Optical fibre biosensor for oxygen and glucose
monitoring based on Ruthenium/ORMOCER®/Enzyme
Layers
V Matejec1, J Mrazek1, S.V. Dzyadevych,2,3, O. Podrazky1,4, K. Rose5, G.
Kuncova4, L. Sasek6, N. Jaffrezic-Renault3, P.J.Scully7, J, Young7.
1
Institute of Radio Engineering and Electronics, Academy of Sciences of the
Czech Republic, Chaberska, 57, 182 51, Prague 8, Czech Republic
2
Laboratory of Biomolecular Electronics, Institute of Molecular Biology &
Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnogo St.,
Kiev 03143, Ukraine,.
3
CEGELY, UMR CNRS 5005, Ecole Centrale de Lyon, 36 avenue Guy de
Collongue, 69134 Ecully Cedex, France.
4
Institute of Chemical Process Fundamentals, Academy of Sciences of the
Czech Republic, Rozvojova 135, 165 02 Praha 6, Czech Republic
5
Fraunhofer Institut Silicatforschung, Neunerplatz 2, D-97082, Wurzburg,
Germany
6
SAFIBRA s.r.o., Politickych veznu 1233, 251 01, Ricany, Czech Republic
7
School of Chemical Engineering and Analytical Sciences, The University of
Manchester, Sackville Street, Manchester M60 1QD.
p.scully@manchester.ac.uk
Abstract. This paper describes the preparation and performance of sensing layers
formed from ORMOCER®s and xerogels (TEOS), combined with Ruthenium
complexes and coated onto declad polymer optical fibre (POF), polymer clad silica
fibre (PCS) and on the inner surface of special silica capillaries. The sensitised fibres
were characterized by measuring angular distributions of the output power. The
sensor response to gaseous oxygen, dissolved oxygen and dissolved glucose was
measured via fluorescence intensity changes. A best detection limit of 0.5%(vol.) has
been determined for gaseous O2 with selected ORMOCER® sensing layers. Glucose
concentrations were measured to an accuracy of 0.3 mmol/l over a range up to 2
mmol/l when POFs were sensitized with TEOS layers overcoated with the GADimmobilised glucose oxidase.
1.
Introduction
Coatings for novel enzyme based fiber optic sensors were developed for in-situ continuous
monitoring of biotechnological production processes in the European Commission funded
project MATINOES. This project comprised three strands: optical coatings, optical
instrumentation, and optical sensors. Novel inorganic-organic hybrid coating materials
(ORMOCER®s) were combined with Ruthenium complexes and enzymatic transducers for
the detection of species such as glucose, fructose or glycerol, to form sensitized coatings for
optical substrates and claddings for optical fibers [1,2]. The optimisation of the coating
development is presented elsewhere in this conference [3]. Instrumentation for on-line
monitoring of bio-reactants by fluorescence life-time was developed to measure the
fluorescence quenching of Ruthenium complexes by oxygen depletion when the enzyme
reacts with the target species [4].
This paper focuses on the preparation and performance of optical coatings formed from
ORMOCER®s and xerogels (TEOS), combined with Ruthenium complexes to form cladding
layers for optical fibres. The layers were coated onto declad polymer optical fibre (POF),
polymer clad silica fibre (PCS) and onto the inner surface of special silica capillaries to form
hollow optical fibres through which gas or liquid could be circulated. Enzymatic transducers
were immobilized onto the sensing layers by using glutaraldehyde (GAD) vapours.
2. Selection of optical fibres and cladding materials
POF
PCS
PCG
Core
Material Refractive
index
Acrylate
1.50
polymer
1.46
F2 glass
1.60
(Schott)
Diameter
[mm]
1
0.3-0.4
0.3-0.4
PCS
PCG-F2
POF
24
20
10*log(P/P0) [dB]
Fibre
type
SENSITIVITY CURVES
28
16
12
Ormocers
8
4
0
-4
1,36
1,40
1,44
1,48
1,52
1,56
1,60
1,64
Refractive index of immersion
Table.1 Characteristics of optical fibre
cores
Fig. 1 Responses of the investigated fiber-optic
substrates to refractive-index changes of their
cladding
The core refractive index and diameters for various optical fibres are shown in Table. 1.
ORMOCER® sensing layers with a refractive index of 1.5 can be applied to optical fibres in
place of their original cladding to enable evanescent-wave detection of fluorescence excited
from a fluorophore contained within the cladding. Transmission of optical fibres as a function
of cladding refractive-index was measured by coupling an inclined collimated beam at 670nm
into the fibre [5]. For these experiments fibre segments with lengths of 20 -30 cm were used
and the cladding was removed over a length of 4 cm in the centre. The bare core was
immersed in liquids of different refractive indexes (Fig 1). It can be seen that POFs transmit
efficiently when ORMOCER®s sensing layers with refractive index of 1.5 are applied.
The ORMOCER® layers were applied to the optical fibre by dip-coating whilst controlling the
withdrawing velocity. Chemical compositions of the ORMOCER® materials can be found
elsewhere [1,3]. The optical characteristics of the sensing layers applied to the fibres were
characterized by coupling an inclined collimated beam and measuring the angular
distributions of the output power when the sensing layer was exposed to gaseous toluene
(Figs 2a & b). For PCG fibers, the applied ORMOCER® layer caused a small decrease of a
number of guided rays, because the difference between the core refractive index of 1.6 and
the refractive index of the ORMOCER® layer, of 1.5, formed a waveguide with a numerical
aperture of about 0.5. Larger changes of angular distribution were achieved using POF,
because the refractive index of the sensing layer is close to that of the fiber core. The largest
change in angular distribution was observed with PCS fiber, because the transmitted optical
power was guided within the sensing layer, which acted like the fiber core because its
refractive index is greater than that of silica. ORMOCER® clad POFs and PCS fibers
demonstrated a higher detection sensitivity than PCG fibers.
RELATIVE OUTPUT POWER
a) Original fiber
b) Fiber with ORMOCER GU2
1,0
0,8
0,6
0,4
POF
PCS
PCG
0,2
0,0
-40
-20
0
20
40
-40
-20
0
20
40
ANGLE OF INCIDENCE [degree]
Fig. 2 Changes of angular distributions of the output power measured for different types of
optical fibre cores when cladded with ORMOCER® ; a) original fibers and b) fibers cladded with
sensing layer
3. Optical fibre sensor performance
A range of ORMOCER® sensing layers containing the Ru transducer and applied onto fiberoptic substrates prepared in IREE were investigated. These layers were excited by a blue
LED at 480 nm and fluorescence intensity at about 600 nm was measured (Figure 3). The
fluorophore (Ru-tris(4,7-diphenyl-1,10-phenanthroline)2+ complex or RuI was used.
2.1 Sensitivity to oxygen: The sensitivity of detection layers coated on POF and PCS fibres to
gaseous oxygen was determined by measuring fluorescence intensity as a function of
oxygen concentrations in the sensing layer, to form the calibration curves in Figure 4. The
sensitivity of fibre coatings to aqueous oxygen is shown in Figure 5. Changes in fluorescence
intensity of about 2-5% were measured.
Gas phase
ORMOCER KSK 1345 II
ORMOCER GU2 on POF
0,5
3500
LED: cut
Integration time: 120 ms
Average: 1
3000
2500
2000
Transmission
Reflection
1500
Calibration curves
0,4
10*log[P(N2)/P] [dB]
OUTPUT POWER [a.u.]
4000
1000
POF
PCS fiber
0,3
0,2
0,1
500
0,0
0
400
600
800
1000
WAVELENGTH [nm]
Fig. 3
Spectra of the RuI transducer
immobilsed in a
layer of ORMOCER®
measured in the transmission or reflection
arrangement
0
4
8
12
16
20
Oxygen concentration [vol.%]
Figure 4: Calibration curves for detection
of gaseous oxygen/nitrogen mixtures by
ORMOCER® KSK 1345 II coating on POF or
PCS fibers.
2.2 Sensitivity to glucose: Glucose dissolved in buffered (pH~ 6) aqueous solutions was used
to test a double-layer optical fibre sensor composed of an oxygen-sensitive ORMOCER®
layer applied onto the fiber onto which a GAD layer immobilizing glucose oxidase (GOD) was
grafted. Temporal changes of the fluorescence intensity at 600 nm due to changes of
glucose concentrations in solutions were measured (Fig. 6). It was concluded that glucose
concentration can be resolved to about 1 mM using intrinsic fiber-optic sensors in which
GOD is immobilized in GAD layers and oxygen is detected by means of ORMOCER® sensing
layers.
2.3 Comparison of ORMOCER®s to Xerogel coatings: Optic fibre sensors using xerogel
layers based on PhTS or TEOS were also prepared and used for reference measurements.
Xerogel detection layers prepared from PhTS sols were used to immobilize a more efficient
Ru transducer (Tris (4,7-diphenyl-1,10-phenanthroline) ruthenium(II) bis(perchlorate)
(RuII). This novel transducer was also immobilized in ORMOCER® detection layers and
responses of the both types of layers to oxygen in air were tested indicating that the
sensitivity was doubled. The enzymatic transducers were immobilized by using
glutaraldehyde (GAD) vapours.
Double-layer layers based on a PhTS detection layer with the Ru transducer and a GAD
layer with GOD were prepared and tested for glucose detection. The calibration curve is
shown in Figure 7, indicating that glucose can be detected up to concentrations of 1.5 – 2
mM.
Xerogel detection layers based on TEOS and containing the Ru transducer were applied
onto the inner wall of silica capillary, exhibiting very high changes in fluorescence intensity
when exposed to gaseous air (Fig. 8). The arrangement was effectively a hollow optical fibre
with sensitive coating around the inner surface. The sensitivity of these fibres to gaseous
oxygen, dissolved oxygen was measured.
Water
ORMOCER GU2, POF, reflection set-up
1,03
1,01
0,80
Realtive output power
Relative output power
LED: modified
Integration time: 50ms
Average: 5
N2
1,02
air
1,00
Aqueous Solutions
KSK 1349-II layer+Enzyme layer, cured 10 min, POF
air
0,99
0,98
0,97
0,96
removal
addition
of buffer solution
0,78
0,76
+1ml 0.1M solution of glucose
0,74
0,72
0,70
+1ml 0.1M solution of glucose
0,68
0,95
N2
0,94
N2
mass flow 100 sccm
0,93
0
200
400
600
800 1000 1200 1400 1600 1800 2000 2200 2400
Time [s]
Figure 5: Response of a layer of ORMOCER®
applied on POFs to oxygen in air dissolved
in water; reflection set-up.
removal
addition
of buffer solution
BUBBLING AIR
0,66
0
100
200
300
400
500
600
700
800
900
1000 1100
Time [s]
Figure 6: Effect of addition of the phosphate
buffer, effect of air and effects of additions of
glucose solutions to double-layer structure of
ORMOER® layer and a layer containing
glucose oxidase in glutaraldehyde; side
excitation.
4. Conclusion
The performance of POF and PCS optical fibres with ORMOCER® claddings were
characterised by measuring the angular distribution of the output power. The optical fibre
sensor response to gaseous oxygen, dissolved oxygen and dissolved glucose was measured
using fluorescence intensity changes. A best detection limit of 0.5%(vol.) was determined for
gaseous O2 . Glucose concentrations were measured to an accuracy of 0.3 mmol/l over a
range up to 2 mmol/l when POF was sensitised with TEOS layers overcoated with the GADimmobilised glucoseoxidase.
5. Acknowledgements
Financial support from the European Community under Framework 5 ‘Competitive and
Sustainable Growth’ Programme (1998-2002) is acknowledged for GRD1-2001-40477:
MATINOES: Novel Organic-Inorganic Materials in Opto Electronic Systems for the
Monitoring and Control of Bio-Processes.
ORMOCER®: Trademark of Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.
V. in Germany.
Gas phase
TEOS Capillary fibre, side excitation
Calibration curve
PhTS, POF, reflection set-up
0.5
Relative output power
1,02
10*log[P] [dB]
0.4
0.3
0.2
0.1
O2
O2
0,99
tINT=300ms
Average: 10
0,96
0,93
N2
0,90
0,87
0,84
0,81
0,78
0.0
0.0
0.5
1.0
1.5
2.0
Glucose concentration [mM]
Figure 7: Calibration curve for glucose
detection by means of PhTS layers applied
on POF and measured in the reflection
arrangement
N2
N2
0,75
0
500
1000
1500
2000
Time [s]
Figure 8: Response of a silica capillary fiber
with a TEOS sensing layer applied onto the
inner wall to gaseous oxygen; side excitation
was used.
5. References
[1] Patent No.05025177.6: “Novel type of sensor for monitoring of bio-processes using
enzymes and Ru complexes in inorganic-organic hybrid coatings”. Filed 17 November
2005.
[2] L.Betancor, F. López-Gallego, A. Hidalgo, M. Fuentes, O. Podrasky, G. Kuncova, J.M.
Guisán and R. Fernández-Lafuente. ”Advantages of the pre-emmobilization of
enzymes on porous supports for their entrapment in sol-gels”. Biomacromolecules. 6,
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