Download Hybrid Coatings as Transducers in Optical Biosensors

Hybrid coatings as transducers in optical biosensors
for oxygen and glucose monitoring
K Rose1, R Fernández-Lafuente2, S Dzyadevych3,4, N Jaffrezic,
G Kuncová5, V Matějec6 and P Scully7
1
2
3
4
5
6
7
Fraunhofer Institut Silicatforschung, Neunerplatz 2, 97082 Würzburg,
Germany
Institute of Catalysis, Campus UAM, Cantoblanco, 28049 Madrid, Spain
Laboratory of Biomolecular Electronics, Institute of Molecular Biology &
Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnogo St.,
Kiev 03143, Ukraine
CEGELY, UMR CNRS 5005, Ecole Centrale de Lyon, 36 avenue Guy de
Collongue, 69134 Ecully Cedex, France
Institute of Chemical Process Fundamentals, Academy of Sciences of the
Czech Republic, Rozvojova 135, 165 02 Prague 6, Czech Republic
Institute of Radio Engineering and Electronics, Academy of Sciences of the
Czech Republic, Chaberská 57, 182 51, Prague 8, Czech Republic
School of Chemical Engineering and Analytical Sciences, The University of
Manchester, Sackville Street, Manchester M60 1QD, United Kingdom
Corresponding author: [email protected]
Abstract. Sensitive coatings are described for use in 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 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 compatibility is achieved both with glass and
polymer surfaces, with enzymes and ruthenium complexes. The sensitive optical
coating was built up as both a double layer and single layer structure. 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.
1. Introduction
In sensor technology the rapid miniaturization of electronic devices initially resulted in the use
of electrically based sensors, followed by the miniaturized optical components causing
considerable growth of applications of optical sensors in many fields. Optical sensors are
compact, flexible in use, immune to electromagnetic fields and hence, they are suitable for
on-line 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 bio-reactants such as glucose, fructose and glycerol is playing
an important role in industrial sectors such as synthesis of bio-fuels and pharmaceuticals or
in food and beverage industry.
Process control data is often obtained by taking samples for remote analysis. The
resulting time delay can be critical for achieving optimum process control, especially for justin-time-production. The concentrations of bio-reactants to be measured are often low or not
suitable for direct detection, requiring enhancement of detection sensitivity by a suitable
designed transducer. Many transducers are not suited to direct measurement of these bioreactants due to interference by pH or temperature. Usually (bio)molecules or
(bio)compounds (chemical and biological transducers) are immobilized at the detection site
(physical transducer).
The work described in this paper exploits the benefits of optical sensors based on special
enzymes as biochemical transducers, incorporated in special hybrid coatings on the physical
transducer, e.g. an optical lens or a fiber.
2. 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 + O2
glucose oxidase
gluconate¯ + H+ + H2O2
Glucose concentration is related to the concentration and depletion of oxygen, which
quenches the fluorescence signal of metal organic ruthenium complexes. Thus, 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 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 sensitive and reactive
compound as well as the ruthenium complex. Thus, the system glucose
oxidase/ruthenium/matrix can be interrogated as part of an extrinsic sensor using a
transmitting fiber to carry excitation light to the sensor layer in combination with a second
fiber to collect the resulting fluorescent light. Or it can be interrogated as a cladding layer of
an intrinsic fiber sensor using evanescent field excitation.
The sensitive element was developed as a double layer structure and as a single layer
structure. In the double layer structure the optical substrate -glass or an optical polymer- was
coated with a primary layer containing the oxygen sensitive ruthenium complex and a
secondary layer containing the enzyme glucose oxidase (figure 1). In the single layer
structure the substrate was coated with a single layer containing both the ruthenium complex
and the enzyme (figure 1).
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 host matrix for the
chemically sensitive elements.
Double layer structure:
Single layer structure:
enzyme
Secondary layer
Primary layer
Ru complex
Single layer
Ru complex + enzyme
optical substrate
optical substrate
Figure 1 Modifications of the sensor element.
3. Chemical transducers
3.1. Hybrid coating material
Due to their inorganic base 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 (figure 2). 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.
R’
sol gel
O
RO Si
OR
OR
+
X
RO Si
OR
OR
Si
R'
Si
- RO H, - H2O
X
R'
Si
Polymerization
film curing
O
O
O
Si
O
O
O
X' S i O
R
O
O
O
+ H2O
O
X
R'
O
Si
O
O
Si
Si
UV, ΔT
R'
O
XR'
R'
O
O
O
O
O
Si O
Si
Si
O
O
Si
O
O
Si
R'
O
Si
O
R'
O
O
Figure 2 Synthesis scheme of hybrid coating materials.
Enzymes such as glucose oxidase have limited thermal stability. Thermal curing of inorganicorganic hybrid coatings requires temperatures above 100 °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 the double layer structure
and for the single layer structure. Selection criteria included adhesion on the substrate (glass
or optical polymer) and stability under the type of aqueous conditions in a bio-reactor.
The single- or multi-component hybrid layers as they were used in the different sensor
set-ups are shown in figure 3. The 2-layer and the single layer structure were used on optical
lenses to form an extrinsic sensor at the end of an optical fiber. A modified double layer
structure was used in an intrinsic fiber optic sensor. The primary layers could also be used as
single coatings in the single layer structure.
Materials applied as single layer or primary layer:
O
O
O
Si
Si
HS
O
Material applied as secondary layer:
O
O
O
O
O
1
Si
OH
CH3
O
O
O
O
C O
O
O
O
O
O
Si
3
O
O
Si
O
HO O
O C
O
CH3
Si
O
O
O
O
O
Al
O
O
O
O
O
Si
N H2
O
O
Si
O O
O
2
Figure 3 Different hybrid materials used in the different sensor set-ups (shown in
the hydrolyzed form incorporated in the inorganic network).
3.2. Oxygen sensitive components
Chemical oxygen sensors commonly use fluorescence quenching of metal organic ruthenium
complexes [3, 4]. Moreover, it is well known that the permeation of oxygen increases with
decreasing polarity of the permeable material [5]. Therefore, to optimize the oxygen sensor,
the sensitive coating must be highly 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). Simultaneously various oxygen sensitive ruthenium complexes
were investigated (figure 4).
2+
O
N
N
N
2 Cl -
Ru
N
O
Si
C H3
O
N
N
Long chained hydrophobic octylsilane (OTEO)
Ruthenium tris-(1,10-phenantroline) dichloride (Ru-1)
2+
N
CH3
N
2+
Ru
N
N
N
Ru
2 Cl-
3
N
2 Cl -
N
N
CH3
Ruthenium tris-(bathophenantroline) dichloride (Ru-2)
Ruthenium tris-(4,7-diphenyl-1,10phenantroline) dichloride (Ru-3)
Figure 4 Components incorporated and tested in hybrid coatings in order to
create high oxygen sensitivity.
In first screening experiments the complex Ru-1 was entrapped in the coatings 1 and 2 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). However, increasing the amount of hydrophobic octylsilane OTEO reduced the
fluorescence intensity (figure 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, higher oxygen permeation rate will improve the oxygen
sensitivity of the sensor layers.
0 % OTEO
25 % OTEO
Fluoreszensspektrum von KSK/1349 - MEMO/GLYMO/AMEO/Al(OBu)3
Proben für Prag / Manchester (Juni 05)
2200
Counts
1 % Ru
3 % Ru
5 % Ru
2000 2000
1800
1400
1% Ru
3% Ru
5% Ru
Counts
Counts
1 % Ru
3 % Ru
5 % Ru
12001200
1600
50 % OTEO
Fluoreszensspektrum von KSK/1351- MEMO/GLYMO/AMEO/Al(OBu)3/OTEO(50%)
Proben für Prag / Manchester (Juni 05)
Fluoreszensspektrum von KSK/1350 - MEMO/GLYMO/AMEO/Al(OBu)3/OTEO(25%)
Proben für Prag / Manchester (Juni 05)
1% Ru
3% Ru
5% Ru
Counts
1 % Ru
3 % Ru
5 % Ru
600 600
1% Ru
3% Ru
5% Ru
10001000
1400
Counts
400 400
Counts
Counts
800 800
1200
1000 1000
600 600
800
200 200
400 400
600
400
200 200
200
0
0
0
500
500
550
600
650
700
600
700
wavelength [nm] [nm]
Wavelength
750
0
0
500
500
550
600
650
600
wavelength [nm]
Wavelength
700
700
750
[nm]
0
500
500
550
600
600
650
Figure 5 Fluorescence spectra of primary coating type 2 with different amounts of
hydrophobic component (OTEO) and different amounts of Ru-1.
The complex Ru-1 was used in basic investigations because it was commercially
available at low price, important for industrial use of the final sensor. However, other
ruthenium complexes are recommended in the literature with higher oxygen sensitivity, e.g.
Ru-3 [6]. Therefore the fluorescence intensity of three different ruthenium complexes was
compared in the hybrid coatings identified so far. Ru-1 gave the lowest and Ru-3 the highest
signal intensity, with Ru-2 having similar activity to Ru-3 (table 1).
Table 1
Comparison of fluorescence signal intensity of different oxygen sensitive
ruthenium complexes in coating 1. The highest signal is set to 100 %.
Ruthenium complex
Ru-1
Ru-2
Ru-3
700
700
wavelength [nm] [nm]
Wavelength
Fluorescence signal
20
91
100
These results indicate that organic substitution at the aromatic phenantroline ligand
increases the sensitivity of the complex. Moreover, the complex Ru-2 is the most promising
alternative, since its sensitivity is comparable to Ru-3, but it is less expensive. The latter
again is very important with respect to commercial use of the sensor.
750
4. Sensor set-up
4.1. Extrinsic Sensor
In the double layer set-up the primary coatings 1 and 2, both containing 50 % of the
hydrophobizing component OTEO and 1 % 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 UV
cured. 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 (figure 6). Preliminary experiments with a double layer sensor using a multi-channel
device gave a glucose sensitivity over the 0 - 3 mmol concentration range.
Lifetime τ [μs]
1.8
air
1.7
1.6
1.5
3 mmol/l
1.4
1.3
2 mmol/l
N2
0
30
1 mmol/l
60
90
primary layer 1
120 150
primary layer 2
180 210
240
Time [min]
Figure 6 Response of the fluorescence lifetimes of the double layer extrinsic optical
biosensor to additions of glucose and to an air/N2 switch.
Within the single layer, both oxygen and glucose migrate into the coating to the sensitive
elements, i.e. to the ruthenium complex and to the enzyme glucose oxidase. Since 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 single layer
construction, the hydrophobic component for increasing the oxygen sensitivity was not
incorporated. Moreover, in order to protect glucose oxidase against loss of activity during the
entrapment procedure and UV curing process, several procedures were optimized in order to
stabilize the enzyme by immobilization in a polymer matrix. These investigations are
described elsewhere [7]. Stabilized glucose oxidase and 3 % w/w of Ru-1 were entrapped
into the non-hydrophobized hybrid coating 2. This material exhibited the highest stability as a
single coating in the harsh environment of a real bio-reactor 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 < 10 s. The
sensor layer was stable under sterilization conditions (alcohol, UV-light) and during a 6 days’
test in a bio-reactor.
4.2. Fiber optic intrinsic sensor
For an intrinsic fiber optic evanescent field sensor, the fiber refractive index must be similar
to that of the sensing layer. Since the refractive index of the hybrid materials is 1.5, polymer
optical fibers with an index of 1.51 were used in the intrinsic fiber optic sensor in combination
with the double layer set-up. The hybrid coating 2 containing 3 % w/w Ru-1 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
RELATIVE OUTPUT POWER
a hybrid layer. Preliminary laboratory experiments showed that glucose concentrations of 1
mmol were detected by fluorescence intensity measurement in buffered aqueous solutions
using a double layer intrinsic fiber optic sensor (figure 7). Some output signal changes are
caused by the buffer solution and air, but a significant signal is induced by the enzymatic
reaction of glucose.
SIDE ILLUMINATION
POFs, KSK 1349 II+ENZYME layer, cured 10 min
0,80
100 ml of buffer
100 ml of buffer
0,78
Removal of buffer
0,76
+1mM solution of glucose
0,74
0,72
0,70
+1mM solution of glucose
0,68
BUBBLING AIR
0,66
0
100
200
300
400
500
600
700
800
900
1000 1100
TIME [s]
Figure 7 Temporal changes of the fluorescence intensity at 600 nm of a double layer
intrinsic sensor due to addition of glucose to a buffer solution.
5. Conclusion
Inorganic organic coatings were used as host matrix for oxygen sensitive ruthenium
complexes together with glucose oxidase to form chemical transducers to detect glucose in
aqueous solutions. Both a double and a single layer 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 in-line monitoring of glucose and extension of the
principle to biomolecules like fructose, saccharose or glycerol which play an important role in
many biochemical processes.
References
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[2] G Schottner, Chem. Mater. 13 (2001) 3422-3435
[3] B Meier, T Werner, I Klimant, O S Wolfbeis, Sens. Act. B 29 (1995) 240-245.
[4] B D MacCraith, C M McDonagh, G O’Keefe, A K McEvoy, T Butler, F R Sheridan,
Sens. Act. B 29 (1995) 51-57.
[5] M Salame, S Steingiser, Polm. Plast Technol. Eng. 8 (1977) 155-175
[6] B D MacCraith, C M McDonagh, G O’Keefe, E T Keyes, J G Vos, B O’Kelly, J F
McGilp, Analyst 118 (1993) 385-388
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Guisán, R Fernández-Lafuente, J. Biotechnol. 121 (2006) 284-289
Acknowledgment
The authors thank the European Commission for funding this work in the project GRD12001-C40477.