Environ. Sci. Technol. 2005, 39, 5385-5389
Immobilization of Humic Acid in Nanostructured Layer-by-Layer Films for Sensing Applications FRANK N. CRESPILHO,† VALTENCIR ZUCOLOTTO,‡ J O S EÄ R . S I Q U E I R A , J R . , ‡ CARLOS J. L. CONSTANTINO,§ FRANCISCO C. NART,† AND O S V A L D O N . O L I V E I R A , J R . * ,‡ IQSC, Universidade de Sa˜o Paulo, CP, 13560-970, Sa˜o Carlos, SP, Brazil, IFSC, Universidade de Sa˜o Paulo, CP 369, 13560-970, Sa˜o Carlos, SP, Brazil, and FCT, Universidade Estadual Paulista, CP 467, 19060-900, Presidente Prudente, SP, Brazil
Humic acids (HAs), naturally occurring biomacromolecules, were incorporated into nanostructured polymeric films using the layer-by-layer (LbL) technique, in which HA layers were alternated with layers of poly(allylamine hydrochloride) (PAH). Atomic force microscopy (AFM) revealed very smooth films, with mean roughness varying from 0.89 to 1.19 nm for films containing 5 and 15 PAH/HA bilayers, respectively. The films displayed electroactivity, with the presence of only one reduction peak at ca. 0.675 V (vs Ag/AgCl). Such a well-defined electroactivity allowed the films to be used as highly sensitive pesticide sensors, with detection of pentachlorophenol (PCP) in solutions at concentrations as low as 10-9 mol L-1.
Introduction Humic acids (HAs) are organic macromolecules bearing colloidal and polyelectrolytic characteristics, which result from the microbiological decomposition of animals and vegetables (1). They can be found in waters, soils, vermicomposts, and peats. Vermicomposts stem from microbial degradation in the intestine of earthworms, whereas peats represent a type of dark color organic matter with varied decomposition degrees, usually found near rivers and lakes. HAs exhibit large ability to interact with herbicides due to their hydrophilic and hydrophobic sites (1-6). Recent efforts have been directed to understand the chemical properties of HA, and to elucidate the mechanisms involved in its coordination to metallic cations or in the adsorption of herbicides and other water and soil-pollutants (7-12). HAs contain a wide variety of oxygen-containing functional groups, such as carboxyl (COOH), carbonyl (CdO), and hydroxyl (OH), which are capable of complexing metal ions. These substances are the most important complexing ligands for metal ions in natural waters (1-7). Schulten and Schnitzer (2) proposed a structure model for HA, based on pyrolysis mass spectrometry (Py-MS) results, with large predominance of aliphatic groups (1, 2). In the latter work, however, the * Corresponding author phone: +55 16 3373 9825; fax: +55 16 3371 5365; e-mail:
[email protected]. † IQSC, Universidade de Sa ˜ o Paulo. ‡ IFSC, Universidade de Sa ˜ o Paulo. § Universidade Estadual Paulista. 10.1021/es050552n CCC: $30.25 Published on Web 06/15/2005
2005 American Chemical Society
authors did not include quinone groups, which are largely responsible for the redox reactions of the molecule (3, 7). Electrochemical applications of HA include its use in modified carbon paste electrodes for detection of metal ions in aqueous solutions (7), where the performance of the electrodes could be tailored by varying parameters such as the amount and nature of HA in the carbon paste. A new approach for HA manipulation has been reported by Galeska et al. (13), in which HA was immobilized in thin solid films using the electrostatic layer-by-layer technique (LbL) for use as semipermeable membranes. The growth of the HA-based films presented strong dependence on pH and ionic strength of HA solutions, which correlated with the degree of ionization of the carboxyl groups and neutralization-induced surface spreading. In this work, we report on the fabrication of LbL films comprising HA and poly(allylamine hydrochloride) (PAH), where HA is exploited in active layers (Scheme 1). PAH/HA LbL films displayed a well-defined electroactivity, which allowed their use as detectors for pesticides. Pentachlorophenol (PCP) was employed as a model compound for the development of the HA-based electrochemical sensors. PCP is a persistent chemical widely used for wood preservation, still in use in the U.S. (14). The concentration limit for PCP established by environmental councils for waters is generally 10 µg L-1. European regulatory boards compromised on a limit value of 5 mg PCP kg-1 dry substance for waste wood to be recycled (15, 16). The maximum residue limit established for natural waters by the Brazilian Environmental Agency is 10 µg L-1 (17).
Experimental Section PAH, MW ) 65 000 g mol-1, was purchased from Aldrich Co. and used without purification. HA from peat was obtained from a tropical region in Sa˜o Paulo, Brazil. Details concerning the HA extraction, purification, and characterization can be found in ref 18. Briefly, HA was extracted from peat using NaOH solution and purified according to the International Humic Substances Society (IHSS) (19). Nanostructured thin films comprising up to 25 PAH/HA bilayers were assembled using hydrophilic glass, ITO-covered glass, and silicon wafers as substrates. PAH solution was used at a concentration of 0.5 g L-1 and pH 6.0. For HA, a concentration of 0.5 g L-1 and pH ) 9.0 was set to ensure complete dissolution of HA. At this pH, most COOH groups are ionized, and ionized phenolic groups can also participate in the adsorption process. The sequential deposition of multilayers was carried out in a HMS series programmable slide stainer (Carl Zeiss Inc) by immersing the substrates alternately into the polycationic PAH solution for 5 min and in the anionic HA solution for 5 min. After each layer deposition, the substrate/film system was rinsed in the washing solution and dried under a N2 flow. The growth of the multilayers was monitored via UV-vis spectroscopy. Film morphology analyses and thickness determination were carried out with an atomic force microscope (AFM) Nanoscope III (Digital Instruments). Fourier transform infrared spectroscopy (FTIR) measurements were carried out in films deposited onto Si substrates using a Nicolet 470 Nexus spectrometer, with the sample chamber purged with N2 gas. Raman spectra were recorded using a micro-Raman spectrograph Renishaw in-Via with the 514.5 and 785 nm laser lines, whose power at the sample was in the µW level. The equipment contains a Leica microscope and a XYZ automatic stage, which allow collecting the spectra point-by-point with a spatial resolution of ca. VOL. 39, NO. 14, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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SCHEME 1. Schematic Fabrication of LbL Films Comprising Humic Acid (HA) and Poly(allylamine hydrochloride) (PAH)
FIGURE 1. Electronic absorption of PAH/HA LbL films containing different numbers of bilayers. The inset shows the linear dependence of the absorbance at 300 nm as a function of PAH/HA bilayers in the film.
1 µm2 and a spectral resolution of ca. 3 cm-1. Cyclic voltammograms were obtained in films containing 2, 4, 6, or 10 PAH/HA bilayers deposited onto ITO-covered substrates using an EG&G PAR M280 electrochemical analyzer. The electrochemical cell was a three-electrode system. An Ag/AgCl electrode was used as reference, Pt wire as counter electrode, and ITO or ITO covered with a (PAH/AH)n film as working electrode (where n is the number of PAH/HA bilayers). All electrodes were inserted into the electrochemical cell containing a NaCl (0.05 mol L -1, pH ) 4) solution, and the voltammogram was immediately recorded. The potentials were scanned from +1.1 to -1.1 V (vs Ag/AgCl). The scan rates were 20, 50, 100, and 200 mV s-1. After electrochemical characterization, a 6-bilayer PAH/HA film was subjected to PCP detection via cyclic voltammetry, by analyzing the electrochemical response of the film as a function of the amount of PCP in the electrolytic solution.
FIGURE 2. FTIR spectra for PAH and HA cast films and for a 25-bilayer PAH/HA LbL film deposited onto Si substrates.
Results and Discussion The growth of PAH/HA multilayers was monitored using UVvis spectroscopy, and the results are shown in Figure 1. The absorption spectra for the LbL films are basically the same as for HA in solution (not shown). The electronic absorbance of the films increased linearly with the number of bilayers (as shown in the inset of Figure 1), suggesting that the same amount of material was adsorbed at each deposition step. A spectroscopic characterization of the PAH/HA LbL films was performed with FTIR and micro-Raman measurements, carried out for neat PAH and HA cast films and for a 25-bilayer PAH/HA LbL film deposited onto Si substrates. The FTIR spectra are shown in Figure 2. The main vibrational bands of PAH (spectrum A) are assigned as follows (in cm-1): 2950 due to asymmetric and symmetric NH3+ stretching, and 1608 and 1511 due to the N-H bending. The HA powder 5386
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FIGURE 3. Raman spectra for HA powder, PAH/HA 25-bilayer LbL film, and HA cast film. (spectrum B) displayed the main absorption bands at 1715 (CdO from COOH), 1614 (stretching vibration of conjugated CC or H-bonded carbonyl CO), and 1239 cm-1 (C-O and O-H from COOH) (20, 21). It is worth noting that in the HA cast film from a solution at pH 9 (spectrum C), the band at 1715 cm-1 vanishes, indicating that HA is in its nonprotonated state. In addition, the band at 1614 is increased and shifted to 1595 cm-1. In the PAH/HA film spectrum (D), the band at 1595 cm-1 (from COO-) appears as a strong, broad band, and a shoulder at 1720 cm-1 becomes clear. This shoulder
FIGURE 4. AFM images of the 5- and 10-bilayer PAH/HA films.
TABLE 1. RMS Roughness for PAH/HA LbL Films Containing Different Numbers of Bilayers for Three Different Observation Windows roughness (nm) bilayers
200 nm × 200 nm
500 nm × 500 nm
1000 nm × 1000 nm
5 10 15
0.6 0.75 0.85
0.89 1.67 1.19
0.91 1.39 2.17
is probably related to the protonation of the COO- groups by the NH3+ groups from PAH. The latter may be indicative of the formation of COO---NH3+ salt bridges, which act as binding forces (22) between PAH and HA in the LbL films. Another possibility to probe the interaction between NH3 and COO groups would be a stepwise analysis in which the spectrum is taken after deposition of any single layer. However, the resolution is expected to be poor because the noninteracting NH3 and COO groups are present only in the outermost layer of the film. The Raman spectra for HA powder, HA cast film, and a 20-bilayer PAH/HA LbL film are shown in Figure 3. In contrast to the IR spectra, no difference was observed in the Raman spectra for HA powder and HA cast film. All of the spectra, including the one from the PAH/HA LbL film, presented two main bands at 1373 and 1607 cm-1 assigned in the literature as band D and band G, respectively (23). These two bands are related to sp2 sites, attributed to the ring breathing (band D) and bond stretching either in chains or in rings (band G) (23). Because the Raman scattering from carbons is a resonant effect, both bands dominate completely the spectrum for the laser in the visible range. This might prevent any evidence for the formation of salt bridges (COO---NH3+) from being observed, as it was suggested by the FTIR results. PAH/HA LbL films subjected to AFM analyses revealed a globular morphology, similar to what has been reported for other LbL films (24). Figure 4 shows the AFM images for PAH/HA LbL films containing 5 and 10 bilayers. Interestingly, all of the films presented a very smooth surface, with rootmean-square (RMS) roughness varying from 0.89 to 1.19 nm for films containing 5 and 15 PAH/HA bilayers, respectively, at an observation window of 500 nm × 500 nm (see Table 1). All of the films analyzed had HA in the outermost layer. It should be noted that for LbL films containing biomolecules and polyelectrolytes, the surface roughness depends on the
outermost layer. For example, Onda et al. (26) reported a larger roughness for LbL films with protein in the outermost layer, and the roughness was significantly reduced when an additional polyelectrolyte layer was deposited atop. The average thickness for each PAH/HA bilayer was ca. 0.8 nm, as measured by AFM, for a 10-bilayer film. The adsorption of such thin bilayers in LbL films is not surprising. In fact, molecularly thin bilayers are expected to occur if fully charged, weak polyelectrolytes are employed (27). In this work, both PAH and HA are expected to be nearly fully charged at the pH employed (pH 6 and 9, for PAH and HA, respectively). The formation of ultrathin, smooth PAH/HA bilayers can be advantageous for building up electrochemically modified electrodes because most of the electrochemical processes are known to be thickness dependent, as will be shown next. Cyclic voltammograms obtained from PAH/HA films deposited on ITO revealed the presence of only one reduction peak at ca. -0.675 V (vs Ag/AgCl). The current at the reduction peak increased linearly with the number of PAH/HA bilayers, as depicted in Figure 5a. We have not performed a detailed study of film stability. It is worth noting, however, that several films were subjected to cyclic voltammetry more than once, as in the case where the scan rate was varied (Figure 5B), and no changes in the film were observed whatsoever. When the scan rate is increased, the potentials of the cathodic peak shift symmetrically, as shown in Figure 5b, resulting in a constant potential at various scan rates. The correlation between scan rate and the electrochemical mechanism involved is not straightforward in this case, because more than one process is present: film reduction plus hydrogen evolution. Reports from the literature, however, state that the redox process of HA is charge-transport limited, because HA is a stable and efficient electron-transfer promoter (28, 29). Because the increase in the peak current shown in Figure 5a was linear, we may suggest that all of the electroactive material added at each deposition step participated in the electrochemical reaction. According to Schlenoff et al. (30), the latter may occur via an electronhopping mechanism between redox, neighboring sites under the influence of a chemical potential. A 6-bilayer PAH/HA modified electrode was further employed as electrochemical sensor for PCP detection, using cyclic voltammetry. Previous work with sensing units made of nanostructured films indicates that the sensitivity usually VOL. 39, NO. 14, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. (a) Cyclic voltammograms of ITO/(PAH/HA)n with different number of bilayers and dependence of the reduction peak current on the number of bilayers (inset). Scan rate: 50 mV s-1. (b) Influence of scan rate on reduction peak current of a 6-bilayer PAH/AH film onto ITO electrolyte: NaCl 0.5 mol L-1 (pH ) 4). detected with reasonable certainty. In a given analytical method, the detection limit is given by eq 1:
DL )
FIGURE 6. Analytical curve showing the electrochemical response of an ITO/(PAH/HA)6 film obtained in aqueous solution containing different concentrations of PCP. The inset shows the linear dependence of the oxidation peak as a function of the amount of PCP in the electrolytic solution. Electrolyte: NaCl 0.5 mol L-1 (pH ) 4.0). Scan rate: 50 mV s-1. increases with the number of bilayers for very thin films, to guarantee full coverage of the substrate (31). When the number increases and the film becomes thick, then the sensitivity drops because of an increase in the resistivity. This is the reason for our selecting a 6-bilayer LbL film as the sensing unit. The voltammograms were recorded at a scan window from 0 to 1.2 V, for PCP exhibits an oxidation peak at ca. 1.0 V (32). When a PAH/HA film deposited onto ITO was subjected to PCP detection, an irreversible anodic peak (PCP oxidation) is observed at 1.05 V (Ag/AgCl) even at PCP concentrations as low as 10-9 mol L-1. Theses results are shown in Figure 6. It is worth noting that the PCP oxidation peak is not observed if a bare ITO electrode is employed, at the concentrations used here. Indeed, the presence of HA molecules improves the adsorption of PCP at the electrode surface, catalyzing the electron transfer between PCP and the electrode. The calibration curve is shown in the inset of Figure 6, where a linear dependence of the peak current on the PCP concentration is observed. The experimental conditions were basically the same used for electrochemical characterization of the films, except for the presence of PCP, which was systematically added in small amounts to the electrolytic solution. According to the IUPAC definition (33) the detection limit (DL) can be related to the smallest response that can be 5388
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kSB b
(1)
where SB is the standard deviation of the blank measurement, b is the sensitivity of the method (determined as the slope of the calibration curve), and k is a statistical constant (a value of k ) 3 is strongly recommended by IUPAC, based on the confidence interval). Using a least-squares regression program (linear fit and not forcing zero), a slope for the data from PCP voltammetric analysis (inset of Figure 6) was determined as 0.02805. Thus, the detection limit calculated for PCP is 1.6 × 10-9 mol L-1 (0.4 mg L-1). It is known that chromatographic methods have been used to detect pentachlorophenol in urine, with detection limits of 0.20 and 0.01 mg L-1 for HPLC/UV and GC/ECD, respectively (34-36). The method proposed here is an alternative with the advantage of obtaining chemically stable films that are easily prepared. Sensing units made from LbL films do not require a pretreatment stage of the sample, which makes the method of detection less costly than chromatographic methods. If compared with other electrochemical methods for detecting PCP, for example, using carbon paste electrodes, the LbL film offers the advantage of a lesser amount of material employed in the sensing unit, in addition to allowing fine-tuning of film thickness and film architecture. Thus, HAs were successfully immobilized in nanostructured films using PAH in the template layers. The films were very flat, with average roughness varying from 0.8 to 2 nm. The well-defined electroactivity of the films allowed its use as modified electrodes for detecting a pesticide via electrochemical sensing. The high sensitivity achieved made it possible for the PAH/HA modified electrodes to detect PCP in the electrolytic solution with a detection limit of 1.6 × 10-9 mol L-1.
Acknowledgments This work was supported by FAPESP, CNPq, and IMMP/ MCT (Brazil).
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Received for review March 21, 2005. Revised manuscript received May 17, 2005. Accepted May 18, 2005. ES050552N
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