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Hybrid coatings as transducers in optical biosensors.

Abstract Sensitive coatings are described for a novel enzyme-based optical sensor for in-situ continuous monitoring of reactants, such as glucose, in biotechnological production processes. Glucose oxidase, incorporated into suitable coating materials that are applied on lenses or optical fibers, is used to catalyze oxidization of glucose to gluconic acid in the presence of oxygen. The presence and consumption of oxygen is determined by measuring the fluorescence signal of incorporated metal organic ruthenium complexes, which is quenched by oxygen. Inorganic-organic hybrid polymers, synthesized via sol-gel processing, were used as coating material. Due to the hybrid character of the coating, good adhesion is achieved on both glass and polymer surfaces. Good compatibility is also given with enzymes and ruthenium complexes. The sensitive optical coating was built up as double-layer and single-layer structures. The double layer comprised a primary coating containing the oxygen-sensitive ruthenium complex, and a secondary coating containing the enzyme. The single layer comprised a single coating containing both the ruthenium complex and the enzyme.

Keywords Biosensor, Optical sensor, Glucose sensor, Oxygen monitoring, Fluorescence, Hybrid coating


In sensor technology, the rapid miniaturization of electronic devices initially resulted in the use of electrically based sensors, followed by miniaturized optical components, causing considerable growth in the application of optical sensors in many fields. Optical sensors are compact, flexible to use, and insensitive to electromagnetic fields. Hence, they are suitable for online monitoring of processes in harsh environments. (1)

Biotechnological processes can be found in food and pharmaceutical industries, waste processing, or environmental protection, and require affordable and rapid sensing techniques. In particular, the monitoring of bioreactants such as glucose, fructose, and glycerol is playing an important role in industrial sectors, such as the synthesis of biofuels and pharmaceuticals in the food and beverage industries.

Process control data are often obtained by taking samples for remote analysis. The resulting time delay can be critical for achieving optimum process control, especially for just-in-time-production.

The concentrations of bioreactants to be measured are often low or not suitable for direct detection, and require enhancement of detection sensitivity by a suitably designed transducer. Many transducers are not suited to direct measurement of these bioreactants due to interference by pH or temperature. (Bio) molecules or (bio) compounds (chemical and biological transducers) are usually immobilized at the detection site (physical transducer).

In general, a biosensor is a sensitive device in which a biological component and a physicochemical detector component are combined for the detection of unknown analytes or unknown concentrations of certain analytes. The main parts of a biosensor and their modes of operation are depicted in Fig. 1.

The work described in this paper exploits the benefits of optical sensors based on special enzymes such as biochemical transducers, incorporated in special hybrid coatings on the physical transducer--e.g., an optical lens or a fiber.


Sensor principle--mode of operation

The glucose sensor described in this paper used the oxygen-consuming enzymatic conversion of glucose to gluconic acid, as shown in the following equation:

glucose + [O.sub.2] [glucose oxidase.[right arrow] [gluconate.sup.-] + [H.sup.+] + [H.sub.2][O.sub.2]

Glucose oxidase is known to be highly selective toward glucose, and therefore interferences with other carbohydrates such as fructose, saccharose, and maltose, etc. can be excluded. The glucose concentration is related to the concentration and depletion of oxygen. which quenches the fluorescence signal of metal organic ruthenium complexes. Thus, the fluorescence quenching of the ruthenium complex is related to the glucose concentration, and is measured via changes in the fluorescence decay lifetime. The fluorescence is excited using blue LEDs at 470 nm.

The most probable interference that has to be considered is the fluctuation of the oxygen concentration in the environment of the analyte--e.g., in the bioreactor. Dissolved oxygen, however, is commonly measured in any bioreactor and the glucose sensor has to be calibrated both with glucose and oxygen concentration in the reaction vessel prior to starting the reaction to be monitored.

The sensitive element consists of an optical substrate--a glass slide, a glass/plastic lens or an optical fiber--coated with a matrix containing glucose oxidase as a sensitive and reactive compound, as well as the ruthenium complex. Thus, the system glucose oxidase/ruthenium/matrix can work as part of an extrinsic sensor, using a transmitting fiber to carry excitation light to the sensor layer in combination with a second fiber that collects the resulting fluorescent light. It can also be used as a cladding layer of an intrinsic fiber sensor using evanescent field excitation.

The sensitive element was developed as both a double-layer structure and a single-layer structure. In the double-layer structure, the optical substrate--glass or optical polymer--was coated with a primary layer containing the oxygen sensitive ruthenium complex, and a secondary layer containing the enzyme glucose oxidase (Fig. 2). In the single-layer structure, the substrate was coated with a single layer containing both the ruthenium complex and the enzyme (Fig. 2).


The glucose sensitivity of the sensor depends strongly on the activity and homogeneous distribution of both the enzyme and the ruthenium complex fluorophores in the coating material. Therefore, the optimum coating material must be selected as the host matrix for the chemically sensitive elements.

Chemical transducers

Hybrid coating materials

Due to their inorganic network inorganic-organic hybrid polymers are expected to exhibit a high mechanical, chemical and thermal stability in harsh environments. Moreover, the presence of functional organic or polymer organic structural units in the hybrid material can control variability and functionality. Thus, these materials are widely used as functional coatings. (2)

Inorganic-organic hybrid polymers are formed by sol-gel processing via hydrolysis and condensation of organically functionalized alkoxysilanes (Fig. 3). The reaction forms an inorganic prepolymer (siloxane) bearing functional organic groups R' or X. The R' groups act as non-reactive network modifiers suitable for network functionalization, for instance to make the coating hydrophilic or hydrophobic. Reactive groups X form an additional organic polymer network by a UV or thermally induced polymerization process. In general this polymerization is used as curing reaction for the coatings.


Enzymes such as glucose oxidase have limited thermal stability. Thermal curing of inorganic-organic hybrid coatings requires temperatures above 100(degrees) C. Therefore, fast UV curing at room temperature was chosen for curing to minimize enzyme decomposition. UV-induced film curing is based on methacryl or vinyl groups as reactive groups X in the starting silane and in the sol-gel derived intermediate product.

A screening program using a range of UV curable hybrid materials showed that different compositions were suitable as primary and secondary coating for both the double-layer and single-layer structures. Selection criteria included adhesion on the substrate (glass or optical polymer) and stability under the type of aqueous conditions in a bioreactor.

The single- or multi-component hybrid layers as they were used in the different sensor set-ups are shown in Fig. 4. The double lenses to form an extrinsic sensor at the end of an optical fiber. A modified double-layer structure was used in an intrinsic fiberoptic sensor. The primary could also be used as single coatings in the single-layer structure.


Chemical oxygen sensors commonly use fluorescence quenching of metal organic ruthenium complexes. (3), (4) It is also well known that the permeation of oxygen increases with the degreasing polarity of the permeable material. (5) Therefore, to optimize the oxygen sensitivity, the sensitive coating must be non-polar or hydrophobic. A matrix with high-oxygen sensitivity was created by minimizing the polarity of the primary coating by incorporation of a long-chained aliphatic octylsilane (OTEO, Fig. 4).

In the first screening experiments, the complex Ruthenium tris-(1,10-phenantroline) dichloride was entrapped in coatings [1.[bar]] and [2.[bar]] by adding an alcoholic solution of the complex to a coating material solution prior to UV curing. UV-cured films of these materials exhibited a significant fluorescence signal, indicating that the fluorescence activity of the ruthenium complex was unaffected. As expected, the fluorescence signal increased with a higher amount of entrapped ruthenium complex (1-5% w/w). Increasing the amount of hydrophobic octylsilane OTEO also reduced the fluorescence intensity (Fig. 5) Reduction of fluorescence was caused by increased hydrophobicity of the coating, which facilitated oxygen diffusion into the layer and enhanced oxygen-based fluorescence quenching of the complex. Thus, a higher oxygen permeation rate will contribute significantly to the oxygen sensitivity of the sensor layers.


Oxygen-sensitive metal complexes

Different ruthenium complexes are described in the literature that can be used as oxygen sensors due to their fluorescence activity, which is quenched by oxygen. (6) The two complexes Ruthenium trix-(1,10-phenantroline) dichloride [Ru-1.[bar]] and Ruthenium tris-(4,7- diphenyl-1,10-phenantroline) dichloride [RU-2.[bar]] (Fig. 6) were investigated with respect to the oxygen sensitivity in the hybrid coating.


The fluorescence intensity of equal concentrations of the different ruthenium complexes was compared in the hybrid coatings identified so far. Selected glucose concentrations based on fluorescence lifetime measurements could be detected with both complexes. Figure 7 shows the detector responses of both complexes at the same level of detector light sensitivity. [Ru-2.[bar]] exhibited fluorescence signal intensity five times higher than [Ru-1.[bar]] indicating that organic substitution at the aromatic phenantroline ligand increases the sensitivity of the complex. The baseline signal of [Ru-2.[bar]] is higher, and in nitrogen atmosphere, the fluorescence lifetime signal increases significantly whereas it decreases again under aerobic conditions due to quenching caused by oxygen. The comparable signal and the effect of nitrogen/oxygen switch are significantly lower with [Ru-1.[bar]]. For further experiments, the light sensitivity of the detector was also optimized to obtain a significant signal with [Ru-1.[bar]]

Additional entrapment of glucose oxidase into a hybrid layer containing oxygen-sensitive ruthenium complexes completed the sensor layer. By adding glucose to a buffer solution, the fluorescence lifetime signal increase, thus indicating that the consumption of oxygen of the sensor reaction could be monitored with ruthenium complexes in hybrid layers (Fig. 7). Again, with [Ru-1.[bar]] the effect was smaller. Despite the higher sensitivity of [Ru-2.[bar]], further investigations with real sensor set-ups were done with the complex [Ru-1.[bar]] because of its lower cost and better commercial availability.


Sensor set-up

Extrinsic sensor

In the double-layer set-up, the primary coatings [1.[bar]] and [2.[bar]], both containing 50% of the hydrophobizing component OTEO and % w/w of the ruthenium complex Ru-1, were applied on microscopic glass slides and cured by UV radiation. Glucose oxidase was mixed with the secondary coating 3, and the mixture was applied on the primary coating and cured by UV radiation. The coated slides were connected to the detector device via optical fibers. The fluorescence quenching of the complex was measured via fluorescence decay lifetime changes caused by glucose concentration in a measurement cell containing a phosphate buffer (Fig. 8). Preliminary experiments with a double-layer sensor using a multi-channel device gave a glucose sensitivity in the 0-3 mmol concentration range.


Within the single layer, both oxygen and glucose migrate into the coating to the sensitive elements--i.e., to the ruthenium complex and the enzyme glucose oxidase. Because glucose is a very hydrophilic molecule, a hydrophobic sensor layer with high oxygen sensitivity will prevent the diffusion of glucose to glucose oxidase. Therefore, in the singly-layer construction, the hydrophobic component for increasing the oxygen sensitivity was not incorporated. In addition, several procedures were optimized in order to stabilize the enzyme by immobilization in a polymer matrix to protect glucose oxidase against loss of activity during the entrapment procedure and UV curing process. These investigations are described elsewhere. Stabilized glucose oxidase and 3% w/w of [Ru-1.[bar]] were entrapped into the non-hydrophobized hybrid coating [2.[bar]]. This material exhibited the highest stability as a single coating in the harsh environment of a real bioreactor, and was tested on PMMA lenses, connected to the measurement device via silica optical fibers.

The concentration range of the single-layer sensor was also 0-3 mmol/L and response time was <10s. The sensor layer was stable under sterilization conditions (alcohol, UV-light) and during a 6-day test in a bioreactor.

Fiberoptic intrinsic sensor

For an intrinsic fiber optic evanescent field sensor, the fiber refractive index must be similar to that of the sensing layer. Because the refractive index of the hybrid materials is around 1.5, polymer optical fibers with an index of 1.51 were used in the intrinsic fiberoptic sensor in combination with the double-layer set-up. The hybrid coating 2 containing 3% w/w [Ru-1.[bar]] without hydrophobic modification was used as the oxygen-sensitive primary layer. Glucose oxidase was immobilized in a glutaraldehyde layer on the primary layer, and was not incorporated into a hybrid layer. Preliminary laboratory experiments showed that glucose concentrations of 1 mmol were detected by fluorescence intensity measurements in buffered aqueous solutions using a double-layer intrinsic fiberoptic sensor (Fig. 9). Some output signal changes are caused by the buffer solution and air, but a significant signal is induced by the enzymatic reaction of glucose.



Inorganic--organic coatings were used as a host matrix for oxygen-sensitive ruthenium complexes, together with glucose oxidase, to form chemical transducers for detecting glucose in aqueous solutions. Both double and single layers can be used for fluorescence detection of the enzymatic reaction of glucose catalyzed by the enzyme glucose oxidase in the sensor layer. The chemical transducers can be used as extrinsic and intrinsic optical sensors. Further work will include continuous inline monitoring of glucose, and extension of the principle to biomolecules such as fructose, saccharose, or glycerol that play an important role in many biochemical processes.

Acknowledgment The authors thank the European Commission for funding this work in project GRD1-2001-C40477.


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K. Rose

Fraunhofer Institut Silicatforschung, Neunerplatz 2, 97082 Wurzburg, Germany


S. Dzyadevych

Laboratory of Biomolecular Electronics, Institute of Molecular Biology & Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnogo St., Kiev 03143, Ukraine

R. Fernandez-Lafuente

Institute of Catalysis, Campus UAM, Cantoblanco, 28049 Madrid, Spain

N. Jaffrezic

Ecole Centrale de Lyon, CEGELY, UMR CNRS 5005, 36 Avenue Guy de Collongue, 69134 Ecully Cedex, France

G. Kuncova

Institute of Chemical Process Fundamentals, Academy of Sciences of the Czech Republic, Rozvojova 135, 165 02 Prague 6, Czech Republic

V. Matejec

Institute of Radio Engineering and Electronics, Academy of Sciences of the Czech Republic, Chaberska 57, 182 51 Prague 8, Czech Republic

P. Scully

School of Chemical Engineering and Analytical Sciences, The University of Manchester, Sackville Street, Manchester M60, 1QD, UK
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Author:Rose, K.; Dzyadevych, S.; Fernandez-Lafuente, R.; Jaffrezic, N.; Kuncova, G.; Matejec, V.; Scully, P
Publication:JCT Research
Date:Dec 1, 2008
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