Apparatus
added with stirring to the electrochemical cell containing
working buffer (10 mL) and the steady-state current values were
recorded. After each run, buffer solution was refreshed and
electrodes were washed with distilled water. The current signal
obtained with respect to various standard glucose concentrations
were plotted as a calibration curve and glucose concentrations in
G. oxydans cultures were calculated using the calibration curve.
For the amperometric and cyclic voltammetric measurements,
Palm Instrument (PalmSens, Houten, The Netherlands, www.
palmsens.com) with a conventional three electrode configura-
tion was used. A graphite electrode (Ringsdorff Werke GmbH,
Bonn, Germany, type RW001, 3.05 mm diameter and 13%
porosity) served as the working electrode, Ag/AgCl (3.0 M KCl
saturated with AgCl as an internal solution) and a Pt electrode
(Metrohm, Switzerland) were used as reference and counter
electrodes, respectively. The electrodes were placed in an elec-
trochemical cell with internal volume of 10 mL. All the
measurements were performed at room temperature.
1H NMR and 13C NMR spectra were recorded in CDCl3 on
Bruker Spectrospin Avance DPX-400 spectrometer and chemical
shifts (d) were given relative to tetramethylsilane. The mass
spectra were recorded on a Thermo Scientific DSQ II Single
Quadrupole GC/MS. A Waters Synapt MS System HRMS
(High Resolution Mass Spectrometer) was used to confirm the
synthesized materials. JEOL JSM-6400 models SEM (Scanning
Electron Microscope) was used for surface imaging. XPS (X-ray
Photoelectron Spectroscopy) was carried out on a PHI 5000
Versa Probe (F ULVAC-PHI, Inc., Japan/USA) model X-ray
photoelectron spectrometer instrument with monochromatized
Al Ka radiation (1486.6 eV) as an X-ray anode at 24.9 W.
Contact angle measurements of a drop of water (2.0 mL) on the
polymer surfaces were carried out using the sessile drop method
with a CAM 100 KSV (KSV, Finland). Recording the drop
profile with a CCD camera allowed to monitor the changes in
contact angle. All reported data were given as the average of five
measurements ꢃ SD. The experiments were conducted at
ambient temperature (25 ꢂC). HPLC (High Performance Liquid
Conclusions
The electrochemically polymerized SNS-anchored carboxylic
acid performs well as an immobilization matrix for the
construction of a glucose oxidase enzyme biosensor. The
amperometric biosensor based on conducting polymer was
fabricated through the covalent immobilization of the enzyme on
a
functionalized conducting polymer; poly-SNS-anchored
carboxylic acid layer. The presence of the protein and amide
bond between the surface and the protein was confirmed by SEM
and XPS studies. The fabricated biosensor exhibited excellent
kinetic parameters such as Km, Imax, low LOD and high stability.
Moreover, it was successfully applied to G. oxydans cultures for
the detection of glucose content.
Acknowledgements
This work is partially supported by TUBA and TUBITAK
110T580 grants. METU Central Laboratory is acknowledged for
€
the SEM images. Merve Yuksel is gratefully acknowledged for
her contributions in HPLC applications. Dr Selda Keskin is also
gratefully acknowledged for access to the XPS facilities.
Notes and references
Chromatography) with
a refractive index detector (RID)
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This journal is ª The Royal Society of Chemistry 2011