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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
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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
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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
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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
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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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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).
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.
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