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1
Istituto per lo Studio dei Materiali
Nanostrutturati
Universitatea din Bucuresti
FLOW INJECTION SYSTEM BASED ON NEW BIOSENSORS
INTERFERENCE- FREE FOR GLUCOSE, AMINOACIDS AND
LACTATE DETERMINATION
MIHAELA BADEA, ANTONELLA CURULLI, DANIELA
NEAGU, ANDREI-FLORIN DANET, GIUSEPPE
PALLESCHI
Dipartimento di Scienze e Tecnologie
Chimiche, Università di Roma Tor Vergata, Rome,
Italy CNR Istituto per lo Studio di Materiali
Nanostrutturati Sezione Roma2, Rome, Italy
Faculty of Chemistry, University of Bucharest,
Bucharest, Romania
AIM OF WORK
Enzyme entrapment within electropolymerized films
is considered at present a versatile and
powerful immobilisation technique in the
fabrication of biosensors. The purpose of this
work is to demonstrate the versatility of the new
non-conducting film not only as a cut-off
membrane to reject the electrochemical
interferences, but also as an immobilisation
matrix for oxidase based biosensors. For the
first time, in our knowledge, the
electropolymerisation of the new monomer
2-(4-aminophenyl)-ethylamine (AP-EA), which has a
similar structure with tyramine, is reported. The
presence of the free amino-groups on the
poly(AP-EA) surface permits the covalent
attachment of the enzymes via peptide bond
formation Moreover a copolymer between AP-EA and
2,6-dihydroxynaphthalene (2,6-DHN), which
improved the stability and the interference
rejection properties of the poly(AP-EA), was
electrosynthesised. The electropolymerisation
was performed by cyclic voltammetry. The
preparation of poly(2,6-DHN), poly(AP-EA) and the
copolymer (2,6-DHN AP-EA) was optimised in
order to be interference-free. Different scan
rates and scan potential ranges were investigated
and selected according to the monomers used. The
permeability of these sensors were tested for
hydrogen peroxide, ascorbic acid and
acetaminophen by cyclic voltammetry and
amperometry (E 650 mV vs Ag/AgCl reference
electrode). Different strategies for enzyme
(glucose oxidase) immobilisation were used
covalent attaching of the enzyme onto the film
surface by carbodiimide coupling cross-linking
of the enzyme onto the film surface via
glutaraldehyde and BSA immobilisation of the
enzyme throughout the polymer layer during the
electropolymerization step. The copolymeric films
with and without enzyme were characterised with
XPS and SEM techniques. The ability of the
copolymer 2,6-DHN AP-EA to provide a general
system to assemble biosensors was demonstrated by
assembling enzyme electrode probes based on
L-amino acid oxidase and lactate oxidase. The
analytical characterisation of the obtained
probes was first made in batch system in
amperometry (E 650 mV vs Ag/AgCl reference
electrode) by measuring the hydrogen peroxide
produced in the enzymatic reaction. Further
studies were performed using the assembled
biosensors in an original flow injection system
based on solenoidal valves. Technical parameters
of the flow system as flow rates, tubing length,
injected volumes, solenoidal valves programming
were optimised each type of biosensor. Also, was
studied the influence of the composition, ionic
strength and pH of buffers on the biosensors
response. Sensors stability, life time and
dynamic range were also studied and optimised.
Stability of the response of GOD biosensor to
substrate and interferents (ascorbic acid and
acetaminophen)
Reproducibility of the GOD biosensor for 5
different GOD/copolymer/Pt electrodes
EXPERIMENTAL and DISCUSSIONS
Cyclic voltammograms for electropolymerization
of 2,6-DHN, AP-EA and electrocopolymerization
of 2,6-DHN with AP-EA on Pt electrodes
2,6-DHN (0.5-1 mM) and AP-EA (10-100mM) were
electropolymerised on the Pt electrode by cycling
voltammetry. All the monomers were dissolved in
0.1 M phosphate buffer solution, pH 7.4 and the
resulting solutions were deareated for 15 min
with argon. The potential was continuously cycled
from 0 to 1200 mV for 2,6-DHN, from 300 to
1300 mV for AP-EA and from 150 to 1300 mV for
the copolymerisation of 2,6-DHN with AP-EA. The
potential was continuously cycled until a minimum
value of current, which remained constant after
further cycling, was observed. Ascorbic acid and
acetaminophen were used as model molecules to
test the interference rejection of the films. It
was observed that the new synthesised films are
characterised by a high permeability for hydrogen
peroxide and by a very good rejection of the
interferents. For biosensors assembling we
choose to work with the copolymer because its
characteristics are a compromise among presence
of the free amino groups, the stability in time,
and the interference rejection. In our studies
glucose oxidase (GOD) was used as a model enzyme
for the evaluation and optimisation of the new
copolymer (2,6-DHN AP-AE) as an immobilisation
matrix. The best results were obtained for the
GOD immobilised throughout the polymer layer
during the copolymer formation in the presence of
glutaraldehyde. The glutaraldehyde concentration
in the polymerisation media on the retained
enzymatic activity is very important and should
be studied for each enzyme used. For L-lactate
and leucine determination were assembled probes
with L-lactate oxidase (LOD) and L-aminoacid
oxidase (L-AAOD), respectively. FIA measurements
were carried out using a single-channel FIA
system. A Metrohm wall-jet cell was assembled
with the modified Pt electrodes. A peristaltic
pump Minipuls 3 and an injection system based on
solenoidal valves (Bio-Chem Valve Inc) were used.
The injected volume was 50 mL. For FIA
determinations we used a measurement system and a
software produced by S. Kalinowsy (Olsztyn,
Poland). The resulting flow-injection peaks were
well shaped, also we observed a linear response
for glucose within the range 0.1 10 mM. A
comparison with the glucose determinations in
batch showed an extension of the dynamic range
and, as expected a decrease of the sensitivity.
Similar result were obtained for lactate and
leucine determination. The electropolymerised
membranes with and without enzymes showed a good
mechanical resistance and a high operational
stability in flow conditions.
Scanning electron micrograph (SEM) for GOD
immobilised throughout (2,6-DHN - AP-EA)
copolymer
Calibration graphs obtained in different days
from the preparation of the GOD biosensor
Analytical characteristics of the biosensors
based on the new electropolimerized films
10 mM
6.0 mM
4.0 mM
2.0 mM
1.0 mM
0.2 mM
FIA registration for calibration of the
GOD/copolymer/ biosensor carrier 0.1 M phosfpate
buffer, pH 6.5 flow rate 0.4 mL/min injected
volume 50 mL
CONCLUSIONS
High reproducible enzyme electrodes can be
fabricated using the copolymer (2,6-DHN AP-EA),
and glucose oxidase as a model enzyme. The GOD
biosensor based on enzyme immobilisation in the
non-conducting copolymer exhibits good
performances, a rapid response and a low
detection limit. The GOD biosensor is
reproducible, stable and free from common
intereferences like ascorbic acid and
acetaminophen. The ability of the copolymer
(2,6-DHN AP-EA) to provide a generic method
for biosensors fabrication is demonstrated by
fabrication of biosensors based on L-aminoacid
oxidase and lactate oxidase, using essentially
the same preparation procedure as used for
glucose oxidase. The preparation is simple and
sufficiently fast (less than 1 h, including the
deaereation step). The characteristic of this new
procedure for biosensor fabrication is the fact
that the enzyme is, at the same time, entrapped
and covalently attached on the polymeric film
during the electropolymerisation step. Also the
interference rejection and the good stability
showed by the polymer films are maintained during
the biosensor assembling and use. The main
advantage of the new co-polymer poly(2,6-DHN
AP-EA) is the very good resistance and the high
stability in flow conditions, which make this
method robust and reliable. The proposed method
for oxidase based biosensors is all
electrochemical and one-step procedure suitable
for the production of disposable devices,
miniaturised biosensors and multi-analyte
sensing device.
Acknowledgements The authors thank European
Community ( MCFA-2000-000725) and to CNR Target
Project MSTA II for the financial support.