U.S. patent application number 13/609371 was filed with the patent office on 2013-01-03 for process for the preparation of modified electrodes, electrodes prepared with said process, and enzymatic biosensors comprising said electrodes.
Invention is credited to Danila Moscone, Giuseppe Palleschi, Alessandro Poscia, Francesco Ricci.
Application Number | 20130001102 13/609371 |
Document ID | / |
Family ID | 39272068 |
Filed Date | 2013-01-03 |
United States Patent
Application |
20130001102 |
Kind Code |
A1 |
Palleschi; Giuseppe ; et
al. |
January 3, 2013 |
PROCESS FOR THE PREPARATION OF MODIFIED ELECTRODES, ELECTRODES
PREPARED WITH SAID PROCESS, AND ENZYMATIC BIOSENSORS COMPRISING
SAID ELECTRODES
Abstract
A process is described for the preparation of modified
electrodes useful for the measurement of analytes in biological
fluids, comprising the deposition of Prussian blue on screen
printed electrodes, and the modified electrodes prepared via said
process; the enzymatic electrodes and the biosensors comprising
said modified electrodes and the method for the determination of
analytes in biological fluids which uses said modified electrodes
are also described.
Inventors: |
Palleschi; Giuseppe; (Roma,
IT) ; Ricci; Francesco; (Roma, IT) ; Moscone;
Danila; (Fondi, IT) ; Poscia; Alessandro;
(Rignano Sull'Arno, IT) |
Family ID: |
39272068 |
Appl. No.: |
13/609371 |
Filed: |
September 11, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12002168 |
Dec 13, 2007 |
8309362 |
|
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13609371 |
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Current U.S.
Class: |
205/777.5 ;
204/290.01; 204/403.14; 427/126.1 |
Current CPC
Class: |
C12Q 1/001 20130101;
C23C 18/1601 20130101; Y10T 436/144444 20150115; G01N 33/5438
20130101; C12Q 1/003 20130101; C23C 18/1204 20130101 |
Class at
Publication: |
205/777.5 ;
204/290.01; 204/403.14; 427/126.1 |
International
Class: |
G01N 27/327 20060101
G01N027/327; B05D 5/12 20060101 B05D005/12 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 13, 2006 |
IT |
FI2006A000322 |
Claims
1.-16. (canceled)
17. A screen printed electrode modified with Prussian blue prepared
by a process, which comprises: sequential deposition on the working
electrode surface of said screen printed electrode of a solution
comprising the and at least one surface-active agent in an
appropriate solvent and a solution comprising the ferrocyanide (II)
or ferricyanide (III) ion and at least one surface-active agent in
an appropriate solvent, said solutions having concentrations such
as to obtain the formation of Prussian blue directly on the surface
of the working electrode; wherein said solutions have equal
concentration of between 20 mM and 2 M, and are deposited in equal
volume of between 100 nL and 4 .mu.L for an electrode surface
between 0.314 mm.sup.2 and 3.14 cm.sup.2; after mixing of the two
solutions, the mixture is left to rest for a period of between 2
minutes and 2 hours; the working electrode surface is then washed
with a washing solution; the electrode thus modified are left dried
at a temperature between 50.degree. C. and 200.degree. C.; wherein
said screen printed electrode has not undergone pre-treatment
before said sequential deposition of the solutions; and wherein the
quantity of Prussian blue deposited is between 10 and 200
nmol/cm.sup.2
18. The screen printed electrode as claimed in claim 17, wherein
the quantity of Prussian blue is 100 nmol/cm.sup.2.
19. An enzymatic electrode comprising the screen printed electrode
modified as defined in claim 17 and a suitable enzyme immobilised
on said electrode, chosen so that a product thereof can be oxidised
or reduced electrochemically on said modified electrode.
20. The enzymatic electrode as claimed in claim 19, wherein said
enzyme is the enzyme glucose oxidase.
21. An enzymatic biosensor comprising the enzymatic electrode as
defined in claims 19 as the working electrode, a reference
electrode, a counter-electrode if necessary and a cell for
receiving the biological fluid to be analysed, so that the latter
can come into contact with the enzyme immobilised on said enzymatic
electrode.
22. The biosensor according to claim 21, comprising said screen
printed electrode and said reference electrode as the sole
electrodes.
23. A method for determination of the quantity of an analyte in a
biological fluid comprising the application of an appropriate
potential value between an enzymatic electrode as defined in claim
19 and a reference electrode included in the an enzymatic biosensor
a counter-electrode if necessary and a cell for receiving the
biological fluid to be analyzed so that the latter can come into
contact with the enzyme immobilised on said enzymatic electrode,
and reading of the current signal generated.
24. The method as claimed in claim 23, wherein said analyte is
glucose and said potential applied is between -250 and +200 mV.
25. The method as claimed in claim 24, wherein said potential
applied is equal to -50 mV.
Description
FIELD OF THE INVENTION
[0001] The present invention refers to the field of modified
electrodes for the measurement of analytes in biological fluids,
and in particular a new process for the preparation of electrodes
modified with Prussian blue, modified electrodes prepared as
described, biosensors comprising said electrodes and the method for
the determination of analytes in biological fluids using said
electrodes.
STATE OF THE ART
[0002] The use of Prussian blue to modify amperometric enzymatic
electrodes has been known for some time, and resulted from the
discovery, going back to the 80s, that Prussian blue can be
deposited in layers on electrodes of different materials such as
platinum, vitreous carbon, SnO.sub.2 and TiO.sub.2, and has a
catalytic effect in redUction of the hydrogen peroxide produced
during the enzymatic oxidisation of the analyte.
[0003] With said electrodes modified with Prussian blue, the
concentration of hydrogen peroxide formed can be identified, thus
permitting indirect measurement of the concentration of the
oxidised analyte, which is directly proportional to the quantity of
hydrogen peroxide produced.
[0004] These electrodes modified with Prussian blue have therefore
been used for analytical applications, in particular in
amperometric biosensors for the measurement of glucose levels in
the blood. In addition to acting as an electrochemical mediator in
reduction of the hydrogen peroxide, the layer of Prussian blue can
also be used as a substrate for immobilisation of the oxidase
enzyme.
[0005] The electrodes modified with Prussian blue can be prepared
for example by means of electrochemical deposition of solutions of
ferric ferrocyanide on an electrode consisting of one of the
above-mentioned materials. The U.S. Pat. No. 5,876,581, for
example, describes an electrochemical deposition process in which a
pair of electrodes are immersed in a solution containing the Iron
(III) and hexacyanoferrate (III) ions; performing electrolysis with
one of the two electrodes as cathode and the other as anode, a
layer of insoluble ferric hexacyanoferrate (III), known as Prussian
blue, deposits on the surface of the cathode.
[0006] The electrodes that can be used in this process according to
U.S. Pat. No. 5,876,581 require the presence of a cathode made of
or coated in an inert metal such as platinum, rhodium, gold etc. or
an oxide of a conductive metal or a semi-conductor.
[0007] More recently these traditional electrodes have been
superseded by screen printed electrodes (SPEs), which have numerous
advantages: they are inexpensive, easy to prepare, they are
versatile and suitable for large-scale industrial production.
[0008] As mentioned above, electrodes modified with Prussian blue
have been used for some time now, depositing this product on the
surface of the electrode by means of electrochemical techniques.
Said techniques, however, are not suitable for the large-scale
production of modified electrodes starting from screen printed
electrodes for two main reasons:
[0009] 1) the electrochemical procedures are generally long, and
have to be performed electrode by electrode, with an enormous waste
of time in the case of preparation of a large number of electrodes;
and 2) the flat shape of the screen printed electrodes makes use of
the electrochemical procedures difficult and laborious since the
latter require immersion of the electrode in the solution
containing the ionic species, and this can lead to the formation of
a layer of Prussian blue also on the surface of the reference
electrode, obstructing electrical conductivity and preventing use
of the screen printed electrode for analytical purposes.
[0010] For these reasons, as far as the Applicant is aware,
electrochemical procedures for deposition of Prussian blue on
screen printed electrodes do not currently exist. A process for
chemical deposition of Prussian blue on a screen printed electrode
is described by Ricci et al. in Biosensors and Bioelectronics 18
(2003) 165-174. Said process comprises, prior to chemical
deposition of the Prussian blue, electrochemical pre-treatment of
the electrodes, which are electrochemically treated for 3 minutes
at a potential of 1.7 V. According to this article, said procedure
is essential for obtaining improved reproducibility and response of
the electrode, but on the other hand makes production of the
modified electrodes very laborious, requiring electrochemical
pre-treatment for each of them and thus nullifying the advantages
of the chemical deposition.
[0011] In the process described in the above article, furthermore,
the Prussian blue is deposited on the electrode manually, by means
of a complicated procedure which requires great caution in order to
prevent an increase in the internal resistance of the system: a
mixture must be prepared on the spot, adding a solution of
potassium ferricyanide in HCl to a solution of ferric chloride in
HCl, after which a drop of said mixture is deposited exclusively on
the surface of the working electrode, trying to avoid the reference
electrode and the counter-electrode.
[0012] Even with the above precautions, the electrodes modified in
this way are limited in terms of reproducibility of the deposition
and stability of the layer of Prussian blue; therefore, even though
the screen printed electrodes are in themselves characterised by a
high level of reproducibility of the electrode surface, responsible
for the stability and reproducibility of the electric signal of the
analyte, the poor reproducibility and lack of uniformity in
preparation of the layer of Prussian blue creates serious problems
in measurement of the quantity of analytes in the biological
fluids, and in general in the applications for which the electrode
is intended.
[0013] Given the advantages connected with the use of screen
printed electrodes, it is evident that there is a great need in the
sector for an easily scalable process, via which the surface of
electrodes screen printed with Prussian blue can be modified,
overcoming the drawbacks highlighted above for the known
processes.
SUMMARY OF THE INVENTION
[0014] The Applicant has developed a new process, particularly
inexpensive and simple to produce, which permits modification of
screen printed electrodes with Prussian blue, obtaining electrodes
with a high level of stability and reproducibility, useful in the
production of planar biosensors for the quantitative determination
of analytes in biological fluids.
[0015] The object of the present invention is therefore a process
for the preparation of a screen printed electrode modified with
Prussian blue, characterised in that it comprises sequential
deposition on the surface of said screen printed electrode of a
solution comprising the Iron (III) or Iron (II) ion and at least
one surface-active agent in an appropriate solvent and a solution
comprising the ferrocyanide (II) or ferricyanide (III) ion and at
least one surface-active agent in an appropriate solvent, said
solutions having concentrations such as to obtain the formation of
Prussian blue directly on the surface of the electrode. The screen
printed electrode modified with Prussian blue prepared by means of
the above-mentioned process, the enzymatic electrode and the
enzymatic biosensor which comprise said electrode, and a method for
determination of the quantity of an analyte in a biological sample
comprising contact between said sample and the above-mentioned
enzymatic biosensor constitute a further subject of the
invention.
[0016] Characteristics and advantages of the invention will be
illustrated in detail in the following description.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1: Profile of the modified screen printed electrode
prepared as described in Example 1.
[0018] FIG. 2: Cyclic voltammogram recorded using the modified
screen printed electrode as described in Example 1 (curve a), and
cyclic voltammogram recorded with the same electrode in the
presence of H.sub.2O.sub.2 (curve b), which shows the
electrocatalytic activity of the Prussian blue.
[0019] FIG. 3: Cyclic voltammogram recorded using the modified
screen printed electrode as described in Example 3 for comparison
(curve a), and cyclic voltammogram recorded with the same electrode
in the presence of H.sub.2O.sub.2 (curve b), which shows the
electrocatalytic activity of the Prussian blue.
[0020] FIG. 4: Comparison between cyclic voltammogram recorded
using the modified electrode as described in Example 1 (curve a)
and voltammogram recorded with the modified electrode as described
in Example 3 for comparison (curve b). Note the greater deposition
of Prussian blue obtained with the electrode of Example 1.
[0021] FIG. 5: Comparison between the operative stability of a
modified electrode according to Example 1 (FIG. 5a) and a modified
electrode according to Example 3 for comparison (FIG. 5b), from
which the greater operative stability of the electrode obtained
with the procedure described in Example 1 can be noted.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The solution containing the Iron (III) or Iron (II) ion used
in the process of the invention is for example an acid solution
with pH between 0.5 and 6.0, preferably an acid solution obtained
by addition of HCl, of a compound chosen from the group consisting
of Iron (III) or Iron (II) salts of inorganic acids, for example
ferric chloride, ferric sulphate and ferric nitrate; preferably the
present solution is a solution of ferric chloride in aqueous HCl
0.01 M having pH 2.0. The concentration of the Iron (III) or Iron
(II) ion in the above-mentioned solution can be for example between
20 mM and 2 M, and is preferably 1M. The solution containing the
hexacyanoferrate (III) ion according to the invention is for
example an acid solution with pH between pH 0.5 and 6.0, obtained
preferably by addition of HCl, of a salt containing the
ferrocyanide Fe(CN).sub.6.sup.4- ion or ferricyanide
Fe(CN).sub.6.sup.3- ion, for example sodium, potassium, ammonium or
cobalt hexacyanoferrate; preferably the present solution is a
solution of potassium ferricyanide K.sub.3Fe(CN).sub.6 in aqueous
HCl 0.01 M having pH 2.0.
[0023] The concentration of the Ferrocyanide (II) or Ferricyanide
(III) ion in the above-mentioned solution can be for example
between 20 mM and 2 M, and is preferably 1M.
[0024] According to the invention, the present process comprises
deposition of the solution containing the Iron (III) or Iron (II)
ion on the surface of the working electrode and, immediately after,
deposition of the solution containing the hexacyanoferrate (III)
ion, in equal volume and concentration.
[0025] The volume of the two solutions with concentration between
20 mM and 2 mM is for example between 100 nL and 4 .mu.L for an
electrode surface of between 0.314 mm.sup.2 and 3.14 cm.sup.2,
preferably 3.14 mm.sup.2.
[0026] Electrodes screen printed on inert material, for example
polycarbonate, polyester, polyvinylchloride (PVC) or other plastic
material, with suitable inks of conductive materials, for example
graphite, silver, gold or platinum, are suitable for use in the
present process for preparing the modified electrodes of the
invention; graphite ink is preferred for the working electrode.
[0027] After mixing the two solutions, the mixture is left to rest
for a period, for example, of between 2 minutes and 2 hours, and
preferably 10 minutes; the working electrode surface is then washed
with a washing solution consisting, for example, of an acid aqueous
solution with pH between 0.5 and 6.0, and preferably an aqueous
solution of HCl 0.01M having pH 2.0.
[0028] Following this, the working electrode can be further washed
with distilled water. The electrodes thus modified are then left to
dry in a stove for example at a temperature between 50.degree. C.
and 200.degree. C. for a period of between 10 minutes and 3 hours.
Preferably the electrode is placed in a stove at a temperature of
100.degree. C. for 1 hour 30 minutes.
[0029] The two solutions comprising the Iron (III) or Iron (II) ion
and the ferrocyanide (II) or ferricyanide (III) ion according to
the invention furthermore comprise at least one surface-active
agent, chosen from cationic, anionic, amphoteric surface-active
agents and their mixtures, in quantities for each solution, for
example, of between 0.001 and 10% in weight with respect to the
total volume of the solution. Preferably the surface-active agent
is chosen from the group consisting of sodium lauryl sulphate and
lauryl ethoxy sulphate, benzalkonium chloride, compounds belonging
to the family of products known under the trade name Tween.RTM.,
i.e. polyoxyethylene derivatives of esters of fatty acids with
sorbitol, and their mixtures.
[0030] According to a particularly preferred embodiment of the
invention, the surface-active agent polyoxyethylene (20) sorbitan
monolaurate, sold under the name Tween.RTM. 20 is added to each of
the two solutions comprising respectively the Iron (III) or Iron
(II) ion and the ferrocyanide (II) or ferricyanide (III) ion, in
quantities of 0.05% in weight with respect to the total volume of
the solution.
[0031] With the present deposition procedure, a layer of Prussian
blue can be obtained on the surface of the working electrode which
is extremely reproducible and active from the electrochemical point
of view. Furthermore, with the cyclic voltammetry technique, the
quantity of Prussian blue present on the working electrode
following the deposition has been determined, identifying values of
between 10 and 200 nmol/cm.sup.2 and preferably 100
nmol/cm.sup.2.
[0032] With the present process, therefore, a massive deposition of
Prussian blue can be obtained, never observed previously with
either chemical or electrochemical processes; the high surface
density of the Prussian blue present on the surface of the
electrode allows extremely high operative stability to be obtained
without affecting the catalytic activity of the Prussian blue
vis-a-vis reduction of the H.sub.2O.sub.2. The electrodes modified
with Prussian blue prepared with the process of the invention can
be used for the preparation of enzymatic biosensors with both two
and three electrodes, which are also the subject of the present
invention. In said biosensors, the modified electrode is used as a
support for immobilisation of a suitable enzyme, chosen on the
basis of the type of analytical determination for which the
biosensor is intended, i.e. such that the analyte to be detected
functions as a substrate for the enzyme, generating a product that
can be oxidised or reduced electrochemically on the modified
electrode, varying the quantity of current detected in proportion
to the quantity of analyte present in the fluid analysed.
[0033] The present biosensor comprises an enzymatic electrode, i.e.
the modified electrode of the invention as described above, on
which the enzyme has been immobilised by means of procedures
commonly used and known to any expert in the sector, and a cell for
receiving the biological fluid to be analysed, so that the latter
can come into contact with the enzyme.
[0034] According to a preferred embodiment of the invention, the
present biosensor comprises a modified electrode of the invention
on which the glucose oxidase enzyme has been immobilised, and is
used for determination of the glucose in biological fluids such as
blood, serum or plasma; in this case the analyte is the glucose
which, oxidised by the enzyme, produces H.sub.2O.sub.2 which is
reduced on the modified electrode due to the potential applied
between this electrode and the reference electrode, generating a
current signal proportional to the quantity of H.sub.2O.sub.2
produced and therefore to the quantity of glucose present in the
fluid contained in the cell.
[0035] A further subject of the invention is the method for
determination of the quantity of an analyte in a biological fluid,
comprising the application of an appropriate potential value
between a modified electrode of the invention as described above
and the reference electrode contained in the present biosensor, and
reading of the generated current signal.
[0036] Preferably, the present method is used for determination of
the quantity of glucose on biological fluids and the potential
applied is low, for example between -250 mV and +200 mV, preferably
-50 mV.
[0037] The present process for the preparation of electrodes
modified with Prussian blue, as described above, has a high level
of reproducibility of the deposition stage, which positively
affects reproducibility of the measurement performed with the
electrodes modified via this process; furthermore, it provides a
modified electrode with greater operative stability than any
electrode modified with Prussian blue prepared so far with the
known processes. It has been observed that in the electrodes
modified by means of the present process, the layer of Prussian
blue is still active and presents a reduction in its
electrochemical activity of only approximately 35%, after 150 hours
of continuous use.
[0038] In addition to greater operative stability, the electrodes
modified with Prussian blue prepared with the process of the
invention also have greater long-term stability during storage.
[0039] These stability values, both operative and storage, and
reproducibility values, in the case of biosensors with both two and
three electrodes, are furthermore achieved by a process which does
not require, contrary for example to the process described by Ricci
et al. in Biosensors and Bioelectronics 18 (2003) 165-174,
electrochemical pre-treatment of the electrode before deposition of
the Prussian blue, and is therefore much less laborious and easy to
automate, and therefore scalable at industrial level.
[0040] A further advantage of the modified electrodes of the
invention, in the case of biosensors with two electrodes which can
be used, for example, for determination of the glucose, is that of
comprising a working electrode and a reference electrode, while
they do not require the counter-electrode, contrary for example to
the electrodes modified with Prussian blue described in the
above-mentioned article by Ricci et al.
[0041] The following examples provide a non-limiting illustration
of the invention.
Example 1
Preparation of the Modified Electrode
[0042] Following the known procedure, a screen printed electrode
was prepared on a polyester sheet, comprising a circular-shaped
working electrode with diameter of 2 mm prepared with a graphite
ink and a silver reference electrode; the surface of the working
electrode is delimited by an ink made of insulating material. FIG.
1 shows the profile of the electrode obtained in this way.
[0043] The following two solutions were then prepared: 1) 1 M
solution of potassium ferricyanide K.sub.3Fe(CN).sub.6 in HCl 10 mM
and 2) 1 solution of ferric chloride in HCl 10 mM. Tween.RTM. 20
was added to each of the two solutions in a quantity of 0.05% in
weight with respect to the total volume of the solution.
[0044] With an automatic dispensing machine, 1 .mu.l of the
solution 1) was deposited on the surface of the working electrode
and, immediately after, with the same automatic dispensing
technique, 1 .mu.l of the solution 2) was deposited on the same
surface of the working electrode, thus resulting in 2 .mu.l of a
solution of ferric chloride and potassium hexacyanoferrate on said
surface.
[0045] After 10 minutes, the electrodes thus modified were washed
with a few millilitres of a 10 mM solution of HCl, then placed in a
stove at 100.degree. C. for 1 hour.
[0046] The procedure described above resulted in deposition on the
electrode of an extremely compact and stable layer of Prussian
blue, with a high surface density of the Prussian blue on the
working electrode.
[0047] The latter is confirmed by the results of the cyclic
voltammetry shown in FIG. 2, which permitted calculation of the
quantity of Prussian blue on the surface of the working electrode:
the surface density value measured is approximately 100
nmol/cm.sup.2, much greater than reported in the literature so far
using different deposition techniques.
Example 2
Determination of the Catalytic Activity of Prussian Blue for the
Electrode Prepared in Example 1
[0048] The properties of the modified electrode prepared as
described above in Example 1 were verified by means of cyclic
voltammetry, in the potential range -0.4 to 0.4 V.
[0049] FIG. 2 shows the voltammograms obtained for the screen
printed electrode not yet modified and for the same electrode
modified with Prussian blue, prepared as described above in Example
1. For the latter electrode, the increase in the cathodic wave to
approximately 0.05 V in the presence of hydrogen peroxide can be
seen in the voltammogram, showing the catalytic activity of the
layer of Prussian blue deposited on the electrode.
Example 3 (Comparison)
Preparation of the Modified Electrode as Described by Ricci et al.
in Biosensors and Bioelectronics 18 (2003) 165-174
[0050] Following the procedure described previously in the article
by Ricci et al., Biosensors and Bioelectronics 18 (2003) 165-174,
an electrode screen printed on a polyester sheet, comprising a
circular shaped working electrode with diameter of 2 mm prepared
with a graphite ink and a silver reference electrode, was modified;
the surface of the working electrode is delimited by an ink made of
insulating material.
[0051] The following two solutions were then prepared: 1) 0.1 M
solution of potassium ferricyanide K.sub.3Fe(CN).sub.6 in HCl 10 mM
and 2) 0.1 M solution of ferric chloride in HCl 10 mM.
[0052] Before deposition of the two solutions by means of the
electrochemical method, a procedure unsuitable for automation and
much more laborious and longer than that of Example 1, the
electrode was pre-treated by application of a constant potential
equal to 1.7 V vs. Ag/AgCI for 3 minutes.
[0053] Using an automatic dispensing machine, 20 .mu.l of the
solution 1) were deposited on the surface of the working electrode
and, immediately after, using the same deposition technique, 20
.mu.l of the solution 2) were deposited on the same surface of the
working electrode, thus obtaining 40 .mu.l of a mixture of ferric
chloride and potassium hexacyanoferrate.
[0054] After 10 minutes, the electrodes thus modified were washed
in a few millilitres of a 10 mM solution of HCl, then placed in a
stove at 100.degree. C. for 1 hour. The electrocatalytic activity
of the layer of Prussian blue thus deposited was assessed following
the same procedure as the one described in Example 2. FIG. 3 shows
the cyclic voltammograms obtained for the modified screen printed
electrode as described above.
Example 4
Determination of the Analytical Performances of the Modified
Electrodes According to the Invention and According to the Prior
Art
[0055] The analytical performances of the electrodes modified with
Prussian blue prepared as described above in Examples 1 and 3 were
tested considering the response to H.sub.2O.sub.2, the operative
and non-operative stability and the reproducibility. As regards
reproducibility, the reproducibility values, calculated as Relative
Standard Deviation percentage (abbreviated below to RSD %), for the
modified electrode as described in Example 3 are approximately 20%,
incompatible with any industrial type of application, whereas an
RSD % of 5% was calculated for the electrode of the invention,
modified as described in Example 1.
[0056] As regards the thickness of the Prussian blue deposited on
the electrodes, with the procedure of the prior art described in
Example 3, a layer of Prussian blue is obtained with a surface
density of between 1 and 10 nmol/cm.sup.2, therefore much lower
than the one obtained for the electrode of the invention of Example
1 (approximately 100 nmol/cm.sup.2) as shown by comparison of the
cyclic voltammetries (FIG. 4).
[0057] This results in greater operative stability of the Prussian
blue, up to a maximum of 200 hours. FIG. 5, for example, shows
continuous monitoring with a flow technique, obtained with the two
electrodes prepared following the procedures described in Examples
1 and 3. As can be clearly seen, with a 5 mM concentration of
H.sub.2O.sub.2, both the electrodes provide a reduction current due
to the catalytic activity of the Prussian blue, but the electrode
of Example 1 shows greater operative stability in the long term,
with a reduction in the initial signal of only 20% after 200
hours.
[0058] In the case of the electrode of the prior art prepared in
Example 3, a more marked reduction is observed equal to
approximately 70% after 200 hours.
Example 5
Preparation of the biosensor
[0059] The modified electrode prepared as described above in
Example 1 was used as a support for immobilisation of the glucose
oxidase enzyme, for the purpose of obtaining a biosensor useful for
continuous monitoring of glucose in the blood. For this purpose 200
mL of a solution of glutaraldehyde (0.025% v/v in H.sub.2O) were
deposited on the surface of the working electrode, previously
modified with Prussian blue as described above in Example 1. After
approximately 25 minutes, 200 mL of a mixture obtained by
dissolving 10 mg of the glucose oxidase enzyme in 1 ml of an
aqueous solution of Nafion.RTM. (0.1%) were deposited on the same
surface of the working electrode. Here again, it is left to rest
for approximately 25 minutes so as to obtain complete drying of the
solution deposited.
* * * * *