WO2004058994A1 - Process for preparation of enzyme electrode - Google Patents
Process for preparation of enzyme electrode Download PDFInfo
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- WO2004058994A1 WO2004058994A1 PCT/IB2002/005684 IB0205684W WO2004058994A1 WO 2004058994 A1 WO2004058994 A1 WO 2004058994A1 IB 0205684 W IB0205684 W IB 0205684W WO 2004058994 A1 WO2004058994 A1 WO 2004058994A1
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- WO
- WIPO (PCT)
- Prior art keywords
- cholesterol
- enzyme
- electrode
- sol
- gel
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
Definitions
- the present invention relates to a process for the preparation thereof.
- the present invention primarily also provides a process for the preparation of an enzyme electrode by coating an immobilized cholesterol oxidase (ChOx) and mediator on a silicate sol gel by microencapsulation.
- ChOx cholesterol oxidase
- Cholesterol and its fatty acid esters are important compounds for human beings as they are components of nerve and brain cells and are precursors of other biological materials, such as bile acid and steroid hormones (P. L. Yeagle, Biology of Cholesterol, CRC Press: Its function and metabolism in biology and medicine: Plenum: New York, 1972). Cholesterol determination in blood is clinically important for the diagnosis of heart diseases since accumulation of cholesterol and its fatty acid esters in blood due to excessive ingestion can be fatal (D. Noble, Anal. Chem., 1993, 65, 1037A-41A). The normal range of blood serum values extends from 3to 6mm for total cholesterol while in the hyperlipidamic condition the level can increases to lOmM. It is therefore desired to develop techniques that allow convenient and rapid determination of cholesterol.
- glucose oxidase is first adsorbed on the surface of the carbon powder and then used for the preparation ofthe sol-gel film on a glassy carbon electrode.
- Kurokawa et al report a similar method where fabricated glucose oxidase doped sol-gel composite is made of various composite fibers such as cellulose or titanium propoxide [Biotechnol. Bioeng., 42 (1993) 394; Biotechnology 7 (1993) 5].
- the co-immobilisation of cholesterol oxidase and horse radish peroxidase in a sol gel film is disclosed for example in Analytica Chimica Acta Nol 414, 23 pp, 2000.
- the method of this disclosure comprises physical adsorption, physically entrapped sandwich and the use of microencapsulation technique for the immobilization of cholesterol and horse radish peroxidase on tetra ortho silicate derived sol gel films.
- the response time for cholesterol estimation is more than 100 minutes.
- a response time of 50 seconds was observed amperometrically with a physically entrapped enzyme sandwich sol gel film.
- the enzyme electrode is reported to be stable for a period of 8 weeks only. Biosensors used in the art suffer from several drawbacks in terms of stability and shorter shelf life.
- US Patent 6,342,364 provides a sensor that electrochemically determines cholesterol in low density lipoprotein by only one feed of a sample.
- the sensor has: an electrode system that is mounted on an electrically insulating base plate and includes at least a working electrode and a counter electrode; an enzyme layer formed on the base plate with the electrode system; and a reagent layer that is arranged before the enzyme layer in a sample solution supply path to the electrode system.
- the enzyme layer includes at least an oxidoreductase and an electron mediator.
- the reagent layer includes a reagent that depresses reactivity of cholesterol in lipoproteins other than the low density lipoprotein with the oxidoreductase, for example, a reagent that attaches to lipoproteins other than the low density lipoprotein to form a water-soluble complex.
- a cholesterol sensor for quantitative determination of cholesterol is provided containing an electrode system and a reaction reagent system.
- the electrode system contains a measuring electrode such as a carbon electrode and a counter electrode, and the reaction reagent system contains cholesterol dehydrogenase, nicotinamide adenine dinucleotide and an oxidized electron mediator.
- Electron mediators include ferricyanide, l,2-naphthoquinone-4-sulfonate, 2,6-dichlorophenol indophenol, dimethylbenzoquinone, l-methoxy-5-methylphenazinium sulfate, methylene blue, gallocyanine, thionine, phenazine methosulfate and Meldola's blue.
- Diaphorase, cholesterol esterase and a surfactant may also be present.
- the electrode system is on an insulating base plate, and the base plate has a covering member containing a groove that is a sample supplying channel which extends from an end of the base plate to the electrode system.
- a reaction layer containing the reagent system in dry form and a layer of a hydrophilic polymer is provided on the base plate or the covering member, or on both the electrode system and covering member so as to be exposed to the sample supplying channel.
- the electron mediator is reduced in conjunction with oxidation of cholesterol in a sample by cholesterol dehydrogenase, and an amount of current required to electrochemically re-oxidize the electron mediator is directly proportional to a quantity of cholesterol present in the sample.
- the sensor has a low shelf life and also potentially shows leaching of both the mediator and the enzyme.
- US Patent 6,071,392 discloses a cholesterol sensor which comprises comprises an electrode system having a measuring electrode and a counter electrode formed on an electrically insulating base plate, an electrode coating layer for covering the electrode system and a reaction reagent layer formed on or in the vicinity of the electrode coating layer, wherein the reaction reagent layer comprises at least an enzyme for catalyzing cholesterol oxidation, an enzyme having a cholesterol ester hydrolyzing activity and a surfactant, the electrode coating layer comprises at least one selected from the group consisting of water- soluble cellulose derivatives and saccharides and is contained at such a concentration that imparts sufficient viscosity to a sample solution for enabling it to hinder invasion of said surfactant into said electrode system when said electrode coating layer is dissolved in said sample solution supplied to said sensor.
- US Patent 6,117,289 discloses a cholesterol sensor which comprises an electrode system composed of at least a measuring electrode and a counter electrode and disposed on an electrically insulating base plate and a reaction layer formed on or in the vicinity of the electrode system.
- the reaction layer contains cholesterol esterase for catalyzing the conversion of cholesterol ester into cholesterol, cholesterol oxidase and a surfactant.
- the response time was up to nine minutes. Additionally the presence of a surfactant can result in electrode degradation.
- Electrochemically polymerised conducting polymers have also received considerable attention over the last two decades.
- the remarkable switching capacity of these materials between the conducting oxidised (doped) and the insulating reduced (undoped) state is the basis of many applications.
- polyconjugated conducting polymers have been proposed for biosensing applications because of advantageous characteristics such as direct and easy deposition on the sensor electrode by electrochemical oxidation of monomer, control of thickness by deposition of charge and redox conductivity and polyelectrolyte characteristics ofthe polymer useful for sensor applications. It is therefore highly desirable to develop biosensors that allow conventional and rapid determination of cholesterol. Objects ofthe invention
- the main object ofthe present invention is to provide a novel sol gel based enzyme electrode useful for the estimation of cholesterol in aqueous medium.
- Another object ofthe invention is to provide a process for the preparation of a novel enzyme electrode, which allows an accurate and rapid estimation of cholesterol in solution.
- the present invention relates to an enzyme electrode useful for estimation of cholesterol in aqueous medium, said electrode comprising: i. An electrically conductive base plate, ⁇ . a film of sol gel derived material deposited thereon, iii. said sol gel derived material of step b) being microencapsulated cholesterol oxidase with an electron mediator, said enzyme electrode showing zero leaching ofthe encapsulated enzyme and ofthe electron mediator, a response time of 30 seconds, being reusable at least five times and a shelf life of six months.
- the electrically conductive base plate used is selected from indium tin oxide coated glass plate and a silver coated non-conducting polymer surface.
- the non-conducting polymer surface used is selected from a film and a sheet.
- non-conducting polymer surface used is selected from the group consisting of polyacrylamide, polyvinyl chloride and polyethylene.
- the sol material used is silica sol.
- the silica sol used is selected from tetraethyl orthosilicate and tetramethyl orthosilicate.
- the electron mediator used is selected from potassium ferricyanide, ferrocene and Prussian blue.
- the enzyme electrode has a sensitivity of 0.4 volt.
- the strength of cholesterol oxidase used is in the range of 3-5 IU per lxl cm 2 of surface area.
- the enzyme electrode works at a pH in the range of 6.5 -7.2.
- the present invention also relates to a process for the preparation of enzyme electrode useful for estimation of cholesterol in aqueous medium, which comprises the steps of: a. preparing a silicate solution by known methods, b. immobilizing an enzyme cholesterol oxidase and an electron mediator by slowly adding 0.05-0.1 M phosphate buffer containing 3-5 IU of cholesterol oxidase and about 0.01M of electron mediator on to the above said silicate solution of step a), c. allowing the resultant mixture to stand till the complete encapsulation of enzyme and mediator by observing turbidity, d spreading the resultant turbid mixture on a conductive base plate by conventional methods, e. drying the conductive base plate with the spread mixture for at least one day at a temperature in a range of 25-30°C to obtain the enzyme electrode.
- the phosphate buffer used has a pH in a range of 6.5-7.2.
- Figure 1 shows the response of the enzyme electrode as a function of the concentration of cholesterol solution.
- the present disclosure essentially involves the stages of preparation of sol and simultaneous addition of mediator in buffer solution added to sol along with the cholesterol oxidase enzyme.
- the mixture of sol and immobilized enzyme is allowed to stand till the complete encapsulation of the enzyme is achieved. This stage is judged by observing the onset of turbidity ofthe mixture. Once the mixture turns turbid, it is usable for deposition on a substrate to prepare the desired electrode.
- the spread mixture when allowed to dry for long time of about 24 hours at a temperature of about 25- 30°C, results in a thin film which has the cholesterol sensing property.
- the preparation of sol is accomplished by using preferably tetraethyl silicate in pure water and HC1.
- tetramethyl silicate may also be used.
- the water used in the preparation ofthe sol is preferably pure water and more preferably a deionized water of more than 15Mohms.
- the preparation of sol maybe accomplished by any conventional known means known to a person skilled in the art.
- a stock sol-gel solution is prepared by mixing 4.5 ml of tetra ethyl orthosilicate (TEOS), 1.4 ml of H 2 O and lOOul of 0.1 M HC1 in a glass vial. The mixture was stirred regularly until a clean solution was obtained. This solution was used throughout the experiment and was diluted as required. Specific casting solution was prepared by mixing 0.5 ml of the stock solution with 0-0.2 ml of deionized water.
- TEOS tetra ethyl orthosilicate
- the next critical step involves preparation of the sol gel containing the immobilized cholesterol oxidase enzyme.
- the speciality in this is the simultaneous encapsulation and immobilization ofthe enzyme while the mediator is being added gradually to the sol with the buffer containing the enzyme.
- the enzyme used is cholesterol oxidase of a concentration in a range of 3-5 IU per square cm of surface area.
- the mediator used is preferably potassium ferricyanide.
- ChOx cholesterol oxidase
- 20 ⁇ l of 0.01M potassium ferricyanide solution made in 0.1 M phosphate buffer (pH 7.0) containing 3U of ChOx for simultaneous entrapment of the enzyme and potassium ferricyanide as a mediator in the growing hydro lyzed gel forming silica network.
- the solution was kept aside until the enzyme and mediator was encapsulating completely with in the growing network.
- the conducting substrate may be a glass plate coated with a conducting film like Indium Tin Oxide (ITO) or may also be any other substrate like a polymer film or a sheet. These may have a deposited silver film for use as a conducting surface for the deposition of film ofthe sol gel containing encapsulated enzyme.
- ITO Indium Tin Oxide
- Prior to film casting indium tin oxide (ITO) coated glass plates were first treated with HNO 3 , for about 2 hrs and were subsequently rinsed thrice with MiUipore water. The glass slides were finally washed with n-propanol prior to film coating technique.
- the film may be prepared by any conventional means known to a person skilled in the art and is preferably kept in air for drying at a temperature in a range of 25-30°C. Films of varying thickness doped with ChOx were then cast onto the ITO glass using the water sol — gel dilution scheme. The film was dried at 25°C and was stored at 4°C.
- a standard cholesterol solution was prepared by dissolving 3mg of Cholesterol in 12.8 ml of propan-2-ol and was mixed with 5.85 ml of Triton X -100 surfactant. After homogenization the volume was made up to 100ml with 0.1M phosphate buffer (pH 7.0) and thermostat at 35°C. This standard solution was further diluted with water to make different cholesterol solutions.
- the characteristics ofthe enzyme coated substrate are measured using Amperometric response studies using the standard cholesterol solution prepared above. Amperometic techniques are well known to a person skilled in the art. In this method, essentially a three- electrode cell configuration is used. The electrodes used are the working electrode i.e. the enzyme electrode ofthe present invention. Typically the enzyme electrode was made on an ITO coated glass. The second electrode is the reference electrode of Ag/AgCl. In actual measurement, cholesterol solution of strength varying between 0.5-lOmM in a phosphate buffer of pH of 7.0 was used with the two electrodes as described above. The current due to enzymetrically produced H 2 O 2 was measured every 100 seconds.
- the bio molecules are immobilized in sol-gel and have comparatively enhanced shelf lives.
- a variety of enzymes may be encapsulated in sol-gel matrices giving optical transparent glasses
- the enzymes are remarkable stable in such matrices
- these enzymes undergo characteristics reversible reaction in sol-gel glasses and
- spectroscopic changes occurring in sol-gel glasses can readily be quantified by optical spectroscopy.
- the sol-gel technique is advantageous since little or no heating is required.
- Such enzyme molecules become entrapped in the covalent network rather then being chemically bound to the inorganic matrix as chemical bonding of the substrate may perturb the activity of the molecule.
- Porous inorganic xerogel such as terra ethyl orthosilicate (TEOS) derived sol-gels are particularly attractive matrices for electrochemical biosensors since they combine physical rigidity, negligible swelling in aqueous solution, chemical inertness and thermal stability. These biosensors in principle have sensitivity and rapid response time and are also free from the problem of any detrimental effects on enzyme activity.
- TEOS terra ethyl orthosilicate
- the electrode ofthe invention also has a reduced response time of 30 seconds and is reusable. It is also observed that the shelf life of the electrode is enhanced and is about six months at ambient temperature of 25-30°C.
- the inventive step of the present invention resides in the immobilization of the enzyme cholesterol oxidase (ChOx) and electron mediator in silicate sol-gel by micro- encapsulation technique and depositing the above said microencapsulated enzyme and mediator sol-gel film onto a conducting indium tin oxide (ITO) coated glass plate for the preparation of an enzyme electrode useful for the dete ⁇ nination of cholesterol in solution.
- ChOx cholesterol oxidase
- ITO conducting indium tin oxide
- Example 1 Enzyme activity measurements A solution of 0.05 cm 3 of 6mm cholesterol dissolved in propane-2-ol and volume of 3 cm 3 of 0.1 M phosphate buffer (pH 7.0) were mixed and kept in a thermostat at 35°C. The ChOx immobilized sol-gel film coated ITO glass plate was immersed and incubated for 2 minute, the plate was removed and the absorbance of the solution was measured at 240 run using a double beam spectrometer to determine the cholesterol produced by the enzymatic reaction. The apparent enzyme activity (Ucm 3 ) was evaluated by the following procedure based on the difference in absorbance before and after incubation ofthe enzyme immobilized sol-gel glass plate.
- ⁇ (Ucm -2 ) AN/ ⁇ ts app
- A is a deference in absorbance before and after incubation
- V is the total volume (3.05cm3)
- ⁇ is the millimolar extinction coefficient of cholesterol (12.2)
- t is the reaction time (min)
- s is the surface area (cm 3 ) of sol-gel film.
- One unit of enzyme activity (U cm 3 ) is defined as the activity that results in the production of 1 ul mol of cholesterol per minute.
- the enzyme activity measurements were made on the enzyme (ChOx/HRP) immobilized sol-gel film. No enzyme (ChOx/HRP) leaching was observed from the enzyme immobilized sol-gel film.
- the oxidation current for H 2 O 2 is recorded as the sensor response in the amperometric biosensor.
- the sensor properties such as time and sensitivity are the reflection of the immobilized enzyme .
- the cyclic voltammetry experiments were carried out in 0.1 M phosphate buffer (pH 7.0) containing different concentration of cholesterol ( 0.5mM to lOmM) using enzyme immobilized sol-gel film cast on ITO glass plate as a working electrode a Ag/AgCl reference electrode and a Pt wire as a counter electrode. The above experiment was conducted in the absence and in the presence of 0.1 niM ascorbic acid and 0.5 mM glucose as interfering reagents.
- the cyclic voltametry shows an oxidation peak at 750 mV which keeps on increase in anodic current with an increase in concentration from 0.5 mM to 10 mM cholesterol.
- the rise is attributed to the direct oxidation of H 2 O 2 on the surface ofthe ITO coated glass plate.
- the oxidation peak at 0.75V shifts anodically by 150mN to 0.9 V Vs Ag/AgCl with increase in anodic current in the presence of 0.1 mM ascorbic acid.
- Example 3 Amperometric response studies A three electrode cell configuration similar to the one used in cyclic voltameteric experiment was used for the amperometric determination of cholesterol in phosphate buffer (pH 7.0).
- the working electrode (comprising cholesterol oxidase ChOx immobilized sol-gel at ITO glass) was polarizing at 0.9V versus Ag/AgCl and amperometric response to cholesterol (0.5-lOmM) was measured by using amperometric calibration for enzymeriiatically produced H 2 O 2 .
- the current was monitored every 100 sec after dispensing different concentration of cholesterol solution (2mM-10mM) into the cell. A maximum current of 5.0 uA was obtained for 10-mM cholesterol above which no significant change in current could be observed .
- the response time to total cholesterol was found to be 90 sec.
- a three electrode cell configuration similar to the one used in cyclic voltameteric experiment has been used for the amperometric determination of cholesterol in phosphate buffer (pH 7.0).
- the working electrode (comprising cholesterol oxidase ChOx immobilized sol-gel at ITO glass) was polarized at 0.4V versus Ag/AgCl and amperometric response to cholesterol of concentration varying from 2mM to lOmM was measured. The current was monitored every 100 sec after different concentration of cholesterol solution (2mM to lOmM) into the cell ( Figure 1).
- Enzymatic electrodes prepared by the invention shows negligible enzyme leaching.
- the enzyme electrode prepared shows fast response to cholesterol in solution
- the enzyme electrode prepared is stable for a longer time. 4.
- the enzyme electrode prepared is highly sensitive to cholesterol.
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- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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Abstract
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP02790640A EP1578986A1 (en) | 2002-12-31 | 2002-12-31 | Process for preparation of enzyme electrode |
CA2512282A CA2512282C (en) | 2002-12-31 | 2002-12-31 | Process for preparation of enzyme electrode |
PCT/IB2002/005684 WO2004058994A1 (en) | 2002-12-31 | 2002-12-31 | Process for preparation of enzyme electrode |
CNB028301870A CN100342029C (en) | 2002-12-31 | 2002-12-31 | Process for preparation of enzyme electrode |
JP2004563412A JP2006512574A (en) | 2002-12-31 | 2002-12-31 | Enzyme electrode preparation method |
AU2002368515A AU2002368515A1 (en) | 2002-12-31 | 2002-12-31 | Process for preparation of enzyme electrode |
Applications Claiming Priority (1)
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PCT/IB2002/005684 WO2004058994A1 (en) | 2002-12-31 | 2002-12-31 | Process for preparation of enzyme electrode |
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WO2004058994A1 true WO2004058994A1 (en) | 2004-07-15 |
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PCT/IB2002/005684 WO2004058994A1 (en) | 2002-12-31 | 2002-12-31 | Process for preparation of enzyme electrode |
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EP (1) | EP1578986A1 (en) |
JP (1) | JP2006512574A (en) |
CN (1) | CN100342029C (en) |
AU (1) | AU2002368515A1 (en) |
CA (1) | CA2512282C (en) |
WO (1) | WO2004058994A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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KR101490186B1 (en) | 2013-05-08 | 2015-02-05 | 건국대학교 산학협력단 | Electrochemical sensor with conducting polymer modified electrodes for cholesterol detection and manufacturing method the same |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
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US7816025B2 (en) * | 2006-08-23 | 2010-10-19 | Canon Kabushiki Kaisha | Enzyme electrode, enzyme electrode producing method, sensor and fuel cell each using enzyme electrode |
CN105675677B (en) * | 2016-01-26 | 2018-03-20 | 南京工业大学 | A kind of preparation method of the Cholesterol Biosensor based on spiro material |
CN111624244B (en) * | 2019-02-28 | 2021-12-21 | 中国科学院大连化学物理研究所 | Glucose oxidase nano capsule sensor and preparation and application thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999019507A1 (en) * | 1997-10-16 | 1999-04-22 | Abbott Laboratories | Biosensor electrode mediators for regeneration of cofactors |
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2002
- 2002-12-31 WO PCT/IB2002/005684 patent/WO2004058994A1/en active Application Filing
- 2002-12-31 CN CNB028301870A patent/CN100342029C/en not_active Expired - Fee Related
- 2002-12-31 CA CA2512282A patent/CA2512282C/en not_active Expired - Fee Related
- 2002-12-31 JP JP2004563412A patent/JP2006512574A/en active Pending
- 2002-12-31 AU AU2002368515A patent/AU2002368515A1/en not_active Abandoned
- 2002-12-31 EP EP02790640A patent/EP1578986A1/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999019507A1 (en) * | 1997-10-16 | 1999-04-22 | Abbott Laboratories | Biosensor electrode mediators for regeneration of cofactors |
Non-Patent Citations (6)
Title |
---|
CHAUBEY ASHA ET AL: "Mediated biosensors.", BIOSENSORS & BIOELECTRONICS, vol. 17, no. 6-7, June 2002 (2002-06-01), June, 2002, pages 441 - 456, XP002251006, ISSN: 0956-5663 * |
KUMAR ARUN ET AL: "Co-immobilization of cholesterol oxidase and horseradish peroxidase in a sol-gel film.", ANALYTICA CHIMICA ACTA, vol. 414, no. 1-2, 1 June 2000 (2000-06-01), pages 43 - 50, XP002251005, ISSN: 0003-2670 * |
LIU ZHENJIU ET AL: "A new tyrosinase biosensor based on tailoring the porosity of Al2O3 sol-gel to co-immobilize tyrosinase and the mediator.", ANALYTICA CHIMICA ACTA, vol. 407, no. 1-2, 29 February 2000 (2000-02-29), pages 87 - 96, XP002251007, ISSN: 0003-2670 * |
P. AUDEBERT ET AL.: "Electrochemical Probing of the Activity of Glucose oxidase embedded Sol-gel matrices", CHEM. MATER., vol. 5, 1993, pages 911 - 913, XP009015713 * |
TAN S N ET AL: "Mediated amperometric glucose sensor modified by the sol-gel method", SENSORS AND ACTUATORS B, ELSEVIER SEQUOIA S.A., LAUSANNE, CH, vol. 40, no. 2-3, 15 May 1997 (1997-05-15), pages 135 - 141, XP004088710, ISSN: 0925-4005 * |
WANG JOSEPH: "Sol-gel materials for electrochemical biosensors.", ANALYTICA CHIMICA ACTA, vol. 399, no. 1-2, 8 November 1999 (1999-11-08), pages 21 - 27, XP002251371, ISSN: 0003-2670 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101490186B1 (en) | 2013-05-08 | 2015-02-05 | 건국대학교 산학협력단 | Electrochemical sensor with conducting polymer modified electrodes for cholesterol detection and manufacturing method the same |
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Publication number | Publication date |
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CN1738906A (en) | 2006-02-22 |
CA2512282C (en) | 2010-04-27 |
CA2512282A1 (en) | 2004-07-15 |
AU2002368515A1 (en) | 2004-07-22 |
EP1578986A1 (en) | 2005-09-28 |
CN100342029C (en) | 2007-10-10 |
JP2006512574A (en) | 2006-04-13 |
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