilliant cresyl blue-Single Walled Carbon Nanotube modified glassy carbon electrode (PBCB/SWNT/GCE)
The Single Walled Carbon Nanotube modified glassy carbon electrode is soaked in contains 0.5mM BCB and 0.1M KNO
3, control pH value of solution=8.CONTROLLED POTENTIAL+0.9V polarized 60～90 seconds, cleaning electrode,, remove surface residue, dry, 4 ℃ of refrigerators are deposited standby.
(3) preparation of alcohol dehydrogenase electrochemical sensor (ADH/PBCB/SWCNT/GCE).
(V: V) mixed, the adding alcohol dehydrogenase got 30mg/mL enzyme colloidal sol in 3: 1 for 0.4% (W/W) carragheen aqueous solution and dimethyl sulfoxide.Getting 10 μ L enzyme colloidal sols drips and is applied to PBCB/SWNT/GCE, 4 ℃ of following 4 hours slow curings.The gained alcohol dehydrogenase sensor can be stored in 4 ℃ of PBS.
(4) will gather brilliant cresyl blue-Single Walled Carbon Nanotube modified glassy carbon electrode and place 5mL pH=7.5PBS solution, control electrode current potential 0V continues interval 45s and adds 5 μ L 3.0mM NADH, detects NADH with electric current-time standard measure.The range of linearity is that 3.0～104.2 μ M, response time＜5s, detection sensitivity are 9.884nA/ μ M, detect and be limited to 1.0 μ M (S/N=3).
(5) the alcohol dehydrogenase electrochemical sensor is placed contain 3.0mM NAD
+5mL pH=7.5PBS in, CONTROLLED POTENTIAL 0V continues at interval that 45s adds 5 μ L0.4M ethanol, detects ethanol with electric current-time standard measure.The concentration of alcohol range of linearity is 0.4～2.4mM, detects to be limited to 0.1mM (S/N=3), Michaelis constant K
App MBe 2.3mM, relative standard deviation＜3.9%.
Claims (5)
1. alcohol dehydrogenase electrochemical sensor, it is characterized in that: be expressed as alcohol dehydrogenase/poly-brilliant cresyl blue/Single Walled Carbon Nanotube/glass-carbon electrode (ADH/PBCB/SWCNT/GCE), promptly with the poly-brilliant cresyl blue of constant potential single stage method preparation/Single Walled Carbon Nanotube modified electrode, control of Electric potentials is at+0.9V, time 60s～90s uses 0.2%～0.4% (W%) polysaccharide hydrogel at the fixing alcohol dehydrogenase of electrode surface again.
2. the preparation method of alcohol dehydrogenase electrochemical sensor as claimed in claim 1 is characterized in that: its step is as follows:
(1) commercially available Single Walled Carbon Nanotube is dispersed into uniform black colloidal sol through 40 ℃ of nitration mixture constant temperature are ultrasonic, filter purifying, clean, vacuum drying, make the Single Walled Carbon Nanotube aqueous solution, drip and to be coated with 2 μ L～10 μ L Single Walled Carbon Nanotube aqueous solution to clean glass-carbon electrode surface, drying at room temperature makes the Single Walled Carbon Nanotube modified electrode;
(2) this electrode is put into brilliant cresyl blue solution, adopt three-electrode system, potassium nitrate is supporting electrolyte, and phosphate buffer solution is regulated the pH value, and electrode is taken out in the CONTROLLED POTENTIAL polarization, cleans, and makes poly-brilliant cresyl blue/Single Walled Carbon Nanotube modified electrode;
(3) get the polysaccharide hydrogel solution, add a little organic molecule, add alcohol dehydrogenase again, make enzyme colloidal sol.Get 10 μ L enzyme colloidal sols and drip and be applied to brilliant cresyl blue/Single Walled Carbon Nanotube modified electrode surface, 4 ℃ dry down, makes alcohol dehydrogenase sensor.
3. the preparation method of alcohol dehydrogenase electrochemical sensor as claimed in claim 2 is characterized in that: in the step (2), described pH value of solution value is 8.0, and CONTROLLED POTENTIAL is+0.9V that the polarization time is 60s～90s.
4. the preparation method of alcohol dehydrogenase electrochemical sensor as claimed in claim 2, it is characterized in that: in the step (3), the polysaccharide hydrogel solution uses a kind of in carragheen, agarose, sodium alginate, the methylcellulose etc., and concentration is 0.2%～0.8% (W/W).
5. the preparation method of alcohol dehydrogenase electrochemical sensor as claimed in claim 2, it is characterized in that: in the step (3), it is dimethyl sulfoxide that micromolecule is arranged, dimethyl formamide, a kind of in the glycerine etc., addition is the polysaccharide hydrogel: organic not molecule=3: 1 (V/V).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106415246A (en) * | 2013-09-16 | 2017-02-15 | 麻省理工学院 | Near infrared fluorescent single walled carbon nanotubes as tissue localizable biosensors |
| CN111896600A (en) * | 2020-02-07 | 2020-11-06 | 山东省科学院生物研究所 | Glucose dehydrogenase electrode and preparation method and application thereof |
-
2009
- 2009-09-11 CN CN200910063973A patent/CN101825602A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106415246A (en) * | 2013-09-16 | 2017-02-15 | 麻省理工学院 | Near infrared fluorescent single walled carbon nanotubes as tissue localizable biosensors |
| CN111896600A (en) * | 2020-02-07 | 2020-11-06 | 山东省科学院生物研究所 | Glucose dehydrogenase electrode and preparation method and application thereof |
| CN111896601A (en) * | 2020-02-07 | 2020-11-06 | 山东省科学院生物研究所 | Lactate dehydrogenase electrode and preparation method and application thereof |
| WO2021155735A1 (en) * | 2020-02-07 | 2021-08-12 | 山东省科学院生物研究所 | Lactic dehydrogenase electrode, and preparation method therefor and use thereof |
| WO2021155734A1 (en) * | 2020-02-07 | 2021-08-12 | 山东省科学院生物研究所 | Malate dehydrogenase electrode, and preparation method therefor and use thereof |
| CN111896600B (en) * | 2020-02-07 | 2022-09-02 | 山东省科学院生物研究所 | Glucose dehydrogenase electrode and preparation method and application thereof |
| CN111896601B (en) * | 2020-02-07 | 2022-09-02 | 山东省科学院生物研究所 | Lactate dehydrogenase electrode and preparation method and application thereof |
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Application publication date: 20100908 |