This document describes a sensor array that can detect small concentrations of humic substances and heavy metals like copper in water. The sensor uses nanostructured films of poly(o-ethoxyaniline) and sulfonated lignin deposited on gold electrodes. Interactions between these films and humic substances or copper ions were analyzed using spectroscopy and microscopy. The sensor array was able to distinguish samples containing varying concentrations of humic substances or copper based on measurements of the film capacitance. Both the polymer films and bare metal electrodes in the array contributed to its ability to detect trace amounts of target analytes in water samples.

1. Sensors for Detecting Humic Substances and Heavy
Metal Complexes in Waters
Alessandra Firmino" 2, Carlos E. Boratol2, Fabio L. Leite" 2, Osvaldo N. Oliveira Jr.3, Wilson T. L. Silva2 and Luiz H.C. Mattoso2.
I - Pos-Gradua,ao em Ciencia e Engenharia de Materiais - USP - Sao Carlos/SP - Brazil
2 - Embrapa Instrumenta,co Agropecuaria - CNPDIA - Sao Carlos/SP - Brazil
3- Instituto de Fisica de Sao Carlos - USP - Sao Carlos/SP - Brazil
Abstract- The interaction between poly(o-ethoxyaniline)
(POEA) adsorbed onto solid substrates and humic substances
(HS) and Cu2+ ions has been investigated with UV-VIS.
Spectroscopy and atomic force microscopy. Both HS and Cu2+
cause the POEA film to be further doped, and alter the film
morphology. This interaction was exploited in a sensor array
made of nanostructured films of POEA, sulfonated lignin and
HS, which could detect small concentrations of HS and CU2+ in
water.
I. INTRODUCTION
The detection ofhumic substances (HS) in waters is important
due to their role in the interaction and complexation with
heavy metals such as Cu2+. Heavy metals are not chemically
or biologically degraded, and therefore pose a potential danger
to the environment [1,2]. Upon reacting with HS, heavy
metals can be found either in solution or in the solid phase,
being particularly toxic. Metal mobility in soils depends on the
concentration of the metal and on the acidity and buffer
capacity of the soil. If metals bound to HS are released, waters
would acquire large amounts of free metal and have low pH,
thus possessing high toxicity [3]. HS can also alter the
complexation of metallic cations and immobilize them in the
environment [4,5]. In this paper, we analyze the interaction of
solutions containing HS and Cu2+ with nanostructured films of
poly(o-methoxyaniline), a polyanilines derivative whose
optical and electrical properties depend strongly on its degree
of protonation. Such interaction is exploited in a sensor array
made of POEA and sulfonated lignin films adsorbed onto
interdigitated gold electrodes, which is able to detect trace
amounts of HS and Cu2+ ions.
II. EXPERIMENTAL
The conducting polymers used in this work were poly(o-
ethoxyaniline) (POEA) and sulfonated lignin (SL). Poly(o-
ethoxyaniline), POEA was chemically synthesized according
to the procedures in reference [6], in the emeraldine salt form.
Sulfonated lignin (LS) is a polyelectrolytic lignin derivative
obtained from cellulose pulp extraction. Lignin is sulfonated
in acidic media by breaking the bonds in the a carbon of the
phenol units and incorporating HSO3- groups, which causes
the solubility of the material in water to increase [7]. For the
film fabrication, a POEA solution was prepared in a
concentration of 10-3M with pH adjusted to 5.0. The solution
of SL was prepared under the same conditions of
concentration, volume and pH of the POEA solution. The
aquatic humic substances were extracted from water samples
collected in Joao Pereira River, in Bertioga, south littoral of
the Sao Paulo state. They were prepared initially in a
concentration of20 mg L-'. The CuS04 5H20 initial solution
was prepared with a concentration of 50 mgL-'. The sensor
array was developed with nanostructured polymer films
deposited onto interdigitated gold electrodes using the layer-
by-layer (LbL) technique [8-10]. The array contained 6
sensing units made from different materials including poly(o-
ethoxyaniline), sulfonated lignin and humic substances.
Another sensor array, containing 5 sensorial units based on
chrome electrodes with no films was also used. The chemical
interaction between POEA (film and aqueous solution), HS
and Cu2+ ions was evaluated by UV-Vis spectroscopy.
Atomic force microscopy measurements were carried out in
the tapping mode using a Topometrix TMX 2010 atomic
force microscope to investigate the surface topography of
POEA films after interacting with HS and Cu2+. The detection
of HS in water and the complexation with Cu2+ ions were
carried by additions of: I) HS in pure water; ii) CUS04 5H20
in pure water and iii) CuS04 5H20 in HS solution (20 mg L-
1). Impedance spectroscopy was employed to characterize the
water and HS samples. Measurements were taken at 1 kHz
and 200 Hz for chrome electrodes, and the film capacitance
calculated using an equivalent electric circuit for the
film/electrolyte system. The data were treated with Principal
Component Analysis (PCA).
III. RESULTS
Figure 1 shows the UV-Vis spectra of POEA in solution
(a) and in the form of a one-layer film (b). Two peaks appear
in the spectra, the first one at 300 nm due to n-Tt* transitions
of the aromatic rings, and the second at larger wavelengths,
which may denote the doping state. POEA is already partially
doped, particularly in solution. Both in solution and in the
film, interaction with HS or Cu2+ causes further doping,
which is indicated by the shift to higher wavelengths in the
polaronic band at -
720-770 nm. For HS the additional
doping is explained by the acid groups (phenolic and
carboxylic). In the case of CuS04' 5H20 salt, doping may be
attributed to a screening effect by the counterions of the
420

2. positive charges in the conducting polymer, which facilitates
protonation ofPOEA [l 1].
1.5 -
^.1.0 -
0
c)
.0
< 0.5 -
0.0 -
immersed in HS/Cu2+ complex in Figure 2(c). When only Cu2+
interacts with POEA, in the absence of HS, there is apparently
the formation of Cu2+ crystals over the film surface. This
analysis illustrates how the film morphology may be affected
by analytes interacting with the film.
(a) monolayer POEA
(20x20 pm)
monolayer POEA 3D
(2x2 p.m)
x (nm)
(a)
(b) POEA/HS
(c) POEA/HS/Cu2+
POEA/HS 3D
POEA/HS/Cu2+ 3D
(b)
Fig - (a) UV-Vis absorption spectra for interaction POEA (a) solution and
(b) film.
Figure 2 shows AFM micrographs of POEA films, as
deposited and after being immersed for 20 min. into solutions
containing HS, HS/Cu2+ and Cu2+ ions and then dried. For the
as-deposited POEA film adsorbed during 3 minutes, the image
in Figure 2(a) displays a globular morphology with grains
distributed all over the sample surface, similar to images from
the literature [12]. The appearance of smaller domains in
Figure 2(b) may be due to the incorporation of counterions
from HS in the polymeric matrix of POEA, causing
morphology changes [13]. The same occurs for the film
(d) POEA/Cu2+ POEA/Cu2+ 3D
Fig 2 - AFM images for as-deposited POEA film and of POEA films after
being immersed into solutions containing HS, HS/Cu2' and Cu2+.
421
ao
6i
.0
-0
k(nm)
J
,Vwwvw-------. i..rivpX-
,,. 1 12.30so't

3. TABLE I - Average height (Z) and roughness for POEA films: as-deposited
and after being immersed into solutions containing HS and HS/Cu2+ (20x20
Films Z (nm) Roughness (nm)
POEA monolayer (pH =5) 19.4 3.5
POEA monolayer (pH =
5)/ HS (20 ppm; 272 20.7
pH =5)
POEA monolayer (pH = 5)/ HS/Cu2+ (20 567 67.9
ppm; pH = 5)
-. X ,
POEA (pH = 5) + Cu2+ (I ppm) 8000 983
One could hypothesize that the changes in morphology should
also affect the electrical response of sensing units produced
from nanostructured POEA films. In order to verify this point,
we used a sensor array containing the following sensing units:
bare gold electrode, POEA film, POEA/SL film, POEA-LS
(complexed) film, POEA/HS film and HS film. Note that HS
is now used in nanostructured films obtained by physical
adsorption. In a subsidiary experiment, we employed a sensor
array made of five bare chrome electrodes. Measurements
were carried out with the sensing units immersed into pure
water or aqueous solutions containing HS and Cu2+ ions. The
electrical response was analyzed using an equivalent electric
circuit, whose film capacitance was taken as the figure of
merit [14]. The PCA plot in Figure 3 shows that a distinction
can be made of the capacitance data for solutions with various
concentrations of HS (0.5; 1; 5; 10 and 20) mgL-, and pure
water. One can note that the first principal component
increases monotonically with the HS concentration.
1 Pare Water
1i I | |HS20magL
1-
0
= 065 =E
-2 l] HS HSSmgL
0
N
1 .5
AHS1mg/L ~
-2 -165 -1 -0.5 0 0.5 1 1.5
1st Principal Com ponent p8-4%)
Fig 3 - PCA for HS solutions and pure water.
4
° 2
cO
0
CL
E -2
0
i -4
.-
u
N
-10
-128
-14
-6
Fig 4
0 Cu4mg/l
-4 -2 0 2 4 6
1st Principal Com ponent 97.74%)
- PCA for Cu2+ solutions and pure water.
The effects from adding Cu2+ into a HS aqueous solution can
also be monitored with electrical measurements, as indicated
by the PCA plot in Figure 5. The latter contains data for
various aliquots of CUSO4 5H20 added to a HS solution (20
mg L-l). Now, even a concentration of 1 mg LU' of Cu2+ could
be distinguished.
0
E
0
U0.
E0
R'I
3 1,,10Z zm
,HS20mg/L+CulO0mgIL HS 20
mgIL C
2 +H
1 ~~~~~DHS 20 mg/L+ Cu 8mgL
HS 20mg/L Cu 6mglL
HS20mgIL+C Img/L
-2
HS 20mgIL Cu 2mgL
0 HS20mgW+Cu4fL
-6 -4 -2 0 2 4 6 8 1'
lst Principal Component 879A%)
Fig 5 - PCA plot for solutions containing Cu2+ and HS.
The data for the capacitance values when the sensing units are
immersed in CUSO4 5H2O solutions with final concentrations
of 1; 2; 4; 6; 8 and 10 mgL-1 of Cu2+ are shown in the PCA
plot of Figure 4. With the exception of 1 mg L-1, whose signal
almost coincided with pure water, concentrations of 2 mgLU'
or higher could be detected.
422
Cu 2mML +
CulOmglI
Pure Water
Culmgit = = CulSuegL
u Img IC ==
11Cud &i
-
6
8
0
0
2

4. 1-
S
0
E
0
0
Q
C)
u
c.
C4
X 1012
4 ~-ICul
1 Pure Water 1
1' -
Cu 2 muL
- 1- +
0 Pure Water2
-2 O -
-3 X 3
11Pure Water3
-1 -0.5 0 0.5 1 1.5 2 2.5 3
1 st P rincipal Corm ponent 95Mf9%) x 10
Fig 6 - PCA for Cu2+ solutions and pure water, obtained with
the sensor array made up of5 chrome electrodes (no film
deposited).
Interestingly, distinction among the various samples could
also be obtained using a sensor array with 5 sensing units
made of bare chrome electrodes. The electrical response of
these electrodes differed from each other owing to the distinct
morphologies of the electrodes. Fig. 6 illustrates this finding
for detection of Cu2+ ions down to a concentration of 2 mgLJ1.
That a sensor array with no films could be equally efficient in
detecting trace amounts of ions indicates the predominance of
interfacial phenomena governing the electrical response. It is
clear therefore that as far as the sensing ability is concerned,
one may merely employ bare metal electrodes. However, the
distinct interactions with POEA films demonstrated here point
to a further avenue to explore, in which non-specific
interactions may be combined with specific interactions.
Indeed, this appears to be the case of highly efficient sensors
made of nanostructured films of humic acid, which are
capable ofdetecting pentachlorophenol down to 10-10 M [15].
IV. CONCLUSIONS
The interaction between a POEA film in the sensing units and
HS/Cu2+ complexes in liquid samples was investigated using
atomic force microscopy and UV-VIS. spectroscopy. With the
latter we observed that the absorption spectra of POEA films
are altered when in contact with HS and Cu2+ ions. PCA plots
indicated that the sensor array could distinguish between the
water samples containing humic substances and different
concentrations ofCu2+.
ACKNOWLEDGMENTS
This work was supported by Embrapa Instrumentacao
Agropecuairia, Sao Carlos, Fapesp, CNPq, Rede Nanobiotec
and CT-HIDRO (Brazil).
REFERENCES
[1] Branco, S.M., In: A Agua e o Homem. In Porto, R.L.L. Et
Al., Hidrologia Ambiental. Editora Da Universidade De Sao
Paulo: Associacao Brasileira De Recursos Hidricos: V.3.
414p., (1991).
[2] Meybeck, M., River Quality Global Ranges, Time And
Space Variabihtes, Proposal For Some Redefinitions. Verth
Internat. Verein Lininol. Stuttgart. P. 81-96, (1996).
[3] Zhang, M. and Florence, T.M., Anal. Cuim. Acta, V. 197,
P. 137 -148, (1987).
[4] Lombardi, A. T. and Jardim, W.F., Chem. Speciat.
Bioavail, 9, P. 1-8 (1997).
[5] da Silva, W. T. L.; Thobie-Crouter, C.; Rezende, M.O.O.,
Electroanalysis, 14(1), p. 71-77, (2002).
[6] Mattoso, L.H.C.; Manohar, S.K.; MacDiarmid, A.G.;
Epstein, A.J., J. Polym. Sci: Part A: Polym. Chem., 33, 1227-
1234 (1995).
[7] Instituto de Pesquisas Tecnologicas de Sao Paulo. Celulose
e Papel. Tecnologia de fabrica,co de pasta celulosica, ed.2,
Sao Paulo, 1, 84 (1988).
[8] Paterno, L. G.; Mattoso, L. H. C.; Oliveira Jr., 0. N.,
Quimica Nova, 24 (2), P. 228-235, (2001).
[9] Paterno, L. G.; Mattoso, L. H. C., Polymer, 42, P. 5239-
5245, (2001).
[10] Decher, G.; Hong, J. D., Ber. Bunsengs. Phys. Chem., 95
(11), P.1430-1434, (1991).
[11] Patemo, L. G.; Constantino, C. J. L.; Oliveira Jr., 0. N.;
Mattoso, L. H. C., Colloids And Surfaces B: Biointerfaces, 23,
P. 257-262, (2002).
[12] Paterno, L. G.; Mattoso, L. H. C., Journal of Applied
Polymer Science, 83, P.1309-1316, (2002).
[13] Xie, L. K.; Josefowicz, L. J., Journal of Materials
Science, 29, P. 4200-4204, (1994).
[14] Riul Jr., A.; Dos Santos, D. S.; Wohnrath, K.; Di
Tommazo, R.; Carvalho, A. C. P. L. F.; Fonseca, F. J.;
Oliveira Jr., 0. N.; Taylor, D. M.; Mattoso, L. H. C.,
Langmuir, 18, P. 239-245, (2002).
[15] Crespilho, F.N.; Zucolotto, V.; Siqueira Jr., J.R.;
Constantino, C.J.L.; Nart, F.C.; Oliveira Jr., O.N.;
Environmental Science & Technology, in press.
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