Thin Solid Films 518 (2010) 44504457

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Thin Solid Films

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t s f

Solgel planar waveguides for improved uorescence microarrays

Mlanie Bedu a,c, Gabriel Sagarzazu a, Thierry Gacoin b, Pierre Audebert c,
Claude Weisbuch b, Lucio Martinelli a,
a

Genewave, 172 rue de Charonne, 75011 Paris, France

Groupe de chimie du solide, Laboratoire de Physique de la Matire Condense, Ecole Polytechnique, CNRS UMR 7643, 91128 Palaiseau Cedex, France
Laboratoire de Photophysique et Photochimie Supramolculaire et Macromolculaire, CNRS UMR 8531, Ecole Normale Suprieure de Cachan, Btiment d'Alembert,
61 avenue du Prsident Wilson, 94235 Cachan Cedex, France

b
c

a r t i c l e

i n f o

Article history:
Received 13 August 2009
Received in revised form 8 February 2010
Accepted 9 February 2010
Available online 16 February 2010
Keywords:
Microarray
Planar waveguides
Solgel
Titanium oxide
Fluorescence
Optical materials

a b s t r a c t
The real-time monitoring of the hybridization signal, giving access to the reaction kinetics, can widen the
results of a microarray experiment. Nevertheless, the presence of a strong uorescent mix often degrade the
experimental sensitivity, limiting the interest of this technique: the implementation of an evanescent wave
excitation scheme can represent in this case a real advantage. In this paper, we propose high refractive index
waveguides fabricated by solgel process for evanescent wave microarray applications. The inuence of the
sol composition and annealing parameters on materials microstructure are carefully studied to obtain good
optical properties and high refractive index (n = 1.82.1) using thermal treatments below 250 C.
Monomode TiO2 planar waveguides chelated by acetic acid are used as substrates for waveguide-based
microarray in real-time experiments, demonstrating a signicant increase of the signal to noise ratio.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Microarrays are a very powerful tool in molecular biology, allowing
for the simultaneous measurement of up to hundreds of thousands of
molecular interactions in one experiment. This capability is of particular
benet for example in genotyping, gene expression measurements, or
competitive genomic hybridization experiments [1,2]. The hybridization events are usually detected by labeling the sample using uorescent
or chemiluminescent molecules, or radioactive isotopes. A large effort
has been done in recent years in developing label-free techniques
(imaging ellipsometry [3], surface plasmon microscopy [4]), but
uorescent labeling is certainly the most widely used [1,5] giving a
favorable balance between sensitivity and simplicity of manipulation.
For uorescence microarrays, data acquisition is done by taking a
uorescence image of the microarray using a confocal scanner or a wide
eld imager.
Usually, only the so called endpoint hybridization signal (i.e. after
post hybridization washings and slide drying) can be measured [6].
Endpoint experiments provide however only qualitative information:
the differences in hybridization kinetics between probes in multiplexed
experiments may signicantly alter hybridization signals [7]. The realtime detection of hybridization processes proved to be a real breakthrough [69] in exploiting the potentialities of microarray analysis,

Corresponding author.
E-mail address: lucio.martinelli@genewave.com (L. Martinelli).
0040-6090/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.tsf.2010.02.026

giving access to association and dissociation constants (ka, kd) and

molecular afnities, which can be used to improve the reliability of the
results, to make quantitative measurement of the targets' concentration
in the sample, and also to allow the fast optimization of the experimental
protocols (temperature, washing stringency, ) [6,7,10].
One of the reasons which limited the development of real-time
applications for uorescent microarrays compared to non-labeling
techniques come from the fact that spots must be detected while the
surface is still immersed in a mix containing uorescent targets in a
large concentration. Using conventional uorescence imaging congurations, both hybridized molecules at the surface and ungrafted
species from the mix are similarly excited leading to a strong
background that signicantly reduces the detection limit.
As already suggested in the mid-70s, a strategy to circumvent this
problem is the use of planar optical waveguides to ensure the
excitation of the spots by evanescent waves [811] (Fig. 1). In those
experiments, light is injected in the waveguide (using a prism, a
grating or through end-coupling) instead of directly lighting the
microarray surface and the excitation is ensured by the evanescent tail
of the guided modes. As the intensity of the evanescent eld decreases
exponentially with the distance from the surface of the waveguide,
with a decay length of the order of the wavelength [10], molecules
bonded to the surface can be efciently and selectively excited
compared to supernatant species and the background should thus be
signicantly reduced.
When choosing materials for waveguide-based microarrays,
in addition to the requirements for the microarray itself (possibility

M. Bedu et al. / Thin Solid Films 518 (2010) 44504457

4451

Fig. 1. Schematics of a waveguide-based uorescence microarray (left part) and conventional microarray (right part) surrounded by the hybridization mix.

of chemical functionalization, resistance to hybridization and washing solutions, ) two parameters should be considered in order to
ensure the best excitation: i) the refractive index and ii) propagation
losses.
Refractive index is the main parameter in establishing the
extension of the evanescent wave at the lm/solution interface and
then the ability to excite selectively the spots with respect to the
hybridization mix. The refractive index of the lm must be higher than
the one of the surrounding media (reagent mix and substrate), which
are typically in the 1.33 to 1.52 range in the visible. The penetration
depth dp of the evanescent wave in a medium of refractive index
nmedium for a guided mode of effective index neff at wavelength can
be dened as:
dp =

1 = 2
 2
2
n nmedium
:
2 eff

The shorter the penetration depth is, the more the surface will be
selectively excited compared to the supernatant (uorescent probe
molecules are typically no further than a few tens of nanometers from
the microarray surface) so the refractive index of the waveguiding
layer should be as high as possible. Besides, for higher refractive index,
the power of the excitation at the interface is increased. Since the
relative power at the interface respect to the total power carried by a
given guided mode is maximal for the mode closest to the cut-off [10],
it's convenient to set the waveguide thickness in order to obtain
monomode or few mode waveguides.
The second parameter to be considered in evaluating waveguides
are its optical losses. Losses can degrade the performance of the
waveguide-based microarray at two levels: rst, scattering losses are
responsible for unwanted excitation of the hybridization mix, due to
light that is deviated towards the hybridization chamber. The
quantication of the effect of scattering losses on the background
shows that this is not a real issue even for losses of several tens of
dB cm 1, since the evanescent excitation take advantage of an
enhancement factor [12] which is of the order of 104. Light which is
scattered outside the guide no long takes advantage for this effect, so
unwanted excitation remains several order of magnitude lower than
evanescent eld excitation. A more important effect is that losses are
responsible for an exponential decay of guided power along the
propagation direction, and hence for a non-uniform excitation of the
uorescence over the microarray surface. This can make uncertain the
quantication of the spot intensities, and is responsible for a reduction
in signal dynamics. A manner of circumventing these problems is to
choose a geometry for excitation and detection that limits the
waveguide travelling length. Travelling length can be easily reduce
to 1 cm or less, making waveguides with losses of a few dB cm 1

acceptable for the application to microarrays by using the appropriate

image analysis tools.
Thin lms of high refractive index metal oxides can be adapted to
this kind of applications. Thin lms of SiO2 [13], TiO2 [13], ZrO2 [14] and
Ta2O5 [15] are now widely used for optical devices such as waveguides,
antireective coatings or Bragg mirrors. Many different deposition
technologies can be used such as sputtering [16], chemical vapor
deposition [17], atomic layer deposition [18] or ion-implantation [19].
Most of these techniques are well adapted for Bragg mirrors or
antireective coatings, for which the main requirement is to obtain the
largest possible refractive index, but does not provide lms with
acceptable optical losses for the specic case of planar waveguides,
even if accessible refractive indexes are sufciently high. For this
application, the solgel process appears to be a more appropriate
deposition technique. Its basic principle relies on the deposition of a
thin liquid lm from a precursor solution using spin- or dip-coating.
After deposition, thermal treatments are performed in order to allow
precursor reactions and tuning the lm properties (refractive index,
porosity, mechanical properties). Annealing temperatures generally
range between 300 C and 600 C in order to achieve the inorganic
polymerization and the removal of organic species [14,2027]. The
main advantage of this type of process is that deposition is easy to
implement on various substrates with a low cost. Films exhibit good
homogeneity, good mechanical and chemical stabilities and nally
good optical quality. Besides, the lm characteristics are also easily
tunable by changing the composition of the sol and adding functional
species.
Three main strategies can be used for the preparation of high
refractive index lms using the solgel process: i) hybrid organic/
inorganic materials, ii) nanoparticles/polymer nanocomposites and
iii) pure inorganic materials. Most of the reported works have
concerned ZrO2 or TiO2 because the chemistry of their molecular
precursors is well known and the corresponding bulk oxides have
high index of refraction (2.2 for ZrO2, 2.52 and 2.76 for TiO2 for
anatase and rutile respectively).
In the rst approach, hybrid materials are prepared using metal
alkoxide (zirconium or titanium) mixed with silicium alkoxides
[13,28,29] or organosilanes (eg. -glycidoxypropyltrimethoxysilane,
3-trimethoxysilylpropylmethacrylate [30,31]). According to the literature, these hybrid materials exhibit excellent optical losses
(b1 dB cm 1). Nevertheless, their refractive index is inherently low
(typical values range between 1.40 and 1.65) due to the very low
density of the oxide phase, and thus these materials are not effective
for evanescent wave excitation microarrays.
In the second approach, the high refractive index oxide material is
incorporated into a polymeric matrix as preformed colloidal nanoparticles. Such lms are commonly used for Bragg mirror elaboration

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M. Bedu et al. / Thin Solid Films 518 (2010) 44504457

[32,33] but rarely for waveguiding applications [34] as these lms

usually exhibit high optical losses resulting from inhomogeneities
linked to the size of the particles themselves of from the unavoidable
presence of aggregates. Moreover, the polymeric matrix limits the
refractive index. As an example, Yoshida et al. [34] obtained optical
losses of 1.4 dB cm 1 with a refractive index of 1.56.
The two previous methods are certainly the easiest to implement
and are indeed widely used for the elaboration of dielectric mirrors or
multimode optical waveguides with a thickness that is typically in the
micrometer range [2831]. Their optical properties are nevertheless
not adapted for the elaboration of high index, single mode planar
waveguides with low optical losses. In the third approach we chose,
these requirements can be fullled by homogeneous (i.e. either fully
amorphous or monocrystalline) inorganic oxide layers. The elaboration of such lms has been reported through the direct deposition
from solutions of metal alkoxide (zirconium or titanium) precursors
diluted in alcohol. A chelating agent (acetylacetone [22,35], acetic acid
[20,21,24,26] or diethanolamine [27]) is usually added to prevent
solution gelation and precipitation of oxide particles due to
uncontrolled hydrolysis/condensation reactions after addition of
water or through aging under ambient atmosphere [36]. Films with
refractive indices in the 1.7 to 2.1 range are thus obtained depending
on the annealing conditions.
Optical losses are rarely reported but values spanning a very wide
range from 0.3 and 5 dB cm 1) are noticed [14,2026]. The annealing
step is probably the most critical for obtaining given properties since
the microstructure, which plays an important role in establishing the
optical properties, depends in a large extent on the post-deposition
treatments.
The material is totally amorphous just after deposition, with an
important fraction of organics still present. Then, depending of sol
composition, deposition and annealing conditions, the condensation
of the oxide network go forward, eventually with the nucleation of a
crystalline phase. Considering the densication process, we can see
that the two material properties we are looking for (high refractive
index and low losses) are not compatible unless elaborating
monocrystalline lms, which requires very peculiar conditions (rst
of all the use of a monocrystalline substrate) which are far from the
purpose of this study. The achievement of a high refractive
index through an almost complete densication of the lm implies
the formation of a polycrystalline structure which is detrimental for
scattering losses [2224]. In order to fulll our requirements is it
then necessary to preserve the amorphous structure of the material
during its densication and consequently to limit the total thermal
budget.
Surprisingly, almost no studies have been reported on the
characterization of the lms optical properties for moderate annealing
treatment temperatures [37,38] and the annealing temperatures
generally range between 300 and 600 C. Langlet [38], Burgos [39] and
co-workers developed TiO2 lms with refractive index of 1.90 at very
low temperature (110 C) but does not report corresponding optical
losses. The objective of this study is to investigate the evolution of lm
microstructure during moderate temperature annealing (from 50 C
to 350 C), their impact on the optical properties and nally to
optimize the waveguide fabrication process for realizing waveguides
adapted to evanescent wave excitation microarrays. On the basis of
previously reported processes, we focused our attention on solgel
processes using titanium and zirconium alkoxides stabilized with
acetic acid and acetylacetone. We optimized the corresponding
protocols (composition of the initial solutions of precursors, annealing
treatment) to obtain lms with appropriate thickness (about 100 to
150 nm), refractive index (N1.80) and optical losses as low as
possible. Special attention has been put on the process reproducibility
as well. Such lms are nally used for the design of waveguide-based
microarrays. The low temperature of processing is a major requirement for a number of applications, including deposition on plastics

substrates, on electronic devices for integrated systems [2,40] or for

active planar waveguides elaboration [41].
2. Experimental details
2.1. Solutions preparation
In this work, standard formulations were used in accordance with
previous studies [24,25]. They consist in an alcoholic solution of the
titanium or zirconium alkoxide modied with a chelating agent that is
either acetic acid or acetylacetone. The lm thickness is mainly
determined by the deposition conditions (i.e. rotating speed in the
present case of spin-coating deposition, environmental moisture,),
the concentration and the viscosity of the sol. For that reason, the
alkoxide concentration has been kept larger than needed, and the sol
is further diluted to obtain the exact desired thickness just before
deposition.
Transition metal alkoxides are indeed well known to be very
sensitive toward hydrolysis/condensation reactions that occur when
they are in contact with water either intentionally added into the
solution or from ambient moisture. Chelating agents such as diketones or carboxylic acids are commonly added to stabilize the
growing oxide particles [4244] and then prevent solution gelation
and oxide precipitation. The amount of chelating agent must be
carefully optimized for obtaining a material with the required
properties. The chelating agent/alkoxide molar ratio has to be high
enough to ensure the appropriate stabilization of the sols but it should
also be also kept as low as possible since part of the organics remains
in the lm especially in case of a low temperature process. This may
limit the nal refractive index, but also increase optical losses and
uorescence background since some titanium or zirconium chelates
(such as the acetylacetonate) exhibit a yellow to orange color.
The amount of water added in the sol is another important
parameter of the formulation. Water is required to ensure a partial
condensation of the alkoxide into small particles or polymeric entities.
Here again, the amount of water should not be too high to prevent the
evolution of the sol into a gel, or even the precipitation of a scattering
solid phase. In order to control low amount of water addition, Bahtat
et al. developed a process using titanium alkoxide and acetic acid so
that water is only produced in-situ through esterication reactions [25,26]. But, as we observed in preliminary experiments using
processes with low amount of water, the ambient moisture in contact
with the precursor solution during long time storage or even during
the deposition itself might represent a signicant and uncontrolled
source of water. As a consequence, the condensation state of the sol
prior to the lm formation is not controlled leading to nonreproducible results.
According to the above discussions, systematic investigation of the
amount of reactants leads us to three optimized formulations
(indicated in Table 1). Reproducibility was checked by measuring
the thickness, the index of refraction and the optical losses of lms
from different stock solutions with similar composition, or from the
same solution stored at 4 C for up to several weeks after its
elaboration. The variations are about 0.04 for the refractive index
Table 1
Optimized compositions of the solutions (alkoxide concentration, chelating agent/
alkoxide and water/alkoxide molar ratios) and lms characteristics of the
corresponding lms measured by spectroscopic ellipsometry (refractive index,
thickness) for curing at 100 C for 20 min.
Solution

Film
Refractive
Water/
Chelating
Alkoxide
thickness
index
concentration agent/alkoxide alkoxide
molar ratio (at 605 nm) (nm)
molar ratio

ZrO2/AcOH 0.20
TiO2/AcOH 0.45
TiO2/Acac
0.42

6
9
2

9
9.34
7

1.57
1.87
1.75

151
123
111

M. Bedu et al. / Thin Solid Films 518 (2010) 44504457

and about 10 nm for the thickness after similar annealing treatments.

All solution preparations were achieved under ambient atmosphere.

2.1.1. TiO2 stabilized by acetic acid

For the preparation of TiO2 stabilized with acetic acid, titanium
butoxide (Ti(OBu)4), butanol (BuOH), acetic acid (AcOH), de-ionised
water (H2O) and ethanol are used. The molar ratios: AcOH/Ti = 9 and
H2O/Ti = 9.34 have been optimized to have a good stability and
reproducibility. Using lower values lead to poor reproducibility as
optical losses appeared to be strongly sensitive to atmospheric
moisture during sol elaboration and lm deposition. Butanol (2.98 g,
40 mmol) and titanium butoxide (6.75 g, 20 mmol) are rst mixed
under vigorous stirring at room temperature. Acetic acid (10.72 g,
179 mmol) is then introduced dropwise (the reaction is exothermic as
mentioned by Sanchez et al. [45]) and stirred 30 min at 50 C to
ensure a complete reaction of complexation. The complexation is
usually carried out at room temperature but we assume that the
corresponding nal state is more reproducible and less sensitive to
the environmental conditions at 50 C. The mixture is cooled down to
0 C before dropwise addition of water (3.33 g, 185 mmol) previously
diluted in ethanol (11.12 g, 241 mmol) in order to increase the
homogeneity of the hydrolysis reaction over the sample volume and
thus to avoid the formation of scattering particles. The solution
becomes white and cloudy during the addition but after heating at
50 C for 1 h, the resultant sol is clear, non-scattering, and colorless.
The solution, nally diluted in ethanol at [Ti] = 0.45 mol l 1 is stable
against gelation for more than 4 weeks at 4 C.
2.1.2. TiO2 stabilized by acetylacetone
The solution of TiO2 stabilized with acetylacetone is prepared
using titanium butoxide (Ti(OBu)4), butanol (BuOH), acetylacetone
(Acac), de-ionised water (H2O) and nitric acid (HNO3). Corresponding
solutions are yellow colored due to the presence of Acac/Ti complexes
[36]. To decrease the absorption, the chelating agent to alkoxide ratio
was reduced while maintaining a good stability and reproducibility.
The optimal molar ratios are Acac/Ti = 2, H2O/Ti = 7 and HNO3/
Ti = 0.4. Butanol (50 ml) is rst mixed with acetylacetone (5.88 g,
59 mmol) under magnetic stirring. Titanium butoxide (10.00 g,
29 mmol) is then added dropwise and the resulting solution is kept
under continuous stirring for 15 min. The solution becomes yellow
due to complexation of titanium butoxide by acetylacetone [36]. Deionised water (3.36 g, 206 mmol) and nitric acid (0.55 g, 12 mmol)
are mixed together and then added dropwise to the solution. The
mixture is stirred at 60 C overnight to get a reproducible nal state.
The resulting solution is clear with a yellowish color, and is stable for
several months at 4 C against precipitation.

2.1.3. ZrO2 stabilized by acetic acid

The sol of ZrO2 stabilized with acetic acid is prepared using
zirconium n-propoxide (Zr(OPr)4) 70% in n-propanol, acetic acid
(AcOH) and de-ionised water (H2O) with the following molar ratios
[24]: AcOH/Zr = 6 and H2O/Zr = 9. As zirconium alkoxides are less
reactive than titanium alkoxides, the chelating agent/alkoxide molar
ratio is reduced. The zirconium n-propoxide solution (10.44 g,
22.3 mmol) is rst mixed with acetic acid (8.04 g, 133.86 mmol)
under stirring. The reaction, which is exothermic, is allowed to
continue for 30 min. The solution is rst clear but a white precipitate
appears at the end of the complexation reaction. In the second step,
de-ionised water is added dropwise into the mixture and the
precipitate progressively disappears. After 15 min under continuous
stirring, the solution is clear and transparent. The resulting solution is
diluted three times with ethanol (v/v). This solution is transparent,
colorless and stable against gelation and precipitation for more than
4 weeks at 4 C.

4453

2.2. Film deposition

Thin lms were deposited on microscope slides (25 75 mm,
Ultraclean slides Erie Scientic Company) by spin-coating. All
solutions are ltered through 0.2 m lters prior to lm deposition
by spin-coating under a moisture controlled nitrogen ow. Spinning
speed is 2000 rpm for a time period of 40 s.
Annealing treatments (or curing) are performed using a hotplate
under ambient atmosphere with temperatures ranging between 50
and 350 C for 2 to 60 min.
2.3. Characterization
Thermal Gravimetry Analysis (TGA) was done using a Nietzsch STA
409 PC, heating from 30 to 800 C at a rate of 1 C/min in a ow of both
nitrogen and oxygen. Powder samples were prepared by evaporation
of the solvent under nitrogen ow at room temperature.
The refractive index and the thickness of the coatings were
measured by spectroscopic ellipsometry (HORIBA Jobin Yvon MM16
Mueller Matrix spectroscopic ellipsometer). The data were analyzed
using thin-lm-analysis software (Delta Psi 2) with the New
Amorphous dispersion formula (derived by Horiba Jobin Yvon on
the basis of ForouhiBloomer formula) in order to obtain the
refractive index (n) and the lm thickness (t). The refractive index
values are given at = 605 nm.
The waveguiding properties (guided modes and optical losses)
were investigated by imaging the propagation of light injected into
the waveguide. A prism (high index glass (n = 1.91) is used for
coupling light of a linearly polarized HeNe laser ( = 632.8 nm) in
transverse electric mode. For a given angle of incidence, the incoming
light is coupled to a well dened mode of the waveguide. Details can
be found in Ref. [46]. Scattered light is observed by imaging the
waveguide surface on a CCD camera. As scattering is proportional to
the guided light intensity, the optical losses are evaluated by
measuring the scattered light decay along the propagation direction.
3. Results and discussion
The main objective of this work is to determine the process
conditions leading to thin lms with high refractive index and low
losses using moderate annealing temperatures, compared to the
300 C to 600 C range commonly explored.
We rst identied the best sol candidate for obtaining the desired
properties by comparing the lm properties for TiO2/AcOH, TiO2/Acac,
and ZrO2/AcOH using a low temperature of annealing (100 C,
20 min). The refractive indices and the lm thicknesses are given in
Table 1. The lm thicknesses lie in the targeted range (100 to 150 nm)
for all the formulations. Unsurprisingly, the refractive indices are
much lower than the corresponding bulk materials due to the
presence of organic species that reduce the oxide volume fraction
and density. The difference is nevertheless much more important in
the case of the Zr/AcOH lms, probably due to the lower reactivity of
zirconium alkoxides. In the case of titania lms the refractive indices
are important (larger than 1.75) using either AcOH or Acac as
chelating agent: the acetylacetone formulation leads to a slightly
lower refractive index, which might be explained by the strongest
afnity of the acetylacetone ligand with the titanium ion, preventing
the densication of the TiO2 oxide network.
On the basis of these results, zirconium formulations were
abandoned and attention was focused on the effect of the thermal
annealing process on the optical properties of the lms for the Ti/
AcOH and Ti/Acac formulations.
The processes involved in the densication of the lms, and thus
determining their refractive indices, are complex and occur more or
less simultaneously with a large distribution of activation energies.
The evaporation of volatile compounds present in the sol (free organic

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M. Bedu et al. / Thin Solid Films 518 (2010) 44504457

solvent and water molecules) begins during the spin-coating

deposition. At the same time, the hydroxo species start to condensate
together leading to the densication of the oxide network and the
elimination of water molecules. Then, the organic complex decomposes either through evaporation or calcination of the ligands,
allowing further condensation of the oxide network during the
annealing step. If the thermal treatment temperature is sufciently
high (at least 350 C), crystallization of the oxide phase takes place
followed by sintering of the crystallites and elimination of the residual
porosity [22,27,41]. The complete densication of the lms may
require annealing temperatures of more than 1000 C for overcoming
the large activation barrier of diffusion in the solid phase. The
evolution of the Ti/AcOH and Ti/Acac lm thicknesses (t) and
refractive indices (n) determined by spectroscopic ellipsometry as a
function of the annealing temperature is presented in Fig. 2a. The
annealing duration in this set of experiments was xed to 15 min. The
main evolution of the refractive index occurs between the ambient
temperature and 200 C with an almost constant slope and an
inexion point at about 150 C. The index variation from 50 C to
200 C is of around 0.25 units for both Ti/AcOH and Ti/Acac. The Ti/
Acac lms exhibit a lower refractive index than that of the Ti/AcOH
lms all over the investigated range of temperature. Annealing
treatments at 200 C lead to lms with refractive indices of 2.0 and
1.94 for Ti/AcOH and Ti/Acac lms respectively. Raising the annealing
temperature up to 350 C, further increases the refractive index by
only 0.06 and 0.08 units for the Ti/AcOH and Ti/Acac lms
respectively. At the same time the lm thickness reduces with almost
the same (but opposite) behaviour as the refractive index.
In order to check the inuence of the duration of the annealing
treatment, i.e. to separate kinetic to thermodynamic effects, the
evolution of refractive index and thickness as a function of the

Fig. 2. a) Inuence of the annealing temperature for 15 anneals on the optical index n
(at 605 nm, squares) and the thickness t (triangles) for TiO2/AcOH (full symbols) and
TiO2/Acac (open symbols). b) Inuence of the treatment time at 100 C (left column)
and 200 C (right column) for TiO2/Acac (open symbols) and TiO2/AcOH (full symbols).

annealing time, from 2 to 60 min, were plotted (Fig. 2b) in the case of
treatments at 100 C (considered as the lowest processing temperature) and 200 C (at which the rst rapid evolution of the material is
complete see Fig. 2a). The Ti/AcOH and Ti/Acac formulations exhibit
a very different behaviour in the case of the 100 C treatment,
explained by the higher stability of Ti/Acac complexes: in the latter
case the complex are sufciently stable at 100 C to prevent any
material evolution during the annealing, while an important variation
for both the thickness and the refractive index is observed for the Ti/
AcOH formulation. For 200 C treatment, the refractive index
increases and the thickness decreases with the annealing duration
for both the chelating agents, with an asymptotic value reached after
about 1520 min.
Since the refractive index of a solgel material depends on a large
extent on the degree of densication of the inorganic oxide network,
on the presence of residual organic compounds, and on porosity, we
focused our attention on the investigation of the physical chemistry of
the densication process and on the structure of the lm.
First of all, X-ray diffraction experiments conrmed that lms
treated up to 350 C are amorphous (i.e. coherence length of the oxide
network is no more than a few lattice parameters), as observed in
previous studies [21,37,47]. From this result, we may infer that the
increase of the refractive index is only a consequence of the increasing
volumetric fraction of the amorphous oxide phase over the organic
one during the lm shrinkage (removal of organic species and
condensation reactions of the oxide network). Besides, the refractive
index of TiO2 lms remains weaker (2.06 for Ti/AcOH and 2.02 for Ti/
Acac) than crystalline phase even after annealing at 350 C.
The removal of the organic compounds in TiO2/AcOH and TiO2/
Acac was studied by TGA on powder, with the assumption that
information is also valid for lms. Results are shown in Fig. 3,
presenting the evolution of the organic mass fraction in the samples as
a function of the temperature. The mass fraction has been calculated
from raw TGA data assuming that all the organic compounds have
been eliminated at 800 C.
The samples used for these experiments were only dried under
nitrogen ow, so that some residual solvent could remain. The
important mass loss at 90 C in the case of the Ti/Acac powder is likely
due to the evaporation of residual butanol. Most of the mass loss
occurs up to 350 C for TiO2/AcOH and 400 C for TiO2/Acac. The
difference between the two compositions is in accordance with the
higher stability of the acetylacetone ligand and explains the lower
refractive index of the corresponding lms. Moreover, a slow mass
loss is still observed up to 800 C for TiO2/Acac which may be due to
the elimination of carboneous degradation species or of water
produced in last stages of the condensation reaction.

Fig. 3. Mass fraction of organics in TiO2/AcOH (full line) and TiO2/Acac (dotted line)
plotted against temperature (C) as deduced from Thermal Gravimetric Analysis. The
mass of the inorganic fraction is set as the mass measured at 800 C, so
MT M 800 C
:
%organics =
M T

M. Bedu et al. / Thin Solid Films 518 (2010) 44504457

The simultaneous analysis of material weight loss and optical

properties can provide precious information on its evolution and help
in identifying the process conditions best suited for obtaining some
specic properties. Qualitatively, we observe the main evolution of
thickness and refractive index between 100 C and 200 C, while the
mass loss is more important in the 250 C to 400 C range. To explain
this difference, we made the hypothesis that up to 200 C, the oxide
network remains soft and exible enough to ll almost all the empty
volume left because of the organics elimination. This leads to the
observed increase of the refractive index and decrease of the lm
thickness. In order to check this interpretation, we set up a
phenomenological model for quantifying the evolution of the
refractive index as a function of the mass loss measured by TGA. Our
rst hypothesis is that lms can be described as composed of two
phases: the inorganic TiO2 phase and an organic phase. The relative
volume fraction of the two phases is deduced from TGA data using the
value of 1 g cm 3 for the organic phase and 3.9 g cm 3 (corresponding
to the anatase phase) for TiO2. The expected refractive index for the
material is then calculated in the effective medium approximation. The
reference value for TiO2 is again assumed to be the one of the anatase
(2.52), while the refractive index of the organic phase is the free
parameter of the model. The value we used is the one providing the
best t for the measurements corresponding to low ( 150 C)
annealing temperatures: we obtain 1.65 in the case of TiO2/AcOH
and 1.67 for TiO2/Acac. These values are quite high for organic phase
but it is due to the fact that, in our model, the partially condensed TiO2
is considered as part of the organic phase. In Fig. 4 the values of the
refractive index calculated from this model are plotted as lines
together with the ellipsometry data. The model provides a good t up
to 200250 C, conrming the validity of the two-phase hypothesis at
least for low temperature treatments.
An important discrepancy between observations and our model
appears as temperature go beyond 250 C. For temperatures ranging

Fig. 4. Comparison between experimental lm thickness (full squares) and refractive

index (open squares) evolution against temperature (C) and computed data
(thickness in full line and refractive index in dotted line) from Thermal Gravimetry
Analysis for a) TiO2/AcOH and b) TiO2/Acac.

4455

from 250 to 350 C, the observed evolution of lm thickness and

refractive index is signicantly lower than expected in our model.
Although this temperature range corresponds to most of the
elimination of the organics form TGA curve, this does not seem to
lead to densication of the lm. It must then be concluded that our
two-phase hypothesis is no longer valid in this range of temperature
and some modication in the lm structure must be taken into
account. We may assume that the oxide network begins to be too rigid
and part of the volume initially occupied by the organics is now
replaced by some porosity. The slow evolution of the lm refractive
indices thus arises from a competition between the pore formation
and a limited densication of the lm.
This change in material microstructure can lead to an important
change in the waveguiding properties. Commonly, high baking
temperatures are preferred to obtain high refractive index material.
However, a few works report the increase of optical losses with the
annealing temperature on solgel ZrO2 and TiO2 lms between 300 C
and 600 C [22,24,26]. The deterioration of optical quality with the
annealing temperature is generally explained by the increase of
surface roughness [25,26] or the presence of crystallization nuclei in
the lm [22,24]. Considering the low process temperature we used in
this study, we can exclude the presence of crystalline precipitates. On
the other hand, the root mean square surface roughness measured by
atomic force microscopy is systematically lower than 1 nm for all the
annealing conditions.
Nevertheless the optical quality measured on TiO2/AcOH lms
(Fig. 5, panels a and b) clearly degrades as the annealing temperature
or time increases.
This behaviour can be explained on the basis of the lm evolution
evidenced from the TGA analysis: the appearance of some porosity in
the lm can explain an increasing in scattering losses due to index
contrast between the pores and the oxide. In Rayleigh approximation,
which is valid in the case of scattering from small particles, the
scattering efciency is proportional to (n22 n21)2, n1 and n2 being the
refractive index of scattering particles and of the surrounding media.

Fig. 5. a) Optical losses are plotted against the annealing temperature ranging from 50
to 350 C with 15 min duration (full triangles) b) Optical losses evolution with
treatment time ranging from 2 to 60 min at 100 C (open squares) and 200 C (full
stars) c) All data from a) and b) are plotted against the corresponding refractive indices.
Symbols are common for all plots.

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M. Bedu et al. / Thin Solid Films 518 (2010) 44504457

Fig. 6. Real-time monitoring of hybridization using evanescent excitation. Backgroundsubtracted uorescence of hybridized spots (triangles) and negative controls (circles) is
plotted against time. The rst hybridization is carried out at 5 nM of Alexa 647 labeled
oligonucleotides for 75 min. Three successive washings are then performed and the
second hybridization mix is injected after 105 min. The second hybridization mix
concentration in Alexa 647 labeled oligonucleotides is 100 nM. This hybridization is
monitored during 60 min. The signal of reference spots is also plotted (dotted line).

As far as some porosity starts to develop, the refractive index of the

surrounding medium suddenly decreases, explaining the sharp
increase in optical losses. A clear conrmation of this effect can be
seen by plotting the waveguide losses as a function of the lm
refractive index (Fig. 5c).
Films with refractive index lower than 1.982.00 (annealed at
100 C or at 200 C for a short time period) exhibit optical losses that
are typically 23 dB cm 1. Then, a sharp increase of the losses is
observed for lms with a refractive index higher than 2. This value
corresponds to the upper limit at which the lm evolution can be
described by a two phase model (inorganic and organic). As soon as
the refractive index exceeds the value of 2, the two phase model was
no longer able to describe the lm evolution and we inferred that the
further condensation of the lm implies the appearance of some
porosity, because of the matrix hardening.
Giving these results, we used in microarray experiments waveguides elaborated using the Ti/AcOH formulation with annealing
treatments at 100 C for 20 min or 200 C for a few minutes, having a
refractive index ranging between 1.80 and 1.95 with sufciently low
optical losses (23 dB cm 1).

As a rst step, the waveguide surface is functionalized for allowing

the linking of molecular probes to the surface. This step is performed in
gas phase with an epoxysilane (3-glycidoxypropyltrimethoxysilane)
using the same conditions as standard glass slide functionalisation [37].
The spotting is then performed using a spotter robot (BioRobotics
Microgrid Compact Plus) equipped with a MicroSpot 2500 Split Pin
96/384. Three different samples are spotted in a spotting buffer
(phosphate, 0.05% sodium dodecyl sulfate (SDS)): control Alexa 647
labeled oligonucleotides (50 base pairs) at 5 M, non-labeled probe
oligonucleotides (50 base pairs) at 10 M either complementary to
the sequence that will be used as target in the hybridization mix
(positive probes, for which an hybridization signal is expected) and
non-complementary (negative probes). The spot diameter is 290 m.
Control spots are printed for easily locating of the spotted area, quality
control of the spotting (e.g. size and homogeneity of the spots), and to
estimate the binding efciency. The control spots also allow for checking
any further probe release during the hybridization and eventually for
correcting the signal intensities accordingly.
After the spotting, the surface is blocked by a 4 sodium saline
citrate (SSC), 0.1% SDS, 0.1% bovine serum albumin mix during 30 min
in order to avoid non-specic binding of oligonucleotides on the
surface and washed in three successive steps: (Wash 1) 1 SSC, 0.2%
SDS, (Wash 2) 0.1 SSC, 0.2% SDS and (Wash 3) 0.1x SSC for 2 min each.
A 100 m thick PDMS hybridization chamber is then xed on the
microarray. Two hybridization mixes having different concentrations
of oligonucleotides (5 and 100 nM) in a 5 SSC, 0.1% SDS, 25%
formamide buffer were injected. The hybridization of 5 nM mix is
observed in real time during 75 min. Then, after the same postblocking washings 1, 2 and 3, the second hybridization mix containing
100 nM of Alexa 647 labeled oligonucleotides is injected and the
hybridization is observed for 60 min. All steps are performed at room
temperature. Fluorescent imaging for real-time monitoring of hybridization are obtained using evanescent excitation (prism coupling of a
HeNe laser at = 632.8 nm). Some reference images are also
acquired using direct excitation in order to compare the performance
of both excitation schemes.
The real-time uorescence signals of controls, positive and
negative spots are plotted in Fig. 6 against time. In the presence of
the 5 nM hybridization mix, the signal of hybridized spots increases
monotonically, and a slight signal decrease is observed during
washings due to the stringency of washing solutions. When the
100 nM hybridization mix is injected, the uorescence signal sharply
increases and reaches an asymptotic value in 2030 min. The signals
of the negative spots remain undetectable during all the experiment
proving that there is no non-specic binding.
The advantage in using evanescent wave excitation for real-time
analysis of hybridization is straightforward when considering the data

Fig. 7. Fluorescent images of the same microarray taken in presence of 100 nM hybridization mix and excited by (left panel) evanescent wave or (right panel) direct excitation. Three
types of spots are present: positive controls, negative controls and hybridized spots. Proles correspond to white dotted arrows.

M. Bedu et al. / Thin Solid Films 518 (2010) 44504457

of 100 nM hybridization. In Fig. 7, uorescence images of the

microarray after the injection of the 100 nM hybridization mix are
shown. The images on left and right panels have been excited by
evanescent and conventional far eld excitations, respectively. When
evanescent eld excitation is used the spots clearly stand out from the
background, which is really close to the camera dark value even for
an exposure time at which spots signal nearly saturate. When
excitation of the uorescence is done by standard, far eld lighting,
the background level increases dramatically. The spots are still
detectable but the dynamics of the image is signicantly reduced
and the signal to noise ratio, and hence the detection limit, degrade.
The background in evanescent eld excitation is reduced by a factor
750 for having the same signal at the spots.
The non-uniformity of the excitation is due to the punctual light
injection in prism coupling conguration. The image quality can be
largely improved by using other light injection schemes, like grating
or end-coupling.
4. Conclusion
In this work, we discuss high refractive index waveguides
fabrication by solgel deposition from metal alkoxides and chelating
agents. The inuence of the sol composition and the post-deposition
annealing treatment on the waveguide properties was investigated
using spectroscopic ellipsometry and propagation losses measurements. The best properties have been obtained by using a titanium
alkoxide solution and moderate temperature of annealing. By
following the material evolution by thermogravimetry analysis, Xray diffraction, and atomic force microscopy for different sol
composition and by linking those data with the optical measurement,
a model of the microstructure and optical properties evolution during
thermal treatments is proposed.
Monomode planar optical waveguides with refractive index about
1.95, 110120 nm thickness and optical losses of about 2 dB cm 1
were obtained for moderate annealing temperature (100200 C)
using the TiO2/AcOH solution. Such waveguides have been used in
evanescent eld excitation, real-time microarray experiment. The use
of the evanescent excitation scheme improves signicantly the signal
to noise ratio respect to a standard far eld excitation. Thanks to such
a device, hybridization can be followed in real-time experiments even
in presence of a strong uorescent mix, simplifying the access to
target/probe hybridization kinetics.
Acknowledgements
The authors acknowledge the help of the Genewave team. This
work has been partly supported by the French ANR 2005 BRIO+
project.
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