Sviluppo di biosensori: modifiche di superfici elettrodiche e sistemi di immobilizzazione enzimatica
Creators: name: Mignani, Adriana <1977>
Publisher: name: Alma Mater Studiorum - Università di Bologna
Year: 2008
Subjects: CHIM/01 Chimica analitica

description: An amperometric glucose biosensor was developed using an anionic clay matrix (LDH) as
enzyme support. The enzyme glucose oxidase (GOx) was immobilized on a layered double
hydroxide Ni/Al-NO3 LDH during the electrosynthesis, which was followed by crosslinking
with glutaraldehyde (GA) vapours or with GA and bovine serum albumin (GABSA)
to avoid the enzyme release.
The electrochemical reaction was carried out potentiostatically, at -0.9V vs. SCE, using a
rotating disc Pt electrode to assure homogeneity of the electrodeposition suspension,
containing GOx, Ni(NO3)2 and Al(NO3)3 in 0.3 M KNO3. The mechanism responsible of the
LDH electrodeposition involves the precipitation of the LDH due to the increase of pH at
the surface of the electrode, following the cathodic reduction of nitrates.
The Pt surface modified with the Ni/Al-NO3 LDH shows a much reduced noise, giving rise
to a better signal to noise ratio for the currents relative to H2O2 oxidation, and a linear range
for H2O2 determination wider than the one observed for bare Pt electrodes.
We pointed out the performances of the biosensor in terms of sensitivity to glucose,
calculated from the slope of the linear part of the calibration curve for enzimatically
produced H2O2; the sensitivity was dependent on parameters related to the electrodeposition
in addition to working conditions. In order to optimise the glucose biosensor performances,
with a reduced number of experimental runs, we applied an experimental design.
A first screening was performed considering the following variables: deposition time (30 -
120 s), enzyme concentration (0.5 - 3.0 mg/mL), Ni/Al molar ratio (3:1 or 2:1) of the
electrodeposition solution at a total metals concentration of 0.03 M and pH of the working
buffer solution (5.5-7.0).
On the basis of the results from this screening, a full factorial design was carried out, taking
into account only enzyme concentration and Ni/Al molar ratio of the electrosynthesis
solution.
A full factorial design was performed to study linear interactions between factors and their
quadratic effects and the optimal setup was evaluated by the isoresponse curves. The
significant factors were: enzyme concentration (linear and quadratic terms) and the
interaction between enzyme concentration and Ni/Al molar ratio.
Since the major obstacle for application of amperometric glucose biosensors is the
interference signal resulting from other electro-oxidizable species present in the real
matrices, such as ascorbate (AA), the use of different permselective membranes on Pt-LDHGOx
modified electrode was discussed with the aim of improving biosensor selectivity and
stability.
Conventional membranes obtained using Nafion, glutaraldehyde (GA) vapours, GA-BSA
were tested together with more innovative materials like palladium hexacyanoferrate
(PdHCF) and titania hydrogels.
Particular attention has been devoted to hydrogels, because they possess some attractive
features, which are generally considered to favour biosensor materials biocompatibility and,
consequently, the functional enzyme stability. The Pt-LDH-GOx-PdHCF hydrogel
biosensor presented an anti-interferant ability so that to be applied for an accurate glucose
analysis in blood. To further improve the biosensor selectivity, protective membranes
containing horseradish peroxidase (HRP) were also investigated with the aim of oxidising
the interferants before they reach the electrode surface. In such a case glucose determination
was also accomplished in real matrices with high AA content.
Furthermore, the application of a LDH containing nickel in the oxidised state was
performed not only as a support for the enzyme, but also as anti-interferant sistem.
The result is very promising and it could be the starting point for further applications in the
field of amperometric biosensors; the study could be extended to other oxidase enzymes.