Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
N. Yildirim et al. / Dyes and Pigments 89 (2011) 144e148
145
most intensively studied enzyme and capable of catalyzing the
oxidation of -glucose by molecular oxygen to -gluconic acid [6].
Automated Microarrayer (England). The optical measurements
were taken with a fluorescence reader from Sensovation AG,
Stockach (Germany). Fluorescence spectral data were recorded on
Perkin Elmer, Luminescence Spectrometer, LS 50B (Germany).
Spectrophotometric measurements were performed with a Perkin
Elmer UV/VIS Spectrometer-Lambda 2 (Germany). JASCO HPLC
(MD-1510 PDA Detector), (Germany) was used as a reference
method for the analysis of glucose content in real samples.
D
D
Furthermore, fluorescence sensing of chemical and biochemical
analytes is the dominant analytical approach in medical testing,
biotechnology, and drug discovery. This method has become one of
the most sensitive and is often used for different bio analytical
purposes applying fluorescence dyes and quantum dots as labels
[7]. Additionally, it is obvious that analytical methods based on the
amalgamation of an enzymatic reaction with molecular fluores-
cence are one of the most interesting and promising analytical
alternatives [8e12]. Previously, GOx was immobilized on the
polyvinyl alcoholepyrene (PVAePy) matrix prepared by “Click”
chemistry approach and used as a water-soluble probe molecule for
the fluorescence glucose sensing by our group. In that work glucose
detection was carried out by following the increase in fluorescence
intensity due to the diminished quenching of the photo excited Py
molecules based on oxygen consumption through the enzymatic
reaction [7]. On the other hand, fluorescent pH probes are also
interesting due to significant advantages over other techniques,
such as non-destructive character, high sensitivity and specificity
[13,14]. Measurement of pH by fluorescence-based techniques is
well established for both imaging and sensing applications.
Therefore, fluorescence offers significant advantages over other
methods for physiological pH measurements and the wide range of
indicator dyes available [15]. Fluorescent pH indicators within the
physiological range are an attractive target in molecular design and
synthesis.
2.3. Set-up of the fluorescence measuring system
The main part of the micro-analytical system is the measuring
cell. Apart from fluorescence detection in transmittive and reflec-
tive mode, the measuring console was suitable for measurements
under different measuring regimes (as fluorescence alone,
absorption combined with fluorescence). The measuring cell is
located in front of the fluorescence camera which is gathering as
much fluorescence light as possible. The excitation light, passing
the filters for wavelength selection, is directed onto the front of the
measuring cell via mirrors. Additional filters in front of the camera
enable a reduction of the stray light and a pre-selection of the
detection wavelength region [18]. The excitation light can be also
directed to the backside of the cell for adsorption measurements.
Such a modular set-up made the measuring console very flexible to
various measuring regimes. The emitted radiation is taken by
a deep cooled CCD camera. The measuring arrangement, a fluores-
cence reader (Sensovation AG, Stockach), consists of a lamp,
a shutter, filters for excitation and emission (Fig. 1) and a 3-stage
Peltier cooled CCD image sensor with integrated micro-lenses and
a high quantum efficiency (up to 90%) for detecting the fluores-
cence signal.
Here we have presented the planar solegel based fluorescent GOx
arrays. A pH sensitive fluorescent dye (2-(4-tolyl)-4-[4-(1,4,7,10-
tetraoxa-13-azacyclopentadecyl)benzylidene]-5-oxazolone), (CPO)
was mixed in tetraethoxysilane(TEOS)/Chitosan (CHIT) together with
the enzyme and spotted onto the glass slides via a microarrayer. The
response signal of the arrays was measured by following of the pH
induced changes in fluorescence intensity of the dye due to the
enzymatic reaction. As well as optimization studies, analytical char-
acterization sample application were carried out.
2.4. Preparation of TEOS/CHIT/CPO/GOx on the surface
of microscope glass
For the preparation of TEOS/CHIT/CPO/GOx; GOx (1 mg), TEOS/
CHIT (100
mL) and CPO (75 mL, 1 mg/mL) were mixed and spotted to
2. Experimental
the cleaned glass surface and allowed to dry at room temperature
for 5 min. After that, spots were treated with glutaraldehyde
solution (2.5% in potassium phosphate buffer, 50 mM, pH 7.5) for
2 min and washed with distilled water and phosphate buffer
solution, respectively.
2.1. Chemicals and materials
Glucose oxidase (GOx; EC 1.1.3.4, from Aspergillus niger e Type
II-S, 50 000 U/mg), chitosan (CHIT, from crab shells, minimum 85%
deacetylated), tetraorthosilicate (TEOS) were purchased from
Sigma (Germany). Tetrahydrofuran (THF) was purchased from
Merck, KH2PO4 was purchased from Riedel-de HaenÒ (Germany),
D-glucose and glutaraldehyde were purchased from VK Labor und
Feinchemikalien (Germany) and Alfa Aesar (Germany), respec-
tively. The CPO derivative was synthesized and purified using the
general procedure according to our previous study [16].
Different beverages, such as Trendy Cola, apple juice, lemon
juice (brand I), lemon juice (brand II), and mixed fruit juice were
used to analyze glucose content and purchased in a local market.
Initially, the samples were degassed and used as stock substrate
solution with dilution by working buffer.
Solegel/chitosan hybrid solution was prepared by mixing
different ratios of TEOS, 60 ml of ethanol, 150 mL of H2O, 600 mL of
0.5 mg/mL CHIT solutions (final concentration 0.3%). This mixture
was stirred for 1 h until a clear solegel composite was formed and
its pH was adjusted to 4.0e6.0 by using 0.1 M NaOH solutions [17].
2.2. Instrumentation
Spotting of TEOS/CHIT/CPO/GOx on the surface of glass slides
was carried out with a BioRobotics MicroGrid, a High Throughput
Fig. 1. Schematic view of the fluorescence measuring set-up.
Yildirim, Nimet
