JP2006512574A - Enzyme electrode preparation method - Google Patents

Abstract

The present invention relates to a method for preparing an enzyme electrode by coating cholesterol oxidase (ChOx) immobilized on a silicate sol-gel by microencapsulation.

Description

  The present invention relates to a method for preparing an enzyme electrode. The present invention also mainly provides a method for preparing an enzyme electrode by microencapsulating immobilized cholesterol oxidase (ChOx) and a mediator and coating the silicate sol gel.

BACKGROUND OF THE INVENTION Cholesterol and its fatty acid esters are important compounds for humans because they are components of nerves and brain cells and are precursors of other biological substances such as bile acids and steroid hormones (PY Yeagle, cholesterol) CRC Press: Its function and metabolism in biology and medicine: Plenum: New York 1972). Measurement of cholesterol in the blood is clinically important in the diagnosis of heart disease. This is because accumulation of cholesterol and fatty acid esters in blood due to excessive eating and drinking can be fatal (D. Noble, Anal. Chem., 1993, 65, 1037A-41A). The normal range of serum values is 3 to 6 mM in total cholesterol, but this level rises to 10 mM in the case of hyperlipidemia. Therefore, it is desirable to develop a convenient and rapid method for measuring cholesterol.

  There are various methods for preparing enzyme electrodes by stabilizing and fixing the enzyme in the carbon paste, covalently bonding to the surface of the glassy carbon electrode, or fixing in the polymer film. Have been used by those skilled in the art. In recent years, retaining an enzyme activity in a sol-gel matrix and immobilizing the enzyme has become an effective means for developing a new biosensor. Avnir et al. Discloses a method for fixing an organic compound to an inorganic support by introducing the organic compound together with a polymerization precursor (J. Phys. Chem. 88 (1984), 5969). Sol-gel processed materials are known for their use in the development of ceramic films for conductive, optical, mechanical and electro-optical applications (Brinker, CJ and Scherer, GW, Sol-Gel Science), Academic Press, New Tork, (1989); Klein, LC, Annu. Rev. Mat. Sci., 23 (1993) 437). Braun et al. Report that alkaline phosphatase retains its activity when immobilized in a sol-gel matrix (Mter. Lett., 10 (1990) 1). There are disclosures in the field of enzyme immobilization, such as glucose oxidase in sol-gel matrices (Yamanaka et al., Chem. Mater. 4 (1992) 495; Shtelzer et al., Biochem. Biotechnol., 19 (1994) 293 Narang et al, Anal. Chem., 66 (1994) 3139).

  Audebert and Sanchez reported the construction of a ferrocene-mediated sol-gel biosensor using a two-step sol-gel preparation method with TMOS and commercial colloidal silica of different particle sizes (Chem. Mater. 5 (1993) 911). . According to this reference, more than 80% of glucose oxidase retains its activity in the gel, and the Faradic response of the electrode is consistent with a theoretical calculation based on this activity. Lev et al discloses the use of composite silica-carbon electrodes obtained with sol-gels, claiming the advantages of both the porosity and rigidity of silica matrix and the conductivity of graphite (Anal. Chem., 66 (1994 1747). In this disclosure, glucose oxidase is first adsorbed on the surface of a carbon powder and then used to prepare a sol-gel film with a glassy carbon electrode. Kurokawa et al. Also reported a similar method in which manufactured glucose oxidase-doped sol-gel composites are made from composite fibers such as cellulose and titanium propoxide (Biotechnol. Bioeng., 42 (1993)). 394; Biotechnology 7 (1993) 5).

  Co-immobilization of cholesterol oxidase and horseradish peroxidase in a sol-gel film is disclosed, for example, in Analytica Chimica Acta Vol. 414, 23 pp. 2000. The disclosed method utilizes the use of physical adsorption, physically trapped sandwich and microencapsulation methods to immobilize cholesterol and horseradish peroxidase with sol-gel films derived from tetra-orthosilicate. Including. Response time for cholesterol estimation is over 100 minutes. A response time of 50 seconds was observed by amperometry in a physically trapped enzyme sandwich sol-gel film. Furthermore, enzyme electrodes have been reported to be stable for only 8 weeks.

  Biosensors used in this field have several problems in terms of stability and short shelf life. Several methods of immobilizing biorecognition elements used by chemical sensing researchers have been reported (RF Tylor, Fundamentals and Applications of Protein Immobilization: Marcel Dicker, New York (1976) Chapter 8, 263-303, And HH Weetal, Immobilized Enzymes: Preparation and Characterization of Antigens, Antibodies and Peptides: Marcel Dicker, New York (1975) Chapter 6, 263-303). Methods reported in the literature can generally be classified into one of the following categories: (1) physical adsorption, (2) covalent attachment, or (3) trap capture, of which the physical adsorption is the simplest This is an immobilization method.

  These immobilization methods have several problems, including problems associated with the large size of biorecognition elements (eg, proteins and enzymes). Physical adsorption results in a range of biorecognition element orientations and apparent biding affinities. Furthermore, physical adsorption generally results in a population of biorecognition elements that are not fully responsive to the target analyte. The immobilized molecular species becomes completely unresponsive to the target analyte. Immobilized molecular species often dissolve / detach from the sensing interface because they are not covalent bonds. Shared schemes generally yield a stable and uniform (interim biorecognition orientation) interface with minimal enzyme leaching. Unfortunately, covalent attachment can involve one or more chemical transformations, which can be time consuming and costly.

  US Pat. No. 6,342,364 provides a sensor that electrochemically measures cholesterol in low density lipoproteins with a single sample pass. The sensor includes an electrode system including at least a working electrode and a counter electrode attached to an electrically insulating base plate; an enzyme layer formed with the electrode system on the base plate; A reagent layer disposed in front of each other. The enzyme layer includes at least an oxidoreductase and an electron mediator. The reagent layer includes a reagent that suppresses the reactivity with oxidoreductase with a lipoprotein other than the low-density lipoprotein, for example, a reagent that adheres to a lipoprotein other than the low-density lipoprotein to form a water-soluble complex. However, the shelf life of this sensor is too low.

  US Pat. No. 6,214,612 discloses a cholesterol sensor comprising an electrode system and a reaction reagent system for quantitative determination of cholesterol. The electrode system includes a measurement electrode such as a carbon electrode and a counter electrode, and the reaction reagent system includes cholesterol dehydrogenase, nicotinamide adenine dinucleotide, and oxidized electron mediator. Electron mediators include ferricyanide, 1,2-naphthoquinone-4-sulfonate, 2,6-dichlorophenol indophenol, dimethylbenzoquinone, 1-methoxy-5-methylphenazinium sulfate, methylene blue, garocyanine, thionine, phenazine, Including methosulfate and Meldola's blue. Diaphorases, cholesterol esterases, and surfactants may also be present. The above electrode system is on an insulating base plate, which has a covering member with a groove that is a sample supply channel, which groove extends from the end of the base plate to the electrode system. A reaction layer comprising a reagent system in dry form and a layer of hydrophilic polymer is provided on the base plate or covering member or on the electrode system and covering member so as to be exposed to the sample supply channel. In operation, the electron mediator is reduced in conjunction with the oxidation of cholesterol by cholesterol dehydrogenase, and the current required to electrochemically reoxidize the electron mediator is directly proportional to the amount of cholesterol present in the sample. However, this sensor has a low shelf life, and both mediators and enzymes can dissolve.

  U.S. Pat.No. 6,071,392 is formed in or near an electrode system having a measuring electrode and a counter electrode formed on an electrically insulating base plate, an electrode coating layer covering the electrode system, and an electrode coating layer Disclosed is a cholesterol sensor that includes a reactive reagent layer, wherein the reactive reagent layer includes at least an enzyme that catalyzes cholesterol oxidation, an enzyme that has a cholesterol ester hydrolyzing activity, and a surfactant, and an electrode coating The layer includes one selected from the group consisting of a water-soluble cellulose derivative and a saccharide, and when the electrode coating layer is dissolved in the sample solution supplied to the sensor, the sample solution is provided with a sufficient viscosity to provide the surface. Can prevent the active agent from entering the electrode system Contained at such concentrations. The sensor of this patent is intended to eliminate the degradation of the sensor response due to electrode degradation due to surfactants that penetrate the electrode system. Although the response time is stated to be low, the shelf life is still not high due to the potential for the enzyme to dissolve.

  US Pat. No. 6,117,289 discloses a cholesterol sensor comprising an electrode system comprising at least a measurement electrode and a counter electrode and disposed on an electrically insulating base plate and a reaction layer formed on or near the electrode system. Disclosure. The reaction layer contains cholesterol esterase that converts cholesterol ester into cholesterol, cholesterol oxidase, and a surfactant. The response time reached 9 minutes. Further, the presence of the surfactant causes electrode degradation.

  Electrochemically polymerized conductive polymers have also received considerable attention over the last 20 years. The remarkable ability of this material to switch between a conductive oxidized (doped) state and an insulating reduced (undoped) state is the basis for many applications. For example, polyconjugated conductive polymers can be deposited directly and easily on the sensor electrode by electrochemical oxidation of the monomer, thickness control by charge deposition, and redox conductivity and sensor It has been proposed for biosensor applications because of its advantageous properties such as the polymer electrolyte properties of the polymers that can be applied.

  Therefore, it is highly desirable to develop a biosensor that can measure cholesterol rapidly by conventional methods.

Objects of the invention The main object of the present invention is to provide a new sol-gel based enzyme electrode which can be used for the determination of cholesterol in aqueous media.

  Another object of the present invention is to provide a method for preparing a new enzyme electrode that allows accurate and rapid quantification of cholesterol in solution.

  Yet another object of the present invention is to provide enzymatically stable, cost-effective and sensitive enzyme electrodes.

  Yet another object of the present invention is to provide an enzyme electrode that allows an accurate measurement of cholesterol within a short period of 30 seconds.

  Yet another object of the present invention is to provide a new sol-gel based enzyme electrode that can be reused at least five times.

SUMMARY OF THE INVENTION Accordingly, the present invention relates to an enzyme electrode that can be used for the determination of cholesterol in an aqueous medium, said electrode comprising:
i. conductive base plate,
ii. a film of a sol-gel derivative deposited thereon,
iii. The sol-gel inducer of step b) is a microencapsulated cholesterol oxidase and an electron mediator,
The enzyme electrode shows no leaching of the encapsulated enzyme and electron mediator, the response time is 30 seconds, it can be reused at least 5 times, and the shelf life is 6 months.

  In another embodiment of the present invention, the conductive base plate is selected from a glass plate coated with indium tin oxide and a non-conductive polymer surface coated with silver.

  In yet another embodiment of the invention, the non-conductive polymer surface is selected from films and sheets.

  In yet another embodiment of the invention, the non-conductive polymer surface is selected from the group consisting of polyacrylamide, polyvinyl chloride, and polyethylene.

  In another embodiment of the invention, the sol material is a silica sol.

  In yet another embodiment of the present invention, the silica sol is selected from tetraethylorthosilicate and tetramethylorthosilicate.

  In another embodiment of the invention, the electron mediator is selected from potassium ferricyanide, ferrocene, and Prussian blue.

  In yet another embodiment of the invention, the enzyme electrode has a sensitivity of 0.4 volts.

In another embodiment of the invention, the cholesterol oxidase used has a strength of 3-5 IU per 1 × 1 cm ² surface area.

  In yet another embodiment of the invention, the enzyme electrode operates at a pH in the range of 6.5 to 7.2.

The present invention also relates to a method of preparing an enzyme electrode that can be used for the determination of cholesterol in an aqueous medium, which method comprises the following steps:
a. preparing a silicate solution by known methods;
b. Slowly add 0.05-0.1 M phosphate buffer containing 3-5 IU cholesterol oxidase and about 0.01 M mediator to the silicate solution of step a) to make enzyme cholesterol oxidase and electron mediator Immobilizing the step;
c. allowing the resulting mixture to stand until the enzyme and mediator are completely encapsulated by observing turbidity;
d. spreading the resulting turbid mixture to a conductive base plate in the usual manner;
e. drying the conductive base plate with the spread mixture for at least one day at a temperature in the range of 25-30 ° C. to obtain the desired enzyme electrode;
including.

  In one embodiment of the invention, the silicate sol used is selected from tetraethylorthosilicate and tetramethylorthosilicate.

  In another embodiment of the invention, the phosphate buffer used has a pH in the range of 6.5 to 7.2.

In yet another embodiment of the invention, the method of preparing the enzyme electrode is a single step method.
Detailed Description of the Invention

  This disclosure essentially relates to the steps of sol preparation and simultaneous addition of mediator to the sol in a buffer solution combined with cholesterol oxidase. The sol and immobilized enzyme mixture is allowed to stand until complete encapsulation of the enzyme is achieved. This stage is determined by observing the occurrence of turbidity in the mixture. If the mixture becomes cloudy, it can be deposited on a substrate and used to prepare the desired electrode. When the spread mixture is dried at a temperature of about 25-30 ° C. for as long as about 24 hours, a thin film with cholesterol sensing properties is obtained.

The preparation of the sol is preferably carried out with pure water and HCl using tetraethyl silicate. However, tetramethyl silicate may be used. The water used for the preparation of the sol is preferably pure water, more preferably deionized water higher than 15 M ohms. The sol may be prepared by any means conventionally known to those skilled in the art. For example, a stock sol-gel solution is prepared by mixing 4.5 ml tetraethylorthosilicate (TEOS), 1.4 ml H ₂ O, and 100 μl 0.1 M HCl in a glass vial. The mixture was stirred constantly until a clean solution was obtained. This solution was used diluted as needed throughout the experiment. Each casting solution was prepared by mixing 0.5 ml stock solution and 0-0.2 ml deionized water.

  The next important step is the preparation of a sol gel containing immobilized cholesterol oxidase enzyme. The special point of this step is the mediator. The enzyme is encapsulated and immobilized simultaneously by gradually adding to the sol together with a buffer containing the enzyme. The enzyme used is cholesterol oxidase with a concentration in the range of 3-5 IU per square centimeter of surface area. The mediator used is preferably potassium ferricyanide. For immobilization of cholesterol oxidase (ChOx), 80 μl of the stock solution is added to 20 μl of 0.01 M potassium ferricyanide solution made with 0.1 M phosphate buffer (pH 7.0) containing 3 U ChOx, and the silica network is added. Simultaneously trap the enzyme and potassium ferricyanide as mediator in the growing hydrolyzed gel that forms. The solution was allowed to stand until it was completely encapsulated within the growing network of enzymes and mediators.

Once the sol-gel containing the immobilized and microencapsulated enzyme is prepared, it can be used at any time to deposit as a film on a conductive substrate. The conductive substrate may be a glass plate coated with a conductive film such as indium tin oxide (ITO), or another substrate such as a polymer film or sheet. These can be used as conductive surfaces for depositing silver films and depositing sol-gel films containing encapsulated enzymes. Prior to casting the film, a glass plate coated with indium tin oxide (ITO) was first treated with HNO ₃ for about 2 hours and then washed three times with Millipore water. Finally, the glass slide was cleaned with n-propanol prior to the film coating procedure. The film may be prepared by any conventional means known to those skilled in the art and is preferably kept in air and dried at a temperature in the range of 25-30 ° C. Thereafter, various thickness films doped with ChOx were cast into ITO glass by a water sol-gel dilution scheme. The film was dried at 25 ° C and stored at 4 ° C.

  A standard cholesterol solution was prepared by dissolving 3 mg of cholesterol in 12.8 ml of prop-2-anol and mixing with 5.85 ml of Triton X-100 surfactant. After homogenization, the volume was increased to 100 ml with 0.1 M phosphate buffer (pH 7.0) and kept constant at 35 ° C. This standard solution was further diluted with water to prepare various cholesterol solutions.

The properties of the enzyme-coated substrate are measured by an amperometric response study using the standard cholesterol solution prepared above. Current measurement methods are well known to those skilled in the art. This method essentially uses a three electrode cell configuration. The electrode used is the working electrode, ie the enzyme electrode of the present invention. Usually, enzyme electrodes were made on ITO-coated sales glass. The second electrode is an Ag / AgCl reference electrode. In actual measurements, a strong cholesterol solution varying between 0.5 and 10 mM in a pH 7.0 phosphate buffer was used with the two electrodes described above. The current due to enzymatically generated H ₂ O ₂ was measured every 100 seconds. Usually response times in seconds are measured against cholesterol concentration. The reaction that produces the current is according to the following scheme.
Cholesterol + O ₂ → Δ-cholesten-3-one + H ₂ O _{2
H 2 O 2 → O 2 +} 2H + + 2e -

  The result of the experiment is shown in FIG. In order to check if there was any detrimental effect on the response of the enzyme electrode when an interfering substance such as glucose or ascorbic acid was added to the cholesterol, the experiment was repeated with the interfering substance mixed in the cholesterol solution. These interfering substances were found to have no effect on the response to the enzyme electrode.

  In order to eliminate the shortcomings of cholesterol measurement disclosed in the prior art, biomolecules were immobilized with sol-gel and shelf life was improved. This is because (i) various enzymes can be encapsulated in a sol-gel matrix into an optically clear glass, (ii) the enzyme is very stable in this matrix, (iii) these enzymes are This is because a characteristic reversible reaction occurs in sol-gel glass, and (iv) spectroscopic changes occurring in sol-gel glass can be easily quantified by optical spectroscopic measurement. The sol-gel method has the advantage that almost no heating is required. Because chemical activity with the substrate disturbs the activity of the molecules, these enzyme molecules are trapped in the covalent network rather than chemically bound to the inorganic matrix. The dried glass fine pore (<10 nm) network does not scatter visible light and allows small molecules to diffuse to the electrode surface. Porous inorganic xerogels, such as tetraethyl ortho silicate (TEOS) derived sol-gel, combine electrochemical rigidity with physical rigidity, negligible swelling in aqueous solution, chemical inertness, and heat resistance. It is a particularly attractive matrix for sensors. These biosensors are in principle sensitive and have a rapid response time, and are free from the problem of harmful effects on enzyme activity.

  Another significant advantage seen when compared to conventional enzyme electrodes is that the elution of enzyme and mediator is zero. The electrodes of the present invention can also be reused with a response time as short as 30 seconds. Also, the shelf life of the electrodes is long, about 6 months at an ambient temperature of 25-30 ° C.

  The inventive step is achieved by immobilizing the enzyme cholesterol oxidase (ChOx) and an electron mediator on a silicate sol-gel by microencapsulation, and the sol-gel film of the microencapsulated enzyme and mediator is made of conductive indium. The purpose is to prepare an enzyme electrode that can be deposited on a tin oxide (ITO) coated glass plate and used to measure cholesterol in solution.

The following examples are given for illustrative purposes and should not be construed to limit the scope of the invention in any way.
Example 1: Measurement of enzyme activity

A solution of 0.05 mM ³ of 6 mM cholesterol dissolved in propan-2-ol and 0.1 M phosphate buffer (pH 7.0) having a volume of 3 cm ³ were mixed and stored in a thermostatic chamber at 35 ° C. An ITO glass plate coated with a sol-gel film immobilizing ChOx was immersed and incubated for 2 minutes. The plate was removed, and the absorbance of the solution was measured by a double beam spectrometer at 240 nm to determine the cholesterol produced by the enzyme reaction. Apparent enzyme activity (Ucm ³ ) was evaluated by the following procedure based on the difference in absorbance before and after incubation of the enzyme-immobilized sol-gel glass plate.

Where A is the difference in absorbance before and after incubation, V is the total volume (3.05 cm3), ε is the millimolar extinction coefficient of cholesterol (12.2), t is the reaction time (minutes), and s is the surface area of the sol-gel film ( cm ³ ). One unit of enzyme activity (Ucm ³ ) is defined as the activity that produces 1 μl mole of cholesterol per minute. The enzyme activity was measured with an enzyme (ChOx / HRP) -fixed sol-gel film. No dissolution of the enzyme (ChOx / HRP) from the appeal fixed sol-gel film was observed.

Example 2
Electrochemical determination of cholesterol containing interfering substances using cholesterol / oxidase immobilized sol-gel ITO (ChOx / sol-gel / ITO) electrodes

Cyclic Voltage Measurement Study When cholesterol comes into contact with an enzyme electrode containing a ChOx-fixed TEOS-derived sol-gel film, the following enzymatic and electrochemical reactions occur.
Cholesterol + O ₂ → Δ-cholesten-3-one + H ₂ O _{2
H 2 O 2 → O 2 +} 2H + + 2e -

The oxidation current of H ₂ O ₂ is recorded as the sensor response of the amperometric biosensor. Since the enzyme is directly immobilized, sensor properties such as time and sensitivity reflect the immobilized enzyme. Cyclic voltage measurement (voltammetry) experiments were performed by attaching an enzyme-immobilized sol-gel film to an ITO glass plate in 0.1 M phosphate buffer (pH 7.0) containing various concentrations (0.5 mM to 10 mM) of cholesterol. Was used as a working electrode, an Ag / AgCl reference electrode and a Pt wire as a counter electrode. The above experiments were performed in the absence and presence of 0.1 mM ascorbic acid and 0.5 mM glucose as interfering substances. Cyclic voltage measurements show an oxidation peak of 750 mV, which continues to increase the anodic current as the cholesterol concentration increases from 0.5 mM to 10 mM. This increase is due to the direct oxidation of H ₂ O _{2 on} the surface of the ITO coated glass plate. However, the oxidation peak at 0.75 V moves anodically 150 mV with increasing anodic current in the presence of 0.1 mM ascorbic acid to 0.9 V Vs Ag / AgCl. The presence of 0.5 mM glucose in cholesterol solution (1 mM) also shows an increase in anodic current, but no appreciable effect on the oxidation potential of H ₂ O ₂ . Thus, the presence of 0.1 mM ascorbic acid and 0.5 mM glucose in cholesterol both significantly affects the observed anodic current.

Example 3: Current measurement response study A three-electrode cell configuration similar to that used in cyclic voltage measurement experiments was also used in amperometric measurements of cholesterol in phosphate buffer (pH 7.0). Working electrode (consisting of cholesterol oxidase ChOx immobilized sol-gel on ITO glass) is polarized with 0.9 V vrsus Ag / AgCl and an amperometric response to cholesterol (0.5-10 mM) is generated enzymatically against H ₂ O ₂ Measured using amperometric calibration. The current was monitored every 100 seconds after dispensing different concentrations of cholesterol solution (2-10 mM) into the cell. A maximum current of 5.0 μA was obtained at 10 mM, above which there was no appreciable change in current. The response time for total cholesterol was found to be 90 sec.

Example 4
Cholesterol is electrochemically quantified using cholesterol oxidase and potassium ferricyanide-fixed sol-gel indium tin oxide (ChOx / Fe ³⁺ / sol-gel / ITO) as electrodes, ascorbic acid (0.1 mM) and glucose (0.5 mM) Investigate the effects of interfering substances such as

Cyclic voltage measurement study Cyclic voltage measurement experiments were performed in the enzyme cholesterol oxidase and potassium ferricyanide fixed sol-gel indium tin oxide (ChOx / Fe ³⁺ ) in 0.1 M phosphate buffer (pH 7.0) containing various concentrations of cholesterol. / Sol-gel / ITO) film as working electrode, Ag / AgCl reference electrode and Pt wire as counter electrode. The following reactions occur:
Cholesterol + ChOx → Cholestenone + ChOx _red
ChOx _red + Fe ³⁺ (ferricyanide) → ChOx + Fe ²⁺ (ferrocyanide)
0.4 V
Fe ²⁺ (ferrocyanide) → Fe ³⁺ (ferricyanide) + e- (at electrode)

  The oxidation current is recorded as the sensor response of the amperometric biosensor. Since the enzyme is directly immobilized, sensor properties such as time and sensitivity reflect the immobilized enzyme. In Example 2, the oxidation peak observed at 0.9 V vs. Ag / AgCl when using an enzyme-immobilized sol-gel film without mediator as the electrode is now cathodically offset by 300 mV to 0.4 V versus Ag. Observed with / AgCl, which increases with increasing cholesterol concentration (2 to 10 mM). The presence of 0.1 mM ascorbic acid and 0.5 mM glucose in the cholesterol solution has no appreciable effect on the oxidation potential.

Example 5: Current measurement response study A three-electrode cell configuration similar to that used in cyclic voltage measurement experiments was also used in amperometric measurements of cholesterol in phosphate buffer (pH 7.0). A working electrode (consisting of a cholesterol oxidase ChOx immobilized sol-gel on ITO glass) was polarized with 0.4 V vrsus Ag / AgCl and the amperometric response to concentrations of cholesterol varying from 2 mM to 10 mM was measured. The current was monitored every 100 seconds after adding different concentrations of cholesterol solution (2 mM to 10 mM) to the cell (FIG. 1). Anode current measured with ChOx / Fe ³⁺ / sol gel / ITO polarized to 0.4 V in 6 mM cholesterol solution (1 mL) results in a steady state in 30 seconds, and this response to cholesterol solution is within 5% Was reproduced. The lower limit of cholesterol detection was found to be 0.5 mM amperometrically.

The main advantages of the present invention are as follows:
1. The enzyme electrode prepared according to the present invention has negligible enzyme dissolution.
2. The prepared enzyme electrode shows a fast response to cholesterol in solution.
3. The prepared enzyme electrode is more stable than before.
4). The prepared enzyme electrode is extremely sensitive to cholesterol.

FIG. 5 shows the response of an enzyme electrode as a function of the concentration of a cholesterol solution.

Claims (9)

A method for preparing an enzyme electrode useful for the quantification of cholesterol in an aqueous medium comprising the following steps:
a. preparing a silicate solution;
b. Slowly add 0.05-0.1 M phosphate buffer containing 3-5 IU cholesterol oxidase and about 0.01 M electron mediator to the silicate solution of step a) to make the enzymes cholesterol oxidase and electron -Immobilizing the mediator;
c. allowing the resulting mixture to stand until the enzyme and mediator are completely encapsulated by observing turbidity;
d. spreading the resulting turbid mixture onto a conductive base plate;
e. drying the conductive base plate with the spread mixture for at least one day at a temperature in the range of 25-30 ° C. to obtain an enzyme electrode;
Including methods.   2. Process according to claim 1, characterized in that the silicate sol used is selected from tetraethylorthosilicate and tetramethylorthosilicate.   The method according to claim 1, characterized in that the phosphate buffer used has a pH in the range of 6.5 to 7.2.   The method of claim 1, wherein the conductive base plate is selected from a glass plate coated with indium tin oxide and a non-conductive polymer surface coated with silver.   The method of claim 1, wherein the non-conductive polymer surface is selected from films and sheets.   6. The method of claim 5, wherein the non-conductive polymer surface is selected from the group consisting of polyacrylamide, polyvinyl chloride, and polyethylene.   2. The method of claim 1, wherein the electron mediator is selected from potassium ferricyanide, ferrocene, and Prussian blue. ^2. The method according to claim 1, wherein the strength of cholesterol oxidase is in the range of 3-5 IU per 1 × 1 cm ^{2 of} sol-gel surface area.   The method of claim 1, wherein the method of preparing the enzyme electrode is a single step method.

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