14-osios Lietuvos jaunųjų mokslininkų konferencijos „Mokslas – Lietuvos ateitis“
2011 metų teminės konferencijos straipsnių rinkinys
ISSN 2029-7149 online
ISBN 978-9955-28-832-9
BIOINŽINERIJA IR BIOINFORMATIKA
THE CREATION OF GLUCOSE BIOSENSOR USING GLUCOSE OXIDASE AND
COLLOIDAL GOLD
J. Voronovič 1, A .Ramanavičius 2, A. Ramanavičienė 1, N. German 1, A. Suchodolskis 2
1
Institute of Immunology of Vilnius University
2
Vilnius University
Annotation. The creation of glucose biosensors using immobilized glucose oxidise (GOx) and gold
nanoparticles (Au-NPs) on the surface of graphite electrode are presented. Immobilized colloidal gold on
graphite rod electrode surface gives the protein and other redox active molecules more freedom in orientation,
thus facilitating the nondirect electron transfer through the conducting tunnels not only of Au-NPs, but of
enzymes too. It is evaluated the sensitivity of determination of glucose (Glu), using amperometric method, are
investigated the stability of modified working electrodes when N-methylphenazonium methyl sulfate (PMS) was
used as an artificial mediator of electron transfer.
Key words: glucose biosensor, glucose oxidase, colloidal gold.
electrode surface (third generation biosensors) (FREIRE et
al 2003, LIU et al 2003-1).
Nanoscale materials provide an ideal remedy to the
usually contradictory issues applied in the optimization of
immobilized enzymes: minimum diffusion limitations,
promotion of electrochemical reaction, maximum surface
area per unit mass and high effective achievement of
enzymes direct wiring to electrode surface by enhancing
the electron transfer between redox centers in proteins
and electrode surface, and have been widely used in constructing electrochemical biosensors (WANG 2005,
PUMERA et al 2007). Metal nanoparticles are generally
defined as isolable particles between 1 and 50 nm in size,
that are prevented from agglomerating by protecting
shells (WANG 2005). Usually nanoparticles, including
metal and oxide nanoparticles, semiconductor and composite nanoparticles, exhibit unique chemical, physical
and electronical properties that are different from those
bulk materials, and can be used for construction of new
electrochemical sensors and biosensors in electroanalysis
(WANG 2005, LUO et al 2006).
Comparing to enzyme labels, nanoparticles are very
stable, offer high sensitivity, wide variety of nanoparticles opens possibilities for multiplexing and miniaturization, and are used as electrochemical label, decreasing
detection limits to several hundreds of biomolecules
(PUMERA et al 2007). Smaller size of gold nanoparticles
(about 5-50 nm) gives the enzyme molecules more freedom in orientation and so increases the possibility that
the prosthetic group is closer to the metal particle surface:
then the distance for electron transfer between the en-
Introduction
Nanotechnology has recently become one of the
most exciting forefront fields in analytical chemistry and
now is rapidly evolving to open new combination of electrochemical biosensors constructions and methods, resolve challenging bioanalytical problems, including
specificity, stability and sensitivity (WANG 2005,
PUMERA et al 2007, WANG et al 2008). Nanobiosensors,
using nanomaterials (gold nanoparticles), were applied
for biochemical, clinical and environment applications: in
areas of cancer diagnostic, the detection of infectious
organisms, the determination of vitamins, amino acids
and sugars (WANG 2005, PUMERA et al 2007, WANG et al
2008, KURNIAWAN et al 2006). Graphite and carbon nanotubes are used for creation of electrochemical biosensors
and are characterized by high surface area, favorable
electronic properties and electrocatalytic effect (PUMERA
et al 2007).
Enzymes are the biological components most usually used in biosensors, while electrochemical transduction
is the most popular method, often employing potentiometric and amperometric techniques (FREIRE et al 2003).
The electronic coupling between redox enzymes and
electrodes for amperometric biosensors can be based on
the electroactivity of the enzyme substrate or product
(first generation biosensors); utilization of redox mediators in solution or immobilized with the biomolecule
(second generation biosensors), or direct electron transfer
between the redox-active biomolecule and the working
© Vilniaus Gedimino technikos universitetas
http://dspace.vgtu.lt
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zyme and electrode will be shorter and transfer of electron easer (LIU et al 2003-2).
Modified by gold nanoparticles electrode can be
prepared by binding gold nanoparticles with functional
groups of covalent self-assembled monolayers, by direct
deposition of nanoparticles onto the bulk electrode surface or by incorporating colloidal gold into the electrode.
The better immobilizing of glucose oxidase or gold nanoparticles could be achieved by cross-linking with bifunctional agents such as glutaraldehyde or by mixing with
components of composite electrodes (YÁÑEZ-SEDEÑO et
al 2005).
The aim of present work was to create GOx and AuNPs based biosensors and to use in the determination of
glucose.
ware in amperometry modes. A conventional threeelectrodes system comprising a prepared working graphite electrode (modified as was described above), platinum
as an auxiliary electrode and silver/silver chloride with
3 mol l-1 KCl as a reference was employed for all electrochemical experiments. All experiments were performed at
room temperature in stirred sodium acetate or sodium
phosphate buffer solutions with KCl. Electrochemical
detection of the analytical signal was performed in the
absent and presence of PMS and different concentration
of glucose. Special conditions in stability test: between
measurements, the GOx-electrode and Au-NPs/GOx- or
GOx/Au-NPs-electrodes were stored at 4°C in a closed
vessel hanging over buffer solution.
Results
Materials and methods
In the work were created two types of the amperometric biosensors for a determination of Glu. One of
them was based on electron transfer between a graphite
electrode and immobilized GOx, which can catalyze the
oxidation of analyte; other – between a graphite electrode, GOx and Au-NPs. Good established and commercially available the GOx-based biosensor is the primary
model system in the development of new sensing materials and methods. In first type of biosensor GOx was deposited on the graphite electrode and in the presence of
Glu and dissolved oxygen GOx started to generate hydrogen peroxide and gluconolactone, which was hydrolyzing to gluconic acid (Fig. 1 (A)). The activity of
enzyme was estimated during measurements by measuring the oxidation current of H2O2 produced by the enzymatic reaction. Coupled enzymatic reactions used in the
Au-NPs-based enzyme electrodes with immobilized GOx
and deposited directly onto the surface of graphite electrode Au-NPs are presented in Fig. 1 (B).
Chemicals. Analytical grade chemicals and triply
distilled water were used to prepare the solutions. It were
used glucose oxidase, N-methylphenazonium methyl
sulfate, D-(+)-glucose, tetrachloroauric acid, tannic acid,
sodium citrate, hydrochloric acid, sodium carbonate,
glutaraldehyde solution, solutions of sodium acetate and
sodium phosphate buffers with KCl.
Pre-treatment of working electrode. Graphite
rod electrodes were sealed into epoxy to prevent contact
of the electrode side surface with the solution. The working surface area of the graphite electrodes was
0.071 cm2. Graphite electrodes were modified with GOx
absent (GOx-electrode) and present gold nanoparticles
(GOx/Au-NPs-electrode and AuND/GOx-electrode, respectively). The synthesis of Au-NPs was described in
details previously in the article.
Working electrode modification by glucose
oxidase and gold nanoparticles. During preparation
of GOx-electrode, GOx solution was deposited on the
electrode and water was evaporated at room temperature
by intensive ventilation. For the preparation of
Au-NP/GOx-electrode additionally gold nanoparticles
colloid solution was deposited on the GOx-electrode. For
the preparation of GOx/Au-NPs-electrodes gold nanoparticles colloid solution was deposited on the working electrode and after evaporation of water additionally solution
of GOx was deposited. After water evaporation all electrodes were stored over 25 % solution of glutaraldehyde
at room temperature in a closed vessel.
Electrochemical measurements. All electrochemical measurements were performed using a computerized
potentiostat Autolab/PGSTAT 30 with GPES 4.9 soft-
Fig. 1. Electrocatalytic oxidation of Glu in the presence of GOx
(A) or GOx and Au-NPs (B) with mediator – PMS.
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It was seen what GOx converts glucose and O2 to
gluconolactone and H2O2, but the electron transfer could
follow by 2 ways: through enzyme and trough Au-NPs.
In such biosensors (Fig. 1(B)) redox center of GOx is
covalently plugging to electrode surface via Au-NPs,
constructing stable structure of the sensor (PUMERA et al
2007). The basic advantage of perfect option for biomolecular immobilization is effectively increase of the
stability and the maintenance of activity, that can be explained because the nanoparticles are chemically more
active than the materials of electrode and are characterized by high surface energy.
Using immobilized by GOx and modified by AuNPs graphite rod electrodes was observed hyperbolic
dependence of amperometric signal on substrate (Glu)
concentration from 0.1 till 100 mmol l-1 (Fig. 2).
The second step of research investigation was the
storage stability of GOx- and GOx/Au-NPs-electrodes
towards the catalytic oxidation of Glu has been examined
as well and it was evaluated the decrease in amperometric
current during time cycling for Glu determination. Compare stability of both electrode types is found what storage of stability of GOx/Au-NPs-electrode decrease faster
when of GOx-electrode, that could be explained by the
removal of Au-NPs during cycling procedures.
Conclusion
GOx can be effectively immobilized on Au-NPs
modified graphite electrode to produce a fast indirect
electron transfer, using PMS as the mediator. Immobilized Au-NPs can facilitate electron transfer between Glu
and electrode surface, then increase the enzyme loading,
and have little effect on the activity of GOx. The creating
electrochemical sensor displays a high sensitivity for
determination of Glu and storage stability.
1
80
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I, A
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Acknowledgement
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This work was financially supported by Lithuanian State Science and Studies Foundation project
number S-6/2007 and COST program D43.
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0
0
20
40
C(Glu)
60
80
100
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Fig. 2. Calibration plot of immobilized by GOx and
Au-NPs graphite rod electrode as a function of glucose
concentrations. Conditions: 1, 2 and 3 curves –
GOx/Au-NPs-, Au-NPs/GOx- and GOx-electrodes.
Using nanomaterials in biosensors, the speed of
electrochemical reaction increase (WANG 2005), and
could suppose that the sensitivity of biosensors could be
higher. It is seen (Fig. 2) that using Au-NPs at the surface
of graphite rod electrodes the sensitivity of amperometric
method increase, what could be explained by higher electron transfer from GOx to electrode. As is investigated
from research results for graphite electrode based on
cross-linking immobilized GOx in sodium acetate and
sodium phosphate buffers the maximal speed of an enzymatic reaction are 1.8 and 1.3 times less, respectively,
compare witch GOx/Au-NPs-electrode; 1.6 and 1.2 times
less, compare witch Au-NPs/GOx-electrode.
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YÁÑEZ-SEDEÑO P., PINGARRÓN J.M., Gold nanoparticlebased electrochemical biosensors, Anal. Bioanal. Chem.,
2005, 382, 884-886.
NEIMAN B., GRUSHKA E., LEV O., Use of gold nanoparticles to enhance capillary electrophoresis, Anal. Chem.,
2001, 73, 5220-5227.
GLIUKOZĖS BIOSENSORIŲ KŪRIMAS NAUDOJANT
GLIUKOZĖS OKSIDAZE IR KOLOIDINĮ AUKSĄ
J. Voronovič, A. Ramanavičius, A. Ramanavičienė, N. German, A. Suchodolskis
Publikacijoje pateikta sukurta ir ištirta savaime
besiorganizuojanti analizinė sistema, paremta fermento
gliukozės oksidazės sąveika su gliukoze. Paruoštas
amperometrinis analizės metodas, pritaikytas anglies elektrodo
paviršiaus modifikavimu, t.y. ištirta, kad acetatiniame
buferiniame tirpale didžiausiu jautrumu pasižymi anglies
elektrodas, kurio sluoksnį sudaro aukso nanodalelės ir gliukozės
oksidazė imobilizuotos glutaro aldehido garais. Įvertintas
elektrodų elektrokatalizinių savybių kitimas laike: stabilesne
laikoma GOx padengta sistema.
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