Surface-enhanced micro-raman detection and characterization of calix[4]arene-polycyclic aromatic hydrocarbon host-guest complexes

Article abstract of DOI:10.1366/0003702054615160

Surface-enhanced micro-Raman spectroscopy (micro-SERS) was used to detect traces of the hazardous pollutant polycyclic aromatic hydrocarbons (PAHs) pyrene and benzo[c]phenanthrene deposited onto a calix[4]arene-functionalized Ag colloidal surface. High spectral reproducibility and very low molecular detection limits (10 8 M) were obtained by using 25,27-carboethoxy-26,28- hidroxy-p-tertbutylcalix[4]arene as host molecule. Films of immobilized aggregated Ag nanoparticles, obtained by chemical reduction with hydroxylamine, were prepared by direct adhesion on a glass surface. The influence of the aggregation degree of the initial Ag nanoparticles on the micro-SERS detection effectiveness was checked. Different relative concentrations of the host (calixarene receptor) and the guest (PAHs) were attempted in order to optimize detection of the pollutant. The obtained results indicated that the detection limit is much lower in the case of benzo[c]phenanthrene than in pyrene when exciting with the 785 nm line of a diode laser. A detailed interpretation of the Raman spectra was accomplished in order to obtain more information about the interaction mechanism of the host-guest complex, which could be useful in the future for the design of powerful-detection systems.

Full text of DOI:10.1366/0003702054615160

Surface-Enhanced Micro-Raman Detection and
Characterization of Calix[4]Arene–Polycyclic
Aromatic Hydrocarbon Host–Guest Complexes
P. LEYTON, S. SANCHEZ-CORTES,* M. CAMPOS-VALLETTE, C. DOMINGO,
J. V. GARCIA-RAMOS, and C. SAITZ
University of Chile, Faculty of Sciences, P.O. Box 653 Santiago, Chile (P.L., M.C.-V.); Consejo Superior de Investigaciones
Cient´ıficas CSIC, Instituto de Estructura de la Materia, Serrano 121, Madrid 28006 Espan˜a (S.S.-C., C.D., J.V.G.-R.); and
University of Chile, Faculty of Chemical and Pharmacological Sciences, P.O. Box 233 Santiago, Chile (C.S.)
troscopy (SERS).9 To accomplish that, we used ca-
lix[4]arene derivatives as self-assembled molecules, ad-
sorbed on Ag nanoparticles. Among a large series of ca-
lix[4]arene derivatives, the 25,27-dicarboethoxy-26,28-
dihidroxy-p-tert-butylcalix[4]arene host molecule
(DCEC) (Fig. 1a) displayed a significant selectivity for
interaction with and detection of PAH molecules bearing
Surface-enhanced micro-Raman spectroscopy (micro-SERS) was
used to detect traces of the hazardous pollutant polycyclic aromatic
hydrocarbons (PAHs) pyrene and benzo[c]phenanthrene deposited
onto a calix[4]arene-functionalized Ag colloidal surface. High spec-
8
tral reproducibility and very low molecular detection limits (10Ϫ
M) were obtained by using 25,27-carboethoxy-26,28-hidroxy-p-tert-
butylcalix[4]arene as host molecule. Films of immobilized aggre-
gated Ag nanoparticles, obtained by chemical reduction with hy-
four benzene rings, mainly pyrene (PYR) (Fig. 1b) and
benzo[c]phenanthrene (BcP) (Fig. 1c). Moreover, a host–
droxylamine, were prepared by direct adhesion on a glass surface.
The influence of the aggregation degree of the initial Ag nanopar-
ticles on the micro-SERS detection effectiveness was checked. Dif-
ferent relative concentrations of the host (calixarene receptor) and
the guest (PAHs) were attempted in order to optimize detection of
the pollutant. The obtained results indicated that the detection limit
is much lower in the case of benzo[c]phenanthrene than in pyrene
when exciting with the 785 nm line of a diode laser. A detailed
interpretation of the Raman spectra was accomplished in order to
obtain more information about the interaction mechanism of the
host-guest complex, which could be useful in the future for the de-
sign of powerful detection systems.
guest interaction mechanism based on a
␲–␲ stacking
interaction was deduced, leading to a charge transfer be-
tween the complex and the metallic surface, which also
induces a notable influence on the surface charge of the
metallic nanoparticle.
Furthermore, in this previous work we carried out mac-
ro- and micro-SERS measurements on Ag colloidal nano-
particles and on immobilized Ag nanoparticles on glass
surfaces, respectively. Since a small surface area is stud-
ied by micro-Raman, the sensitivity of micro-SERS is
much higher than in macro experiments.10 Therefore, the
aim of the present work is to systematize the use of the
micro-Raman technique combined with SERS experi-
ments (micro-SERS) to decrease the molecular detection
limits of PYR and BcP as guest molecular systems and
DCEC as a host molecule. Our ultimate goal is to un-
derstand the structural changes occurring in the molecular
recognition phenomena involved in the interaction of ca-
lixarene derivatives with PAHs, in order to develop a
reproducible measurement tool for single molecule stud-
ies.
Index Headings: Surface-enhanced micro-Raman spectroscopy; Mi-
cro-SERS; Calixarenes; Polycyclic aromatic hydrocarbons; PAHs;
Host–guest complexes; Detection of pollutants.
INTRODUCTION
Molecular recognition with calixarenes capable of size-
selective molecular encapsulation is a topic of current
interest in supramolecular chemistry with promising ap-
plications in sensor design.1–3
No surface-enhanced Raman (SERS) spectra of PAHs
can be obtained in the absence of a host molecule due to
the low affinity of these molecules for adsorbtion on a
metallic surface. However, the situation is different in
functionalized PAHs, such as nitro-PAHs, which can be
attached to the metal surface through the nitro group.4–6
For non-functionalized PAHs another strategy is neces-
sary to bring the analyte to the surface and obtain a SERS
spectrum. One of these strategies is surface functionali-
zation with host molecules such as calixarenes.
EXPERIMENTAL
Materials. Calix[4]Arene. The calixarene employed
for this study was DCEC, since this molecule afforded
the maximum SERS intensification, which was synthe-
sized by us according to described procedures.11 The pre-
cursor, p-tert-butylcalix[4]arene, was synthesized in the
following manner. A mixture of p-tert-butyl phenol, 37%
formaldehyde, and an amount of KOH corresponding to
0.045 mols with respect to the phenol is heated for 2 h
Up to now, few published works have been devoted to
the application of calixarene molecules in the detection
of pollutants through Raman techniques.7,8 In a recent
work, we selectively detected PAH molecules at trace
concentrations by using surface-enhanced Raman spec-
at 110–120 ЊC to produce a light yellow, taffy-like pre-
cursor, which is then added to xylene and refluxed for
1.5–2 h. Filtration of the cooled reaction mixture yields
a crude product that is neutralized and then recrystallized
from chloroform–methanol.
The lower functionalization is achieved by addition of
BrCH2COOEt in alkaline medium afforded by K2CO3.
Received 7 March 2005; accepted 18 May 2005.
* Author to whom correspondence should be sent. E-mail: imts158@
iem.cfmac.csic.es.
0003-7028/05/5908-1009$2.00 /0
2005 Society for Applied Spectroscopy
Volume 59, Number 8, 2005
APPLIED SPECTROSCOPY
1009
᭧

M. This activation is needed in order to increase the
nanoparticle SERS activity by properly modifying the
morphology of the particles.15 Then, 20
␮L of the final
suspension was deposited onto a glass cover slide and
dried at room temperature.
The immobilization of the Ag nanoparticles already
having the host–guest complex afforded a more effective
result, as concerns the SERS signal intensification, than
the a posteriori addition of host and analyte. This is prob-
ably due to the better organization of adsorbates on the
metal surface. An aliquot of the original non-activated
colloid was placed on a glass slide with a shallow groove
(2 cm in diameter and 380 ␮m deep), and then the cover
glass slide containing the dried activated Ag nanoparti-
cles was placed on the groove with the side containing
the dried nanoparticles facing downwards so that the sus-
pension is placed in the groove, as depicted in Fig. 2.
A scanning electron micrograph of an Ag film obtained
by the procedure above is also shown in Fig. 2. As can
be seen, the resulting Ag aggregates have an average di-
Fig. 1. (a) 25,27-dicarboethoxy-26,28-dihidroxy-p-tert-butylca-
lix[4]arene (DCEC), (b) pyrene (PYR), and (c) benzo[c]phenanthrene
(BcP).
ameter of 1–2
several micrometers (1–10
␮
m and are separated by a distance of
m). This distribution is uni-
␮
form throughout the whole immobilized surface. A detail
of an aggregate is also displayed in Fig. 2, showing that
they are integrated with 50–60 nm diameter Ag nano-
particles giving rise to a morphology that is much appro-
priate for the SERS enhancement.
First, 9.25 g of the precursor (8 mmol) were dissolved in
50 cm3 of diphenylether, then 15 g (96 mmol) of
BrCH2COOEt and 13.90 g (96 mmol) of potassium car-
bonate were added to the precursor solution. The DCEC
product was extracted by adding ethyl acetate to the mix-
ture. The precipitate was washed with distilled water and
reprecipitated with acetone.
Spectral Reproducibility. The SERS spectra obtained
from different aggregates are reproducible in what con-
cerns the SERS profiles, although the intensities varied
due to the dependence on the aggregate morphology. This
reproducibility was maintained from batch to batch.
Preparation of Samples for Scanning Electron Mi-
croscopy. The samples obtained by immobilization of na-
noaggregates on glass cover slides were also employed
for SEM observation. All samples were coated at room
temperature with a 3–4 nm layer of gold–palladium (Au/
Pd) using a high-resolution magnetron sputter coater op-
erated at 800 V and 5 mA plasma current to obtain a
coating rate of 0.5 nm/min.
Instrumentation. The micro-SERS spectra were re-
corded with a Renishaw Raman Microscope System
RM2000 equipped with a diode laser (providing a line at
785 nm), a Leica microscope, an electrically refrigerated
charge-coupled device (CCD) camera, and a notch filter
to eliminate the elastic scattering. The spectra shown here
Polycyclic Aromatic Hydrocarbons. Pyrene (PYR) and
BcP were purchased from Aldrich and Merck and used
as received. Solutions of these compounds in acetone
2
(99%) were prepared to a final concentration of 10Ϫ M.
Preparation of the Metal Surfaces and Samples for
Surface-Enhanced Micro-Raman Spectroscopy. Col-
loidal silver nanoparticles were prepared by means of a
novel procedure based on the chemical reduction of Agϩ
using hydroxylamine as the reducing agent.12 These nano-
particles have the advantage of a more uniform distri-
bution of size and shape together with the absence of the
reducing agent (citrate or borohydride) excess and their
oxidation products,13 which could interfere with the
SERS measurements.14 In addition, this colloid could
have better electrical conditions on the surface for the
detection of PAHs, since the number of negative charges,
mainly due to the residual chloride ions, should be much
lower than in the case of citrate and borohydride colloids.
On the other hand, the adherence properties of these chlo-
ride-covered nanoparticles seems to be better, as revealed
by their effective immobilization on glass giving rise to
films by a direct deposition on a glass surface.
were obtained by using a 100ϫ objective. The output
laser power was in the range of 2.0 mW. Spectral reso-
1
lution was 2 cmϪ . A scheme of the Raman system em-
ployed is depicted in Fig. 2.
Scanning electron micrographs were taken in an En-
vironmental Scanning Electron Microscope, (ESEM)
PHILIPS XL30, with tungsten filament operating under
high vacuum mode. The acceleration voltage was 25 KV.
For secondary electrons, the standard Everhard–Thornley
detector was used. Sample coating was accomplished us-
ing a high-resolution magnetron sputter coater Polaron
SC 7640.
Metal films of Ag nanoparticles for micro-SERS mea-
surements were prepared by immobilizing the colloidal
nanoparticles. Previous to this immobilization, an aliquot
of the calixarene in acetone was added to 500
␮L of
silver colloid up to the desired concentration. In the case
of the calixarene/PAH complexes, an aliquot of the PAH
solution, also in acetone, was then added to reach the
final desired host–guest concentrations. Afterwards, the
RESULTS AND DISCUSSION
colloid was activated by addition of 0.5 M aqueous po-
Colloidal Aggregation. Since the Ag nanoparticles
were activated with KNO3, the aggregation effect of the
2
tassium nitrate up to a final concentration of 4
ϫ
10Ϫ
1010 Volume 59, Number 8, 2005

Fig. 2. Raman instrument and sampling device employed for micro-SERS measurements. See text for further details regarding the SEM images.
salt was studied in order to optimize the immobilized Ag
film rendering the most intense micro-Raman signal in
the surface-enhanced Raman spectra. In Fig. 3 the SERS
relative intensity of the most intense bands of PYR (1234
1
1
cmϪ ) and BcP (1376 cmϪ ) are plotted against the nitrate
concentration. A maximum SERS intensity obtained
2
when using a 2
ϫ
10Ϫ M concentration of KNO3 was
the most adequate final concentration, since it induced
the formation of Ag aggregates with optimal morpholog-
ical properties. Thus, this concentration was used to carry
out the micro-SERS experiments.
Below and above that optimal nitrate concentration the
Ag immobilized aggregates do not have as good a mor-
phology due to either a poor aggregation leading to string
aggregates at lower concentrations, or globular-like ag-
gregates at higher concentrations.16
Surface-Enhanced Micro-Raman Spectroscopy of
the Complexes at Different DCEC Concentrations. It
is important to note that no SERS spectra of PAHs can
be obtained in the absence of calixarene. Since the pres-
ence of the host molecule is absolutely necessary for the
observation of any SERS signal coming from the PAHs,
we have obtained the SERS spectra of the DCEC/PAH
complexes by varying both the concentrations of the host
and the guest in order to study the spectral modifications
induced on the calixarene and the PAH molecule as a
consequence of the interaction.
Fig. 3. Relative Raman intensities of the most intense SERS bands of
1
1
PYR (1234 cmϪ ) and BcP (1376 cmϪ ) at different potassium nitrate
concentrations. The relative intensities were calculated by dividing all
the SERS intensities by the SERS intensity observed at the nitrate con-
2
centration at which the maximum SERS intensity was obtained (10Ϫ
The DCEC/PYR and DCEC/BcP complexes were
6
M).
studied with the DCEC concentrations ranging from 10Ϫ
APPLIED SPECTROSCOPY
1011

Fig. 5. Raman spectra of BcP in the solid state (a) and micro-SERS
12
spectra of DCEC/BcP complexes at the following concentrations: 10Ϫ
/
Fig. 4. Raman spectra of PYR in the solid state (a) and micro-SERS
5
5
10Ϫ M (b) and 10Ϫ6/10Ϫ M (c). (Inset) SERS intensity of the 1376
spectra of DCEC/PYR complexes at the following concentrations:
5
5
10Ϫ12/10Ϫ M (b) and 10Ϫ6/10Ϫ M (c). (Inset) SERS intensity of the
cmϪ band at different DCEC concentrations. The intensity measure-
1
PYR 1234 cmϪ band at different DCEC concentrations. The intensity
1
ments were carried out by averaging the values found for the BcP 1376
1
cmϪ band at each DCEC concentration in four different immobilized
measurements were carried out by averaging the values found for the
1
PYR 1234 cmϪ band at each DCEC concentration in four different
aggregates. In the figure the average value as well as the quadratic
standard deviation are displayed.
immobilized aggregates. In the figure the average value as well as the
quadratic standard deviation are displayed.
figure in Fig. 4). Below the latter concentration the SERS
intensity decreases. In the case of BcP, a progressive in-
tensity increase was seen on lowering the calixarene con-
centration such that the SERS intensity at a DCEC con-
centration of 10Ϫ12 M is much higher than at 10Ϫ6 M (see
inset of Fig. 5). This trend could be due to a multilayer
effect of the calixarene when adsorbed on the metal,
which at high DCEC concentrations may avoid the ad-
sorption of the PAH molecule leading to a decrease in
the SERS signal.
The analysis of the PYR SERS bands (Fig. 4) in the
complex also reveals interesting differences with respect
to the solid compound (Fig. 4a) due to the complexation
with the host calixarene, with the trend being similar at
low and high calixarene concentrations (Fig. 4b and 4c,
12
M to 10Ϫ M while keeping the PAH concentration at
10Ϫ M. Figures 4 and 5 show the SERS spectra corre-
5
sponding to the extreme calixarene concentration, i.e., at
10Ϫ6 and 10Ϫ12 M. In the inset figures the SERS intensities
of the most intense bands (1234 and 1376 cmϪ , for PYR
1
and BcP, respectively) are plotted against the DCEC con-
centration. As can be observed, the PAH signal is still
observed at low calixarene concentration (Figs. 4b and
5b). Taking into account the analyzed area (approximate-
ly 1
␮
m2) and the analyte concentration of the added
solution, we have calculated that the total amount of an-
alyzed molecules is about 50, which is in the vicinity of
the single molecule.
We also note the fact that BcP rendered a higher SERS
intensity than PYR at all the calixarene concentrations
analyzed. In a previous work9 we have seen that the
SERS signal of PYR is higher in macro-sampling exper-
iments exciting at 1064 nm. However, the fact that at 785
nm BcP afforded a higher SERS signal could be related
to a resonance effect for the BcP/DCEC complex, which
indicates that the interaction with the calixarene is in
principle different for PYR and BcP. This also points out
the importance of the experimental conditions on the
study of these complex systems. On the other hand, the
SERS intensity of PYR increases on lowering the con-
1
respectively). In general, there is a significant 6–10 cmϪ
1
shift downwards of the SERS bands above 1000 cmϪ ,
even larger than that observed in our previous work,9
probably because of the lower PYR concentration em-
ployed here. The enhancement of in-plane ring bands
such as those at 1616 and 590 cmϪ indicates a perpen-
1
dicular orientation of PYR with respect to the surface.
1
The band at 1400 cmϪ decreases in the spectrum of Fig.
4c, but in other spectra of complexed PYR shown here
this trend is not observed; thus, we suggest that this is a
local effect of the analyzed area.
centration of DCEC from 10Ϫ to 10Ϫ M (see the inset
The BcP SERS spectra also display a significant shift
6
8
1012 Volume 59, Number 8, 2005

Fig. 6. Raman of DCEC in the solid state (a); micro-SERS spectra of
4
DCEC (10Ϫ M) (b); DCEC/PYR complexes at the following concen-
6
4
trations (M/M): 10Ϫ4/10Ϫ (c) and 10Ϫ4/10Ϫ M (d); and micro-Raman
spectra of PYR in solid state (e).
4
Fig. 7. Micro-SERS spectra of DCEC (10Ϫ M) (a); DCEC/BcP com-
downwards for the most intense in-plane ring stretching
plexes at the following concentrations (M/M): 10Ϫ4/10Ϫ (b), 10Ϫ4/10Ϫ
8
6
1
bands at 1600, 1497, and 1384 cmϪ , which is stronger
M (c), and 10Ϫ4/10Ϫ M (d); and micro-Raman spectra of BcP in solid
4
as the calixarene concentration is lowered (Fig. 5b). In
state (e).
1
particular, the band at 1600 cmϪ shifts towards 1574
cmϪ1 when DCEC is at a concentration of 10Ϫ12 M. How-
ever, the relative intensity of the bands does not change
as strongly as in the case of PYR.
the calixarene carboethoxy moiety, such as those ob-
served in the Raman of the solid at 1243 and 1202 cmϪ ,
1
Surface-Enhanced Micro-Raman Spectroscopy of
the Complexes at Different Polycyclic Hydrocarbon
Concentrations. In order to verify the detection limits
imposed by the calixarene concentration, we have ob-
tained the spectra of the PAHs at different concentrations
which are markedly enhanced and shifted downwards in
the complex (Figs. 8c–8e). In particular, the band at 1202
1
cmϪ is very intense in the IR (result not shown here),
thus indicating that it is due to C–O stretching vibrations
in the ester moiety. Other bands that are also sensitive to
the complexation are those seen at 1300, 1122, 1029,
4
by keeping constant the calixarene concentration at 10Ϫ
1
M (Figs. 6 and 7 for PYR and BcP, respectively). We
have employed this relatively high concentration of ca-
lixarene in order to investigate its structural modification
when interacting with the analyte. In this case the bands
ascribed to the PAHs are observed up to concentrations
924, 907, 867, and 749 cmϪ , which are also related to
the ester group and the benzene rings. This suggests that
the spectral modifications induced by PAHs are due to
changes in the carboethoxy side chain and to conforma-
tional changes occurring in the four-benzene calixarene
cycle.
The DCEC ester carbonyl band also undergoes impor-
tant variations upon adsorption and interaction with the
guest, which were commented on extensively in our pre-
vious work.9 In general, the relative intensity of the car-
bonyl band markedly weakens in the complex and is in-
versely proportional to the PAH concentration. This in-
dicates that the interaction strength of DCEC with the
surface increases in the complex.
6
8
of 10Ϫ M in the case of PYR and 10Ϫ M in the case of
BcP. We suggest that the detection limit is not as low at
a relatively high concentration of DCEC (10Ϫ4 M) due to
the larger amount of calixarene molecules existing on the
surface, invoking the multilayer effect described above.
Figure 8 displays the SERS spectra of DCEC (Fig. 8b)
and its complexes with PAHs in the region where the
most important changes were observed, the 1350–600
1
cmϪ region. Many of the changed bands correspond to
APPLIED SPECTROSCOPY
1013

alyte, should be a consequence of the analyte’s own ste-
reochemistry. Since PAHs containing bay-like regions
have been demonstrated to be potent carcinogens,17,18 the
use of calixarene of the DCEC type opens a promising
perspective in the selective calixarene-based detection of
these important pollutants.
CONCLUSION
The micro-SERS technique has been employed in the
detection and characterization of guest–host supramolec-
ular systems at very low concentrations. By using ca-
lix[4]arenes functionalized with a carboethoxy group in
the lower rim under a 1,3 substitution pattern, the detec-
tion limits of PYR and BcP are in the neighborhood of
single molecule detection.
Silver films prepared by immobilizing hydroxylamine-
reduced Ag nanoparticles were demonstrated to be very
sensitive when applied in micro-SERS detection, giving
rise to very reproducible results. The morphological
properties of the Ag nanoparticle aggregates, and thus,
the effectiveness of the enhancement, can be modulated
by changing the amount of potassium nitrate added to the
suspension. The measurements are very reproducible in
terms of spectral pattern, but not in what concerns the
overall intensity, which strongly depends on the optical
properties of the immobilized Ag aggregates.
The SERS spectra of the calixarene/PAH complexes
obtained at different host and guest concentrations indi-
cate that at conditions of high host concentrations the
PAH signal can be dramatically lowered due to a massive
adsorption and probable multilayer formation onto the
surface of the metal. Thus, it is concluded that for ana-
lytical purposes the best micro-SERS spectrum of the an-
alyte should be obtained at relatively low concentrations
of the host molecule rather than the opposite.
The interaction of the PAH with the calixarene host
induces a marked structural change in the analyte and the
host molecule, which seems to depend on the analyte
stereochemistry. While PYR is more affected by the com-
plexation, BcP seems to induce a stronger effect on the
calixarene adsorbed molecule because of its non-planar
structure induced by the existence of bay-like moieties.
Many of the changes induced on the calixarene are re-
lated to the carboethoxy moieties, which are responsible
for the molecule anchorage to the metal surface. Fur-
thermore, the interaction of the PAH molecule with the
calixarene also induces a marked strengthening of the
complex interaction with the metal surface.
Fig. 8. Effect on DCEC of the interaction with PAHs in the 1350–
1
600 cmϪ region: Raman of DCEC in the solid state (a); micro-SERS
4
6
spectra of DCEC (10Ϫ M) (b); DCEC/PYR complex (10Ϫ4/10Ϫ ) (c);
and DCEC/BcP complexes at the following concentrations (M/M):
10Ϫ4/10Ϫ8 (d) and 10Ϫ4/10Ϫ6 M (e). (f) The micro-Raman spectra of BcP
in the solid state.
When comparing the effect on the calixarene spectrum
6
of PYR and BcP at a 10Ϫ M concentration (Figs. 8c and
8e, respectively) a stronger influence was deduced for
BcP. The differences noted between both PAHs in the
complexation with DCEC are probably related to the dif-
ferent stereochemistry of the analytes. In fact, PYR and
BcP exhibit structural differences based on the existence
of a bay-like structure in BcP (Fig. 1).
The steric hindrance between H atoms existing in this
bay moiety induces a non-planar structure in BcP, which
could be in part responsible for the remarkable differ-
ences seen in the distinct interaction of PYR and BcP
with the calixarene host. This non-planar structure could
induce stronger structural changes in the DCEC. Thus,
the accommodation of each adsorbed analyte on the host
molecule is not equivalent. On this basis one can infer
that the observed detection limits, different for each an-
ACKNOWLEDGMENTS
The authors acknowledge project Fondecyt 1040640 from Conicyt
(Chile), the Convenio Conicyt/CSIC 2003/2004, and grant FIS2004-
00108 from Direccio´n General de Investigacio´n, Ministerio de Educa-
cio´n y Ciencia (Spain) and Comunidad Autonoma de Madrid project
number GR/MAT/0439/2004 for financial support. P. Leyton acknowl-
edges project AT 4040084 from Conicyt.
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