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.
1Institute of Radio Engineering and Electronics, Academy of Sciences of the Czech Republic, Chaberska, 57, 182 51, Prague 8, Czech Republic
2Laboratory of Biomolecular Electronics, Institute of Molecular Biology & Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnogo St., Kiev 03143, Ukraine,.
3CEGELY, UMR CNRS 5005, Ecole Centrale de Lyon, 36 avenue Guy de Collongue, 69134 Ecully Cedex, France.
4Institute of Chemical Process Fundamentals, Academy of Sciences of the Czech Republic, Rozvojova 135, 165 02 Praha 6, Czech Republic
5Fraunhofer Institut Silicatforschung, Neunerplatz 2, D-97082, Wurzburg, Germany
6SAFIBRA s.r.o., Politickych veznu 1233, 251 01, Ricany, Czech Republic
7School of Chemical Engineering and Analytical Sciences, The University of Manchester, Sackville Street, Manchester M60 1QD.
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 GAD-immobilised 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
Fibre type / CoreMaterial / Refractive index / Diameter [mm]
POF
PCS
PCG / Acrylate polymer
F2 glass (Schott) / 1.50
1.46
1.60 / 1
0.3-0.4
0.3-0.4
/
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.
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 fiber-optic 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.
Fig. 3 Spectra of the RuI transducer immobilsed in a layer of ORMOCER® measured in the transmission or reflection arrangement / 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.
Figure 5: Response of a layer of ORMOCER® applied on POFs to oxygen in air dissolved in water; reflection set-up. / 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 GAD-immobilised 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.
Figure 7: Calibration curve for glucose detection by means of PhTS layers applied on POF and measured in the reflection arrangement / 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, 1027 – 1030, (2005).
[3] K Rose, R Fernández-Lafuente, S Dzyadevych, N Jaffrezic, G Kuncová, V Matejec and P Scully (2006). Hybrid coatings as transducers in optical biosensors for oxygen and glucose monitoring. Photon 06: Optics and Photonics 2006. The University of Manchester, Sept 4-7 2006.
[4] J.S.Young, P.J.Scully, F.Kvasnik, K.Rose, G.Kuncova, O.Podrazky, V.Matejec, Jan Mrazek (2005). “Optical fibre biosensors for oxygen and glucose monitoring”. OFS-17. 17th International Conference on Optical Fibre Sensors, Voet M, Willsch R, Ecke W, Jones J, Culshaw B, eds, 431-434, (2005).
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