CN100342029C - Process for preparation of enzyme electrode - Google Patents

Abstract

The present invention relates to a process for coating immobilized cholesterol oxidase (ChOx) and a medium on silicate sol gel through a microcapsule packing process to prepare an enzyme electrode.

Description

Preparation method of enzyme electrode

Technical Field

The present invention relates to a method of preparing an enzyme electrode. The present invention generally provides methods for preparing enzyme electrodes by microencapsulating cholesterol oxidase (ChOx) immobilized on silicate sol and a mediator.

Background

Cholesterol and Its fatty acid esters are compounds of great importance for humans, since they are not only constituents of nerve and brain cells, but also other biological substances such as bile acids and precursors of steroid hormones (P.L. Yeast, Biology of Cholesterol, CRC Press: Its function and metabolism: Plenum: New York, 1972). The determination of cholesterol in blood is important for the clinical diagnosis of heart disease because the accumulation of cholesterol and its fatty acid esters in blood caused by excessive ingestion is fatal enough (d.noble, anal.chem., 1993, 65, 1037A-41A). The normal range of total cholesterol values in plasma is 3-6mM, and in the case of hyperlipidemia this level may increase to 10 mM. Therefore, there is a need to develop a technique that can measure cholesterol easily and rapidly.

To prepare enzyme electrodes, various methods have been used in the art to stabilize and immobilize enzymes in carbon pastes, or to covalently attach enzymes to glassy carbon electrode surfaces, or to immobilize them in polymer membranes. In recent years, methods of immobilizing enzymes in sol gel matrices and maintaining their activity have become potential tools for the development of new biosensors. Avinr et al disclose a method of immobilizing an organic compound in an inorganic support by introducing the organic compound with a polymeric precursor [ j.phys.chem., 88(1984), 5969]. Sol Gel treated materials are well known for their use in the development of ceramic membranes for electrical, optical, mechanical and optoelectronic applications [ Brinker, c.j., and Scherer, g.w., Sol-Gel Science, Academic Press, New York, (1989); klein, l.c., annu.rev.mater.sci., 23(1993)437]. Braun et al reported that alkaline phosphatase retained its activity when immobilized in a sol gel matrix [ Mater. Lett., 10(1990)1]. Methods for immobilizing enzymes, including glucose oxidase, in sol gel matrices have been disclosed in the art [ 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 preparation of ferrocene-mediated sol-gel biosensors using a two-stage sol-gel preparation method based on TMOS and commercial silica gels of various particle sizes [ chem. Mater.5(1993)911]. According to this document, more than 80% of the glucose oxidase remains active in the gel and the results of the faradaic reaction of the electrode are in agreement with theoretical calculations based on this activity. Lev et al disclose the use of sol-gel fabricated composite silicon-carbon electrodes and claim to have the dual advantages of silicon matrix porosity and hardness, as well as graphite conductivity [ anal. chem., 66(1994)1747]. In this disclosure, glucose oxidase is first adsorbed onto the surface of carbon powder and then used to prepare a sol gel film on a glassy carbon electrode. A similar approach is reported by Kurokawa et al, in which glucose oxidase doped sol-gel composites are made from various composite fibers such as cellulose or titanium propoxide [ biotechnol.bioeng., 42(1993) 394; biotechnology 7(1993)5].

Co-immobilization of cholesterol oxidase and horseradish peroxidase in sol gel membranes has been disclosed, for example, in analytical Chimica Acta, vol.41423pp, 2000 by methods including physical adsorption, physical entrapment of sandwich structures and immobilization of cholesterol and horseradish peroxidase on tetra-orthosilicate-derived sol gel membranes using microencapsulation techniques. The response time of the cholesterol assay was over 100 minutes. The response time measured amperometrically using a physically entrapped enzyme sandwich sol gel membrane was 50 seconds. In addition, the enzyme electrode was reported to be stable for only 8 weeks.

The existing biosensor has many defects in stability and shelf life. Various methods of immobilizing biorecognition elements for chemosensing studies have been reported [ r.f. taylor; protein immobilization Fundamentals and Applications: marcel dicer, New York (1975) Chapter 8, 263-303 and H.H.Weetall, Immobilized Enzyme; antibodies, Antibodies and Peptides Preparation and Characterization: marcel dicer, New York (1975) Chapter 6, 263-303]. The methods reported in the literature can generally be classified into one of the following categories: (1) physisorption, (2) covalent attachment or (3) entrapment, with physisorption being the simplest means of immobilization.

These immobilization methods have some disadvantages, such as problems associated with the large size of the biorecognition elements (e.g. proteins and enzymes). Physisorption results in a variety of biological recognition element orientations and apparent binding affinities (biding affinity). Furthermore, physisorption often produces a large number of biorecognition elements that are completely unresponsive to the target analyte. The immobilized species is completely unresponsive to the target analyte. Due to the absence of covalent bonds,the immobilized species often leach or desorb from the inductive interface. Covalent methods generally result in a more stable and uniform (transitional biorecognition orientation) interface with minimal enzyme loss. However, covalent attachment can involve one or more chemical changes, and is generally time consuming and can be costly.

U.S. Pat. No. 6,342,364 provides a sensor that can electrochemically measure cholesterol in low density lipoprotein by applying a sample only once. The inductor comprises: an electrode system including at least a working electrode and a counter electrode mounted on an insulating substrate, an enzyme layer formed on the substrate with the electrode system, and a reagent layer disposed in front of the enzyme layer on a path for supplying a sample solution to the electrode system. The enzyme layer includes at least an oxidoreductase and an electron mediator. The reagent layer contains a reagent which inhibits cholesterol activity in a lipoprotein other than the low density lipoprotein containing the oxidoreductase, for example, a reagent which can bind to a lipoprotein other than the low density lipoprotein to form a water-soluble complex. However, the shelf life of such sensors is too short.

U.S. patent No. 6,214,612 discloses a cholesterol sensor including an electrode system and a reaction reagent system for quantitatively determining cholesterol. The electrode system includes measuring electrodes such as a carbon electrode and a counter electrode, and the reagent system includes cholesterol dehydrogenase, nicotinamide adenine dinucleotide and an oxidized electron mediator. The electron mediator includes ferricyanide, 1, 2-naphthoquinone-4-sulfonate, 2, 6-dichlorophenolinium, dimethylbenzoquinone, 1-methoxy-5-methylphenazinium sulfate (1-methoxy-5-methylphenazinium sulfate), methylene blue, gallocyanine, thionine, N-methylphenazinium (monomethyl sulfate) salt, and Meldola's blue. Can also contain diaphorase, cholesterol esterase and surfactant. The electrode system is located on an insulating substrate having a covering member with a recess extending from the end of the substrate to a sample supply channel of the electrode system. A reaction layer containing the reaction reagent system in a dry form and a hydrophilic polymer layer are provided on the base plate or the cover member, or both the electrode system and the cover member, so as to be exposed to the sample-supplying well. During operation, the electron mediator is reduced while cholesterol in the sample is oxidized by cholesterol dehydrogenase, and the amount of current required to electrochemically reoxidize the electron mediator is proportional to the amount of cholesterol in the sample. However, the sensor has a short shelf life and loss of the medium and enzyme may occur.

U.S. Pat. No. 6,071,392 discloses a cholesterol sensor comprising an electrode system including a measuring electrode and a counter electrode formed on an insulating substrate, an electrode coating for covering the electrode system, and a reaction reagent layer formed on or in the vicinity of the electrode coating, wherein the reaction reagent layer contains at least an enzyme for catalyzing oxidation of cholesterol, an enzyme having cholesterol ester hydrolysis activity, and a surfactant, and the electrode coating contains at least one of a water-soluble cellulose derivative and a saccharide at a concentration which gives a sample solution a sufficient viscosity to prevent the surfactant from invading the electrode system when the electrode coating is dissolved in the sample solution supplied to the sensor. The sensor of this patent is directed to eliminating the decrease in sensor response caused by electrode degradation caused by surfactant intrusion into the electrode system. Although the response time of this sensor is said to be shorter, its shelf life is still not long enough, again because of potential enzyme loss.

U.S. patent No. 6,117,289 discloses a cholesterol sensor comprising an electrode system including at least a measuring electrode and a counter electrode on an electrically insulating substrate, and a reaction layer formed on or near the electrode system. The reaction layer includes cholesterol esterase for catalyzing conversion of cholesterol ester to cholesterol, cholesterol oxidase, and a surfactant. The response time was 9 minutes at the maximum. In addition, the presence of surfactants can lead to degradation of the electrodes.

Electrochemically polymerized conductive polymers have also received considerable attention over the last two decades. The extraordinary ability of these materials to switch from a conductive oxidized state (doped) to an insulating reduced state (undoped) is the basis for many applications. For example, a poly-conjugated conductive polymer is considered for biosensing applications due to advantages such as direct and easy deposition on the sensing electrode by electrochemical oxidation of the monomer, control of thickness by charge deposition, and redox conductivity and polyelectrolyte properties of the polymer for sensor applications.

Therefore, there is an urgent need to develop a biosensor that can conveniently and rapidly measure cholesterol.

Object of the Invention

The main object of the present invention is to provide a novel sol-gel based enzyme electrode for the assessment of cholesterol in aqueous media.

It is another object of the present invention to provide a method for preparing a novel enzyme electrode capable of accurately and rapidly evaluating cholesterol in a solution.

It is a further object of the invention to provide a cost-effective, highly sensitive enzyme electrode that is enzyme stable.

It is still another object of the present invention to provide an enzyme electrode which can accurately measure cholesterol in a short time of 30 seconds.

It is a further object of the present invention to provide a novel sol-gel based enzyme electrode that can be used at least five times.

Summary of The Invention

Thus, the present invention relates to an enzyme electrode for use in the assessment of cholesterol in an aqueous medium, the electrode comprising:

i) a conductive substrate having a plurality of conductive layers,

ii) a sol-gel derived material deposited on the conductive substrate,

iii) the sol-gel derivative of step ii) is cholesterol oxidase microencapsulated with an electronic mediator,

the enzyme electrode has the following properties: the encapsulated enzyme and the electronic mediator have zero run-off, a response time of 30 seconds, at least 5 times of repeated use, and a storage time of 6 months.

In another embodiment of the invention, the conductive substrate used is selected from the group consisting of indium tin oxide coated glass plates and silver coated non-conductive polymer surfaces.

In another embodiment of the present invention, the non-conductive polymer surface used is selected from the group consisting of films and sheets.

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

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

In another embodimentof the present invention, the silica sol used is selected from tetraethyl orthosilicate and tetramethyl orthosilicate.

In another embodiment of the present invention, the electron mediator used is selected from the group consisting of potassium ferricyanide, ferrocene, and prussian blue.

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

In another embodiment of the invention, the concentration of cholesterol oxidase used is in the range of 3-5IU per square centimeter of surface area.

In another embodiment of the present invention, the working pH of the enzyme electrode ranges from 6.5 to 7.2.

The invention also relates to a method of preparing an enzyme electrode for use in assessing cholesterol in an aqueous medium, comprising:

a. the silicate solution is prepared using known methods.

b. Immobilizing the cholesterol oxidase and the electron mediator by slowly adding a 0.05-0.1M phosphate buffer solution containing 3-5IU of the cholesterol oxidase and about 0.01M of the electron mediator to the silicate solution described in step a).

c. The resulting mixture was allowed to stand until turbidity was observed, which indicated complete encapsulation of the enzyme and the electronic medium,

d. the resulting cloudy mixture was coated on a conductive substrate using conventional methods.

e. Drying the conductive substrate with the coated cloudy mixture at a temperature in the range of 25-30 ℃ for a period of at least one day to obtain the enzyme electrode.

In an embodiment of the invention, the silicate sol used is selected from tetraethyl orthosilicate and tetramethyl orthosilicate.

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

In another embodiment of the present invention, the method of preparing the enzyme electrode is a one-step process.

Brief description of the drawings

Figure 1 shows the enzyme electrode response as a function of cholesterol solution concentration.

Detailed description of the invention

The invention mainly comprises the following stages: preparing sol, and adding the medium in the buffer solution and the cholesterol oxidase into the sol at the same time. The mixture of sol and immobilized enzyme is allowed to stand until complete encapsulation of the enzyme is achieved. This stage was judged by observing that the mixture started to become cloudy. Once the mixture begins to become cloudy, it can be deposited on a substrate to prepare the desired electrode. After drying at a temperature in the range of 25-30 ℃ for an extended period of about 24 hours, the applied mixture forms a film having the cholesterol-sensing properties.

The preparation of the sol is preferably carried out using tetraethyl silicate in pure water and HCl. But tetramethylsilicate may also be used. The water used to prepare the sol is preferably pure water, more preferably deionized water in excess of 15 Mohms. The sol can also be prepared by any conventional method known to those skilled in the art. For example, by mixing 4.5ml of tetraethyl orthosilicate (TEOS), 1.4ml of H₂O and 100. mu.l of 0.1M HCl were mixed in a glass vial to prepare a stock sol-gel solution. Mixing the obtained extractsThe mixture was stirred untila clear solution was obtained. This solution will be used throughout the experiment and may be diluted as required. A specific casting solution was prepared by mixing 0.5ml of the stock solution with 0-0.2ml of deionized water.

The next key step involves the preparation of a sol gel containing immobilized cholesterol oxidase. The step is characterized in that the encapsulation and immobilization of the enzyme are simultaneously realized in the process of gradually adding the medium and the buffer solution of the enzyme into the sol. The enzyme used was cholesterol oxidase, the concentration of which ranged from 3-5IU per square centimeter of surface area. The medium used is preferably potassium ferricyanide. For the immobilization of cholesterol oxidase (ChOx), 80 μ l of the stock solution was added to 20 μ l of a 0.01M potassium ferricyanide solution prepared in a 0.1M phosphate buffer solution (pH 7) containing 3U ChOx, thereby simultaneously encapsulating the enzyme, and potassium ferricyanide was used as a mediator in the growing silica network-forming hydrogel. The solution was set aside until the enzyme and medium were completely encapsulated in the growing network

Once the sol gel containing the immobilized and microencapsulated enzyme is prepared, a film can be easily deposited on a conductive substrate. The conductive substrate canIn the form of a conductive film such as Indium Tin Oxide (ITO) coated glass plate, or any other substrate such as a polymer film or sheet. These substrates may have deposited thereon a silver film which serves as a conductive surface for the deposition of the encapsulated enzyme-containing sol-gel film. With HNO prior to film casting₃Indium Tin Oxide (ITO) coated glass plates were treated for approximately 2 hours and then washed three times with Millipore water. Finally, the glass plate was cleaned with n-propanol prior to film casting. The membrane may be prepared by any conventional means known to those skilled in the art and is preferably maintained in air for drying at a temperature in the range of 25-30 ℃. ChOx-doped films of different thicknesses were then cast on the ITO glass using the hydrosol gel dilution protocol. The films were dried at 25 ℃ and stored at 4 ℃.

A standard cholesterol solution was prepared by dissolving 3mg cholesterol in 12.8ml 2-propanol and mixing with 5.85ml Triton X-100 surfactant. After homogenization, the total volume of the mixture was diluted to 100ml with 0.1M phosphate buffer (pH 7.0) and thermostated at 35 ℃. This standard solution was further diluted with water to a different cholesterol solution.

The properties of the enzyme-coated substrate were examined using an Amperometric response study (Amperometric response study) with the standard cholesterol solution prepared above. Amperometry is a well known method to those skilled in the art. In this method, a three-electrode battery system is basically used. The electrode used is the working electrode, i.e. the enzyme electrode of the invention. Usually, the enzyme electrode is coated with ITOIs made of glass. The second electrode was an Ag/AgCl reference electrode. In the actual measurement, a cholesterol solution having a concentration varying between 0.5 and 10mM in a phosphate buffer solution having a pH of 7.0 and the above-mentioned two electrodes were used. Detection of H production by enzyme action every 100 seconds₂O₂Resulting in a current. In general, the response time (seconds) is measured for the concentration of the cholesterol solution.

The reaction causes the current to rise based on the following process

Cholesterol + O₂→ DELTA-cholesten-3-one + H₂O₂

H₂O₂→O₂+2H⁺+2e^-

The results of the experiment are shown in FIG. 1. In order to examine whether an interfering agent such as glucose or ascorbic acid, which has any adverse effect on the response of the enzyme electrode, is present in cholesterol, repeated experiments were carried out with a cholesterol solution mixed with the interfering agent. These interfering agents were found to have no effect on the response of the enzyme electrode.

In an attempt to improve the drawbacks of the cholesterol measurement methods disclosed in the prior art, biomolecules are immobilized in a sol-gel method and the storage time is relatively long. This is due to the fact that: (i) the various enzymes are encapsulated in a sol gel matrix to form a transparent glassy material (ii) the enzymes are very stable in such a matrix (iii) the enzymes undergo property-reversible reactions in the sol gel glassy material (iv) the spectral changes occurring in the sol gel glassy material can be readily quantified spectroscopically. The sol-gel technique is advantageous in that no or only a very small amount of heat is required. These enzyme molecules are embedded in a covalent network and are not chemically attached to an inorganic matrix, since the chemical bonds of the matrix may interfere with the activity of the molecules. The network of pores within the dried glass (<10nm) does not scatter visible radiation but allows small molecules to diffuse to the electrode surface. Porous inorganic xerogels such as Tetraethylorthosilicate (TEOS) derived sol-gels are particularly attractive matrices for electrochemical biosensors because they combine the characteristics of being physically stiff, having negligible expansion in aqueous solution, being chemically inert and thermally stable. These biosensors are high in sensitivity, short in response time, and free from any problem of impairing enzyme activity as a whole.

Another significant advantage observed over the enzyme electrodes ofthe prior art is zero loss of enzyme and mediator. The response time of the electrode is shortened to 30 seconds, and the electrode can be repeatedly used. It was also observed that the storage time of the electrode was lengthened and it could be stored at a normal temperature of 25-30 c for about 6 months.

The inventive step of the present invention consists in immobilizing cholesterol oxidase (ChOx) and an electron mediator in a silicate sol-gel using a microencapsulation technique, and depositing the above microencapsulated enzyme and electron mediator sol-gel film on a conductive Indium Tin Oxide (ITO) -coated glass plate, thereby making an enzyme electrode for measuring cholesterol in a solution.

The following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention in any way.

Example 1: determination of enzyme Activity

0.05cm in length³6mM cholesterol solution was dissolved in 2-propanol solution and mixed with 3cm³A volume of 0.1M phosphate buffer solution (pH 7.0) was mixed and thermostated at 35 ℃. An ITO glass plate coated with ChOx-immobilized sol gel film was immersed in the above solution and incubated for 2 minutes, the glass plate was removed and the absorption of the solution was measured at 240nm using a two-beam spectrometer, thereby measuring cholesterol produced by the enzyme reaction. Apparent enzyme activity (Ucm) was evaluated from the change in light absorption of the solution before and after incubation of the enzyme-immobilized sol-gel glass plate by the following method³)。

${\mathrm{\&Sigma;}}_{\mathrm{app}}^{\mathrm{enz}}\left(U{\mathrm{cm}}^{-2}\right)=\mathrm{AV}/\mathrm{\&epsiv;ts}$

Wherein A is the change in light absorption of the solution before and after incubation and V is the total volume (3.05 cm)³) ε is the millimolar extinction coefficient of cholesterol (12.2), t is the reaction time (min), and s is the surface area of the sol-gel film (cm)²). One enzyme Activity Unit (Ucm)³) Defined as the activity to produce 1. mu.l mol cholesterol per minute. Enzyme activity measurements were performed on sol gel membranes on which the enzyme (ChOx/HRP) was immobilized. No enzyme was observed(ChOx/HRP) was eluted from the enzyme immobilized sol gel membrane.

Example 2

Cholesterol containing interfering reagents was measured electrochemically using a cholesterol oxidase immobilized sol-gel-ITO (ChOx/sol-gel/ITO) electrode.

Cyclic voltammetry

When cholesterol is contacted with an enzyme electrode containing ChOx immobilized in a TEOS-derived sol gel film, the following enzymatic and electrochemical reactions occur:

cholesterol + O₂→ DELTA-cholesten-3-one + H₂O₂

H₂O₂→O₂+2H⁺+2e^-

Record H₂O₂As a sensor response in an amperometric biosensor. Because of the direct immobilization of the enzyme, the properties of the sensor, such as time and sensitivity, are a reflection of the immobilized enzyme. Cyclic voltammetry experiments were performed in 0.1M phosphate buffer (pH 7.0) containing various concentrations of cholesterol (0.5mM to 10mM) using an enzyme-immobilized sol gel film cast on an ITO glass plate as the working electrode, Ag/AgCl as the reference electrode, and a platinum wire as the counter electrode. The above experiments were performed in the presence and absence of 0.1mM ascorbic acid and 0.5mM glucose as interfering reagents. The cyclic voltammetry experiment showed an oxidation peak at 750mV and its anodic current increased with increasing cholesterol concentration from 0.5mM to 10 mM. This rise is due to H₂O₂Directly oxidizing on the ITO coated glass surface. However, in the presence of 0.1mM ascorbic acid, as the anodic current increased, the oxidation peak at 0.75V shifted 150mV to the anode to 0.9V relative to the Ag/AgCl electrode. When 0.5mM glucose was present in the cholesterol solution (1mM), there was also an increase in anodic current but for H₂O₂The oxidation potential of (A) had no significant effect, indicating that the presence of 0.1mM ascorbic acid and 0.5mM glucose in cholesterol had a large effect on the measured anodic current

Example 3 Ampere response study

Cholesterol in phosphate buffered solutions (pH 7.0) was measured amperometrically using a three-electrode cell system similar to that used in cyclic voltammetry experiments. The working electrode (sol gel containing cholesterol oxidase ChOx immobilized on ITO glass) was polarized at 0.9V relative to the Ag/AgCl electrode and H generated by amperometric calibration of the enzyme action₂O₂To measure the amperometric response to cholesterol (0.5-10 mM). After various concentrations of cholesterol solution (2mM-10mM) were prepared in the cell, the current was monitored every 100 seconds. A maximum current of 5.0. mu.A was observed at 10mM cholesterol, and no significant change in current was observed beyond this concentration. The observed response time to total cholesterol was 90 seconds.

Example 4

Sol-gel indium tin oxide immobilized with cholesterol oxidase and potassium ferricyanide(ChOx/Fe³⁺sol-gel/ITO) as electrode, cholesterol was measured electrochemically in the presence of ascorbic acid (0.1mM) and glucose (0.5mM) interfering reagents.

Cyclic voltammetry

Cyclic voltammetry experiments were performed using sol-gel indium tin oxide (ChOx/Fe) immobilized with cholesterol oxidase and potassium ferricyanide in 0.1M phosphate buffer solutions (pH 7.0) containing varying concentrations of cholesterol³⁺sol-gel/ITO) membrane as working electrode, Ag/AgCl as reference electrode, platinum wire as counter electrode. The following reactions occur:

cholesterol + Chox → cholestenone + Chox_red

ChOx_red+Fe³⁺(ferricyanide) → ChOx + Fe²⁺(ferrocyanide)

The oxidation current was recorded as the sensor response in an amperometric biosensor. Because of the direct immobilization of the enzyme, the properties of the sensor, such as time and sensitivity, are a reflection of the immobilized enzyme. When the enzyme-immobilized sol gel membrane without mediator was used as the electrode, the oxidation peak observed at 0.9V versus the Ag/AgCl electrode in example 2 shifted 300mV to the cathode in this experiment to 0.4V versus the Ag/AgCl electrode, and it increased with increasing cholesterol concentration (2-10 mM). The presence of 0.1mM ascorbic acid and 0.5mM glucose in the cholesterol solution had no significant effect on the measured oxidation potential.

Example 5 Ampere response study

Cholesterol in phosphate buffered solutions (pH 7.0) was measured amperometrically using a three-electrode cell system similar to that used in cyclic voltammetry experiments. The working electrode (sol gel containing cholesterol oxidase ChOx immobilized on ITO glass) was polarized at 0.4V versus Ag/AgCl electrode and the amperometric response to cholesterol varying from 2mM to 10mM concentration was measured. The current was monitored every 100 seconds after different concentrations of cholesterol solution (2mM-10mM) were dispensed into the cell (FIG. 1). Chox/Fe polarized at 0.4V in 6mM cholesterol solution (1mL)³⁺The anode current measured by the/sol-gel/ITO electrode reached a steady state within 30 seconds, and the response to the cholesterol solution was reproducible within 5%. Ampere derived cholesterol concentrationsThe lower limit of current detection of the intensity was 0.5 mM.

The invention has the main advantages that:

1. the enzyme electrodes prepared according to the invention show negligible enzyme loss.

2. The enzyme electrode prepared by the invention shows rapid response to cholesterol in a solution.

3. The enzyme electrode prepared by the invention can keep stable for a long time.

4. The enzyme electrode prepared by the invention is highly sensitive to cholesterol.

Claims (7)

1. A method of preparing an enzyme electrode for use in assessing cholesterol in an aqueous medium, comprising the steps of:

a. preparing a silicate solution, wherein the silicate solution is selected from the group consisting of tetraethyl orthosilicate and tetramethyl orthosilicate,

b. immobilizing the cholesterol oxidase and the electron mediator by slowly adding a 0.05-0.1M phosphate buffer solution containing 3-5IU of the cholesterol oxidase and about 0.01M of the electron mediator selected from the group consisting of potassium ferricyanide, ferrocene and Prussian blue to the silicate solution in step a),

c. the resulting mixture was allowed to stand until turbidity was observed, at which point the encapsulation of the enzyme and the electronic medium was complete,

d. the resulting cloudy mixture was applied to a conductive substrate,

e. drying the conductive substrate and the coated mixture at a temperature of 25-30 ℃ for at least one day, thereby obtaining the enzyme electrode.

2. The method according to claim 1, wherein the phosphate buffer used has a pH value in the range of 6.5 to 7.2.

3. The method of claim 1, wherein the conductive substrate is selected from the group consisting of indium tin oxide coated glass plates and silver coated non-conductive polymer surfaces.

4. The method of claim 1, wherein the non-conductive polymer surface is selected from the group consisting of a film and a sheet.

5. The method of claim 5, wherein the non-conductive polymer surface is selected from the group consisting of polyacrylamide, polyvinyl chloride, and polyethylene.

6. The method of claim 1, wherein the concentration of the cholesterol oxidase is in the range of 3-5IU per square centimeter of sol gel surface.

7. The method of claim 1, wherein the method of preparing an enzyme electrode is a one-step process.

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