Note: Descriptions are shown in the official language in which they were submitted.
CA 02512282 2005-06-30
WO 2004/058994 PCT/IB2002/005684
PROCESS FOR PREPARATION OF ENZYME ELECTRODE
Field of the invention
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.
Background of the invention
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 inetabolism in biology and medicine: Plenum: New York, 1972).
Cholesterol
determination in blood is clinically iniportant 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 10mM. It is therefore desired to develop techniques
that allow
convenient and rapid determination of cholesterol.
Various methods have been employed in the art for stabilization and
immobilization
of enzymes within carbon paste or covalently linking it to the surface of
glassy carbon
electrode or immobilizing it within a polymer film for the preparation of
enzyme electrode. In
recent years, enzyme immobilization with the retention of enzyme activity
within a sol-gel
matrix has become a potential tool for development of new biosensors. Avnir et
al disclose
the immobilization of organic compounds in inorganic supports by introducing
the organic
compound with a polymerization precursor [J. Pliys. Chem., 88 (1984), 5969].
Sol-gel
processed materials are known for their use in development of ceramic films
for conductive,
optical, mechanical and electro-optic 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 report that alkaline phosphatase retains its activity when
immobilized in a
sol-gel matrix [Mater. Lett., 10 (1990) 1]. There is disclosure in the art of
the immobilization
of enzymes including glucose oxidase within a sol-gel matrix [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 report the construction of a ferrocene mediated sol-gel
biosensor using a two stage sol-gel preparation method based on TMOS and.
commercial
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CONFIRMATION COPY
CA 02512282 2009-05-05
colloidal silica of varying particle size [Chem. Mater. 5 (1993) 911].
According to this
literature reference, more than 80 % of the glucose oxidase retains its
activity in the gel and
the Faradic response of the electrode agrees with theoretical calculations
based on this
activity. Lev et al disclose the use of sol-gel derived composite silica-
carbon electrodes and
claim the dual advantage of both the porosity and rigidity of the silica
matrix and the
electrical conductivity of the graphite [Anal. Chem., 66 (1994) 1747]. In this
disclosure,
glucose oxidase is first adsorbed on the surface of the carbon powder and then
used for the
preparation of the 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 Kumar et al. Analytica Chimica Acta Vol 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.
Further 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. Several have reported methods of immobilization of
biorecognition
elements for use in chemical sensing researchers [R. F. Taylor, Protein
Immobilizing
Fundamentals and Applications: Marcel Dicker, New York (1975) Chapter 8, 263-
303 and H.
H. Weetall, Immobilized Enzyme; Antigen, Antibodies and Peptides Preparation
and
Characterization: Marcel Dicker, New York (1975) Chapter 6, 263-303]. The
methods
reported in literature can generally be classified into one of the following
categories (1)
physisorption (2) covalent attachment or (3) entrapment, among which
physisorption is the
simplest immobilization approach.
Several disadvantages arise with these methods of immobilization such as
problems
associated with the large size of the biorecognition elements (e.g. proteins
and enzymes).
Physisorption produces a range of biorecognition element orientations and
apparent biding
affinities. Besides physisorption generally leads to a population of
biorecognizing elements
that is completely unresponsive to target analyte. The immobilized species is
completely
unresponsive to target analyte. The immobilized species will often
leach/desorb from sensing
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WO 2004/058994 PCT/IB2002/005684
interface because there are no covalent bonds. Covalent schemes generally lead
to more
stable and uniform (interim of,biorecognition orientation) interface and
enzyme leaching is
minimized. Unfortunately covalent attachment can involve one or more chemical
transformation and tends to be time consuming and can be costly.
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. However, the shelf life of this
sensor is too low.
US Patent 6,214,612 discloses 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, 1,2-naphthoquinone-4-sulfonate, 2,6-dichlorophenol indophenol,
dimethylbenzoquinone, 1-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. During
operation, 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. However, the sensor has a low shelf life and also potentially shows
leaching of both
the mediator and the enzyme.
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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. The sensor of this patent is aimed at
eliminating
impairment of sensor response due to electrode degeneration caused by invading
surfactant
into the electrode system. While the response time is stated to be low, the
shelf life is again
not high due to potential enzymatic leaching.
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. For example, 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 of the polymer useful for sensor applications.
It is therefore highly desirable to develop biosensors that allow conventional
and rapid
determination of cholesterol.
Objects of aspects of the invention
An object of an aspect of the present invention is to provide a novel sol gel
based
enzyme electrode useful for the estimation of cholesterol in aqueous medium.
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Another object of an aspect of the 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.
Yet another object of an aspect of the present invention is to provide an
enzymatic
stable, cost-effective high sensitive enzyme electrode.
Still another object of an aspect of the present invention is to provide an
enzyme
electrode, which provides an accurate measurement of cholesterol within a
short time period of
30 seconds.
It is yet another object of an aspect of the invention to provide a novel sol
gel based
enzyme electrode which is reusable at least five times.
Summary of the invention
Accordingly 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,
ii. 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 of the encapsulated enzyme and of
the electron
mediator, a response time of 30 seconds, being reusable at least five times
and a shelf life of
six months.
In another embodiment of the invention the electrically conductive base plate
used is
selected from indium tin oxide coated glass plate and a silver coated non-
conducting polymer
surface.
In still another embodiment of the invention, the non-conducting polymer
surface used
is selected from a film and a sheet.
In a further embodiment of the invention non-conducting 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 silica
sol.
In yet another embodiment of the invention, the silica sol used is selected
from
tetraethyl orthosilicate and tetramethyl orthosilicate.
In another embodiment of the invention the electron mediator used is selected
from
potassium ferricyanide, ferrocene and Prussian blue.
In a further embodiment of the invention the enzyme electrode has a
sensitivity of 0.4
volt.
In another embodiment of the invention the strength of cholesterol oxidase
used is in
the range of 3-5 IU per lxl cmZ of surface area.
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WO 2004/058994 PCT/IB2002/005684
In a further embodiment of the invention 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.O1M 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.
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 in
a
range of 6.5-7.2.
In yet another embodiment of the invention the process of preparation of
enzyme
electrode is a single step process.
Brief description of the accompanying drawing
Figure 1 shows the response of the enzyme electrode as a function of the
concentration of cholesterol solution.
Detailed description of the invention
The present disclosure essentially involves the stages of preparation of sol
and
simultaneous addition of inediator 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 of the 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
fihn which has the
cholesterol sensing property.
The preparation of sol is accomplished by using preferably tetraethyl silicate
in pure
water and HCI. However tetramethyl silicate may also be used. The water used
in the
preparation of the sol is preferably pure water and more preferably a
deionized water of more
6
CA 02512282 2009-05-05
than 15Mohms. The preparation of sol maybe accomplished by any conventional
known
means known to a person skilled in the art. For example, a stock sol-gel
solution is prepared
by mixing 4.5 ml of tetra ethyl orthosilicate (TEOS), 1.4 ml of H?O and 100u1
of 0.1 M HCl
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.
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 of the 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. For the immobilization of cholesterol oxidase (ChOx) 80 1 of
stock solution was
added to 20 1 of 0.01 M 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 hydrolyzed gel forming silica
network. The solution
was kept aside Lmtil the enzyine and nlediator was encapsulating completely
with in the
growing network.
Once the sol gel containing the immobilized and microencapsulated enzyme is
prepared it is ready for use for deposition as a film on a conducting
substrate. 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 of the
sol gel containing
encapsulated enzyme. Prior to film casting indium tin oxide (ITO) coated glass
plates were
first treated with HNO3, for about 2 hrs and were subsequently rinsed thrice
with MilliporeTM
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-100TM surfactant.
After
homogenization the volume was made up to 1 OOm 1 with 0.1 M phosphate buffer
(pH 7.0) and
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WO 2004/058994 PCT/IB2002/005684
thermostat at 35 C. This. standard solution was fiirther diluted with water to
make different
cholesterol solutions.
The characteristics of the 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 of the present invention. Typically the enzyme electrode was
made on an
ITO coated glass. The second electrode is the reference electrode of Ag/AgCI.
In actual
measurement, cholesterol solution of strength varying between 0.5-10mM in a
phosphate
buffer of pH of 7.0 was used with the two electrodes as described above. The
current due to
enzymetrically produced H202 was measured every 100 seconds. Typically a
response time
of seconds was measured for a concentration of
The reaction giving rise to current is due to the following scheme
Cholesterol +02 4 A-Cholesten-3-one + H202
H202 4 02 + 2IT' + 2 e-
The results of the experiments are shown in Fig 1. In order to check if
addition of any
interfering agents in cholesterol, like glucose or ascorbic acid, will have
any deleterious
effect on the response of the enzyme electrode, the experiment was repeated
with cholesterol
solution mixed with the interfering agents. It was found that these
interfering agents did not
show any effect on the response to the enzyme electrode.
In an attempt to improve the shortcomings of the prior art disclosures of
cholesterol
measurement, the bio molecules are immobilized in sol-gel and have
comparatively enhanced
shelf lives. This is because of fact that (i) a variety of enzymes may be
encapsulated in so1-gel
matrices giving optical transparent glasses (ii) the enzymes are remarlcable
stable in such
matrices (iii) these enzymes undergo characteristics reversible reaction in
sol-gel glasses and
(iv) 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. The fine pore network in dried glass (<lOnm)
does not scatter
visible radiation and allows the diffusion of small molecules onto the
electrode surface.
Porous inorganic xerogel such as tetra 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.
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These biosensors in principle have sensitivity and rapid response time and are
also free from
the problem of any detrimental effects on enzyme activity.
Another significant advantage observed over the prior art enzyme electrodes is
that
there is zero leaching of the enzyme and the mediator. The electrode of the
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 determination of cholesterol
in solution.
The following examples are given by the way of illustration and therefore
should not
be constructed to limit the scope of the present invention in any manner.
Example 1: Enzyme activity measurements
A solution of 0.05 cm3 of 61nm cholesterol dissolved in propane-2-ol and
volume of 3
cm3 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 nm
using a double 'beam spectrometer to determine the cholesterol produced by the
enzymatic
reaction. The apparent enzyme activity (Ucm) was evaluated by the following
procedure
based on the difference in absorbance before and after incubation of the
enzyme inunobilized
sol-gel glass plate.
E em (Ucm 2) = AV/sts
app
Where A is a deference in absorbance before and after incubation, V is the
total
voluine (3.05cm3), s is the millimolar extinction coefficient of cholesterol
(12.2), t is the
reaction time (min) and s is the surface area (cm) of sol-gel film. One unit
of enzyme activity
(U cm) is defmed 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 enzylne (ChOx/HRP) leaching was observed from the
enzyine
immobilized sol-gel film.
Example 2
Electrochemical estimation of cholesterol containing interfering reagents by
using
cholesterol oxidase immobilized sol-gel-ITO( ChOx/sol-gel/ITO ) electrode.
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Cyclic Voltametery studies
When cholesterol comes in contact with enzyme electrode containing ChOx
immobilized in a TEOS derived sol-gel film the following enzymatic and
electrochemical
reaction occurs
Cholesterol +02 4 0-Cholesten-3-one + H202
H202 4 02 + 2H+ + 2 e-
The oxidation current for H202 is recorded as the sensor response in the
amperoinetric
biosensor. Owing to the direct immobilization of the enzyme, 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 10mM) using enzyme immobilized so1-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 mM 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 H202 on the surface of the ITO coated glass plate. However
the oxidation
peak at 0.75V shiffts anodically by 150mV to 0.9 V Vs Ag/AgCl with increase in
anodic
current in the presence of 0.1 mM ascorbic acid. The presence of 0.5 mM
glucose in the
cholesterol solution (1mM) also shows an increase in anodic current but does
not show any
significant effect on the oxidation potential of H202, thereby showing that
the presence of
both 0.1 mm ascorbic acid 0.5 mm glucose in cholesterol have a significant
effect on the
observed anodic current.
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/AgCI and amperometric response
to
cholesterol (0.5-10mM) was measured by using amperometric calibration for
enzymematically produced H202. The current was monitored every 100 sec after
dispensing
different concentration of cholesterol solution (2mM-IOmM) 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.
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Example 4
Electrochemical estimation of cholesterol using cholesterol oxidase and
potassium
ferricyanide immobilized sol-gel indium tin oxide (ChOx/Fe3+/sol-gel/ITO) as
electrode and
with influence of interfering reagents such as ascorbic acid (0.1mM) and
glucose (0.5mM)
Cyclic Voltametery studies
The cyclic voltammetry experiments were carried out in 0.1 M phosphate buffer
(pH
7.0) containing different concentration of cholesterol using enzyme
cholesterol oxidase and
potassium ferricyanide immobilized sol-gel indium tin oxide (ChOx/Fe3+/sol-
gel/ITO) film as
a working electrode, a Ag/AgC1 reference electrode and a Pt wire as a counter
electrode. The
following reactions occur
Cholesterol +ChOx -> Cholestenone + ChOxrea
ChOx,~d + Fe3+ (ferricyanide) 4 ChOx + Fe2+ (ferrocyanide)
0.4V
Fe2+ (ferrocyanide) -> Fe3+ (ferricyanide) + e- (at electrode)
The oxidation current is recorded as the sensor response in the amperometric
biosensor. Owing to the direct immobilization of the enzyme, the sensor
properties such as
time and sensitivity are the reflection of the immobilized enzyme. An
oxidation peak
observed earlier in Example 2 at 0.9V vs. Ag/AgCl when enzyme iinmobilized sol-
gel film
without mediator was used as an electrode now shifts 300mV cathodically and is
observed at
0.4V versus Ag/AgCl, which increases with increase in cholesterol
concentration (2 to
10mM). The presence of 0.1mM a.scorbic acid and 0.5mM glucose in cholesterol
solution
does not show any significant effect on the oxidation potential.
Example 5: Amperometric response studies
A three electrode ce11 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/AgCI and amperometric
response to
cholesterol of concentration varying from 2mM to 10mM was measured. The
current was
monitored every 100 sec after different concentration of cholesterol solution
(2mM to 10mM)
into the cell (Figure 1). The anodic current measured in 6mM cholesterol
solution (1mL) at
ChOx/Fe3+/sol-ge1/ITO polarized at 0.4 V yields the stead the state in 30
seconds and this
response to cholesterol solution was reproducible to within 5%. The lower
detection limit of
cholesterol was found amperometrically to be 0.5mM.
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The main advantages of the present invention are:
1. Enzymatic electrodes prepared by the invention shows negligible enzyme
leaching.
2. The enzyme electrode prepared shows fast response to cholesterol in
solution
3. The enzyme electrode prepared is stable for a longer time.
4. The enzyme electrode prepared is highly sensitive to cholesterol.
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