Materials Science and Engineering C 47 (2015) 339–344
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Nanostructured layer-by-layer films containing phaeophytin-b: Electrochemical characterization for sensing purposes Gisele Elias Nunes Pauli a, Felipe B. Araruna b, Carla Eiras b,c, José Roberto S.A. Leite b, Otemberg Souza Chaves d, Severino Gonçalves Brito Filho d, Maria de Fátima Vanderlei de Souza d, Lucas Natálio Chavero a, Maria Luisa Sartorelli a, Ivan H. Bechtold a,⁎ a
Departamento de Física, Universidade Federal de Santa Catarina, Florianópolis, SC 88040900, Brazil Núcleo de Pesquisa em Biodiversidade e Biotecnologia, BIOTEC, Campus Ministro Reis Velloso, CMRV, Universidade Federal do Piauí, UFPI, Parnaíba, Brazil c Laboratório Interdisciplinar de Materiais Avançados, LIMAV, CCN, UFPI, Teresina, PI 64049-550, Brazil d Programa de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos, Universidade Federal da Paraíba, 58051-970 João Pessoa, Paraíba, Brazil b
a r t i c l e
i n f o
Article history: Received 15 July 2014 Received in revised form 27 September 2014 Accepted 6 November 2014 Available online 7 November 2014 Keywords: Phaeophytin-b LbL technique Electrochemical sensor
a b s t r a c t This paper reports the study and characterization of a new platform for practical applications, where the use of phaeophytin-b (phaeo-b), a compound derived from chlorophyll, was characterized and investigated for sensing purposes. Modified electrodes with nanostructured phaeo-b films were fabricated via the layer-by-layer (LbL) technique, where phaeo-b was assembled with cashew gum, a polysaccharide, or with poly(allylamine) hydrochloride (PAH). The multilayer formation was investigated with UV–Vis spectroscopy by monitoring the absorption band associated to phaeo-b at approximately 410 nm, where distinct molecular interactions between the materials were verified. The morphology of the films was analyzed by atomic force microscopy (AFM). The electrochemical properties through redox behavior of phaeo-b were studied with cyclic voltammetry. The produced films were applied as sensors for hydrogen peroxide (H2O2) detection. In terms of sensing, the cashew/phaeo-b film exhibited the most promising result, with a fast response and broad linear range upon the addition of H2O2. This approach provides a simple and inexpensive method for development of a nonenzymatic electrochemical sensor for H2O2. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The search for new materials that can be modified at the molecular level has been objective of numerous studies worldwide. In this regard, the self-assembly LbL technique, proposed by Decher [1–4], in the 90s, has become an interesting choice. Decher's approach makes use of electrostatic interaction of oppositely charged layers, by adsorbing alternatively anionic and cationic polyelectrolytes on solid supports. The main advantage of this technique is the simplicity of the experimental apparatus used in the manufacture of films. Furthermore, the technique can be used for coating surfaces of any shape and size and with film thickness controlled by the number of adsorbed layers. The potential of this method allows fabrication of nanostructured films from a wide range of materials, including polymers [5], biological molecules [6–8] and advanced ceramics [9]. Concerning the fundamental mechanisms involved in the LbL technique, in most cases adsorption of molecules is governed by electrostatic interactions between species bearing opposite charges, but specific interactions or secondary interactions, hydrogen bonding and covalent interactions between the film ⁎ Corresponding author. E-mail address: [email protected] (I.H. Bechtold).
http://dx.doi.org/10.1016/j.msec.2014.11.022 0928-4931/© 2014 Elsevier B.V. All rights reserved.
components have also been shown to be very important, thus opening new alternatives of molecular architectures [4,10–15]. LbL multilayer films are also a versatile platform for numerous applications including sensors [16,17]. A new possibility for formation of nanostructures involves the use of natural gums. The cashew gum is an exudate obtained from the cashew tree (Anacardium occidentale L.), which is known mainly for its nuts that are used as food ingredients, very abundant in the North-eastern Brazil. It is non-toxic, inexpensive, biodegradable, and hydrophilic, characterized as a heteropolysaccharide complex and when solubilized in water, has an anionic nature [18]. The cashew gum has been used in several studies and has shown therapeutic action, emphasized by its antimicrobial [19] and antitumoral [20] activities, biological healing [21], as well as its application in the development of LbL sensors [17,22]. Phaeo-b is a compound derived from chlorophyll, with biomedical properties that have been studied for use in photodynamic therapy [23–25]. Chlorophyll (chl) is the most abundant and widely distributed green pigment in plants and it plays crucial roles in photosynthesis. Chl molecules have cyclic tetrapyrrole moieties as the photofunctional core, having magnesium (Mg) as the central atom, whereas peripheral substituents of the cyclic tetrapyrroles give their structural diversity [24, 26,27]. The demetallization of chlorophyll originates the phaeophytin,
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where the metal ion (Mg2+) is replaced by hydrogen atoms [27,28]. The number of different chls is not so large in nature, with the chlorophyll-a being the most abundant, followed by chlorophyll-b. The chl-a and chlb differ in the C-7 carbon substituent of the chlorin macrocycle. The chla has a methyl group (−CH3) at the C-7 whereas chl-b contains a formyl group (−CHO) at the same position [26]. The phaeo-b used in this work was isolated from the species Turnera subulata belonging to the family Turneraceae [29]. In Brazil its roots are used to treat amenorrhea [30]. The species of Turneraceae family are widely distributed in tropical and subtropical regions of the world. Here, the LbL technique was used to manufacture nanostructured films of phaeo-b containing cashew gum and PAH as polyelectrolytes. Through appropriate characterization techniques (UV–vis and AFM), the film formation and molecular interactions between the materials were investigated. The electrochemical properties and the redox behavior of phaeo-b immobilized in the LbL films were investigated as well. Based on these results, these systems were applied as modified electrodes for the detection of H2O2. Cyclic voltammetry showed that cashew/phaeo-b multilayer films displayed the best catalytic response to the reduction of H2O2. This modified electrode showed a good response time towards H2O2 and the catalytic current was found to be linear to the addition of H2O2 in a wide concentration range. 2. Experimental The samples of cashew gum were collected from the native cashew forest located in the municipality of Ilha Grande, Piauí State (Northeast region of Brazil) and purified as a sodium salt using the method described by Costa and co-workers [31]. Nodules free of bark were selected, triturated and dissolved in ultrapure water at room temperature to give a 5% (w/v) solution that was successively filtered in gaze. The pH of the solution was adjusted to approximately 7.0 by addition of diluted aqueous NaOH (0.05 mol/L). Then, the clear solution was successively filtered through sintered glass (coarse grade) and the cashew gum was precipitated with ethanol (ratio 40:10 alcohol:cashew). After three purification steps, 0.5 g of cashew gum was solubilized in 100 mL of ultrapure water under stirring for 12 h and filtered through sintered glass under vacuum. The final aqueous solution had a concentration of 1.0 mg/mL and pH 7.3. The phaeo-b was solubilized in chloroform at a concentration of 2 mg in 1 mL resulting in 2.5 mL of stock solution. The final solution used in the LbL film formation was obtained by diluting the stock solution to a final volume of 5.0 mL, with 90% alcohol and 10% water resulting in a final phaeo-b concentration of 0.7 mg/mL. Fig. 1 shows the chemical structure of phaeo-b [29]. LbL films were assembled in a monolayer structure only with phaeob and in a bilayer fashion using phaeo-b conjugated with PAH as polycationic solution or cashew gum as polyanionic solution. Phaeo-b and PAH were used without further purification. The PAH was purchased from Sigma-Aldrich and dissolved in aqueous solutions at a
Fig. 1. Chemical structure of the phaeo-b, adapted from [29].
concentration of 1.0 mg/mL and pH 7–8. Therefore, the films were investigated with three distinct architectures: phaeo-b (monolayers), PAH/phaeo-b and cashew/phaeo-b (bilayers). The multilayer films were obtained by immersing the substrates sequentially into the different solutions for 5 min according to the desired architecture and the number of mono/bilayers. According to many examples in the literature of adsorption processes using similar systems, as metallic phthalocyanines [32–34], an immersion period of 5 min assures that the adsorption saturation is completed. After each deposition step the substrates were rinsed in a washing solution (pH 7–8) and dried under N2 flow. The growth of the multilayers was monitored with UV–vis absorption during the deposition steps. The spectra were obtained for solutions (using a quartz cuvette) and also for the variable number of mono/bilayers deposited onto quartz plates, in order to compare the behavior of the material in solution and immobilized as a film. The absorption measurements were performed with an Ocean Optics USB 4000 spectrophotometer. The morphology of the films was analyzed with AFM, Nanosurf model Easyscan2, in tapping mode at 1.0 Hz scanning rate and 512 × 512 lines. Electrochemical measurements were performed using a METROHM AUTOLAB PGSTAT302N potentiostat and a three-electrode electrochemical cell. A Hg/HgCl/KCl (sat.) (SCE) electrode was chosen as the reference electrode; a 7.0 cm2 platinum foil was selected as the auxiliary electrode; and the LbL films deposited with different architectures onto ITO were employed as the working electrodes. All the experiments were performed in a 50 ml electrolytic solution of 0.1 mol/L HCl at room temperature. Amperometric and cyclic voltammetric measurements were employed for detecting H2O2.
3. Results and discussion A wide variety of experimental methods have been employed to characterize the LbL films. One of the most used is UV–vis spectroscopy since most materials processed by the LbL technique absorb light in this wavelength region. In addition, this spectroscopy provides a direct estimation of the amount of material adsorbed during the multilayer deposition. Through this experimental method, firstly, we show the optical characterization of the solutions used in the film formation. Fig. 2 shows the absorbance spectrum of the phaeo-b in solution, where two absorption bands are clearly observed: the first one located between 400 and 450 nm, which is characteristic of porphyrins and their derivatives, corresponding to the Soret band [27]; the second absorption process is observed between 600 and 700 nm, being favorable for photodynamic therapy [24,27]. This result is similar to that obtained by M. Kobayashi et al. [35]. Unlike phaeo-b, PAH and cashew gum solutions (not shown here) do not absorb in the visible region.
Fig. 2. Absorption spectra of the phaeophytin-b in solution.
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The absorption spectra of the phaeo-b films with different numbers of monolayers are presented in Fig. 3, where the profile is very similar to that obtained for phaeo-b in solution. It is shown that at least up to 20 monolayers of phaeo-b could be successfully deposited. The original mechanism of the LbL method is associated with the spontaneous adsorption of oppositely charged polymer layers, and this successfully explains adsorption mechanisms for some polyelectrolytes. However, for multilayered film growth where only one type of material is used (in the case of phaeo-b monolayers), secondary interactions, namely van der Waals forces and H-bonding [11,14,36], prevail over the electrostatic ones and dominate the film formation. An important aspect of LbL films is the linear increase of the thickness with the number of bilayers, meaning that the same amount of material is adsorbed after each deposition step [37]. On the other hand, Fig. 3B shows the dependence of the optical absorbance at 405 nm for phaeo-b LbL films as a function of the number of monolayers and a non-linear increase is observed. It is well established that a non-linear buildup for the first few layers of adsorption can be associated to substrate effects [37]. Films with exponential growth were described by Elbert et al. [38] for poly(L-lysine)/alginate (PLL/ALG) films. Picart and collaborators [39] associated the exponential growth to the ability of at least one of the polyelectrolytes used in the film construction to diffuse in and out of the films. After that, several other studies showed multilayer films exhibiting this type of growth [17,40,41]. From Fig. 3B, it seems that the amount of adsorbed phaeob increases exponentially in the first few layers, indicating that the amount of adsorbed material is not the same at the initial deposition steps, and the linear behavior occurs only after the eighth or tenth monolayer. The absorbance signal is intense for the film containing phaeo-b monolayers, indicating that the system has a large amount of adsorbed material. Therefore, it is assumed that interactions that occur among phaeo-b molecules have a highly attractive character. Fig. 4 displays the UV–vis spectra of the PAH/phaeo-b and cashew/ phaeo-b bilayer structured films. It is worth to underline that the PAH acts as cationic polyelectrolyte while the cashew gum acts as an anionic one. The aim here was to verify the influence of polyelectrolyte charge for the film formation. The results show that PAH is more efficient for production of multilayer films with phaeo-b, since the spectra of Fig. 4A exhibit higher absorbance values and well-defined bands when compared to those obtained with cashew gum, presented in Fig. 4C. It suggests a stronger interaction between PAH and phaeo-b, which can be related to the cationic character of PAH and reveals an anionic character for the phaeo-b. Fig. 4B and D shows the absorbance at the maximum wavelength as a function of the number of deposited bilayers for PAH/phaeo-b and cashew/phaeo-b, respectively. An opposite behavior in relation to phaeo-b monolayer films was observed for the film with bilayers of PAH/phaeo-b, Fig. 4B. The reason for this can be the strong attractive interaction between the glass substrate and PAH molecules. Similar results were obtained by Nunes and collaborators [8] in a dye system.
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The amount of material adsorbed for the PAH/phaeo-b film is constant with the number of deposited bilayers, leading to a linear increase that characterizes a self-regulated [17] process, where the same amount of opposite charge is adsorbed in each deposition step. For the cashew/ phaeo-b film the linearity is not observed, as both substances do not have opposite charges. In this case, the specific secondary interactions prevail over the electrostatic ones, as was also the case for the monolayer films of phaeo-b. AFM measurements were performed in films with 15 layers to investigate the morphological aspects of the different LbL structured films. The results are presented in Fig. 5. From the AFM analyses, the surface mean roughness (RMS) values were obtained by means of the WSxM5.0 software. The film with 15 monolayers of phaeo-b presents a homogeneous covering of the surface with granular characteristics and good monodispersity of the grain sizes (about 200 nm in diameter), which also reflects the low RMS value (9.0 nm), see Fig. 5A. The PAH/ phaeo-b film with 15 bilayers presented in Fig. 5B also displays a granular morphology, but with larger grain domains probably due to agglomerations of the pheo-b molecules in the PAH matrix (RMS of 27 nm). The film with 15 bilayers of cashew/phaeo-b is very flat, with an RMS value of 6.0 nm, without the characteristic granular structure present in the other films, see Fig. 5C. Actually, it seems that a low quantity of phaeo-b molecules is incorporated in this last structure, which correlates well with the reduced values of absorption. After confirming that it was possible to produce LbL films with phaeo-b, and by using cashew gum and PAH, these systems were studied with cyclic voltammetry in order to characterize the immobilized species and also to verify the application feasibility of these films as electrochemical sensors. Fig. 6 shows the voltammograms (50 mV/s) of the films in acidic medium (HCl), where the working electrode containing three monolayers of phaeo-b demonstrated the best electrochemical response in the potential range evaluated, both for the current density (j) as for the definition of the oxidation process of the phaeo-b (at 1.05 V/SCE). Monolayers of cashew and PAH were tested and these films did not exhibit redox processes (not shown here). On the other hand, the bilayer system containing phaeo-b immobilized with PAH showed a higher current value when compared to the system where the PAH was replaced by cashew gum. These effects seem to be driven by the quantity of phaeo-b adsorbed on the films, in accordance with the UV–vis and AFM analysis. With 4 bilayers, absorbance measurements indicate that both phaeo-b and PAH/phaeo-b films have similar quantities of adsorbed materials. However, the presence of PAH seems to have a blocking effect on the oxidation process of phaeo-b. On the other hand, the low absorbance measured for 4 bilayers of cashew/ phaeo-b correlates well with the low quantity of adsorbed material already indicated by absorption and AFM measurements. By comparing with the behavior of a Ni-pthalocyanine (NiTsPc) [34], it is possible to suggest that the oxidation observed at 1.05 V/SCE is related to the phaeo-b macrocycle.
Fig. 3. (A) Absorption spectra of phaeo-b for different numbers of monolayers. (B) Absorbance at 405 nm as a function of the number of monolayers.
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Fig. 4. Absorption spectra at 410 nm, for different numbers of bilayers of (A) PAH/phaeo-b and (C) cashew/phaeo-b. Absorbance as a function of the number of bilayers of (B) PAH/phaeo-b and (D) cashew/phaeo-b. The dashed line at (B) serves only as guide.
In the sequence, the sensing properties of these systems were investigated, where the detection of H2O2 was evaluated. Hydrogen peroxide is a clear, colorless liquid which is completely miscible with water. Industrially, hydrogen peroxide is used mainly in bleaching processes in the industries of paper, textile and cellulose, water purification and, particularly in Europe, in the manufacture of perborate and percarbonate used in detergents [39]. A modest, approximate 10% of the total world production is estimated to be employed in the manufacture of organic chemicals [42]. The reliable and fast determination of hydrogen peroxide is of considerable interest and of great importance in chemical, biological, medicine, food, clinical, pharmaceutical, industrial, and environmental analyses and many other fields [43–45]. Many techniques have been developed to detect hydrogen peroxide as fluorimetry [43] and chemiluminescence [46], but many of these techniques have obvious drawbacks because they are time-consuming and expensive. Due to their simplicity, electrochemical methods [47] have been extensively employed in hydrogen peroxide determination. In order to know the effect of phaeo-b on the reduction of H2O2, when the first is immobilized in different architectures, either forming mono- or bilayers over ITO, voltammetric responses of the different
electrodes are compared and shown in Fig. 7. One observes that both PAH and cashew films enhance the peroxide reduction current in comparison to the plain ITO film. Normalized curves (not shown) indicate, moreover, that the cashew gum film presents a slight catalytic activity towards H2O2 reduction, since the current maximum is detected at lower values of cathodic potential (−0.26 V/SCE). For the pure phaeob film, on the other hand, the reduction current doubles in comparison to the cashew gum film and the current peak occurs at −0.14 V/SCE, indicating an efficient electrocatalytical activity towards H2O2. The most striking result, however, is observed for the cashew/phaeo-b bilayer, which, despite having the lowest quantity of adsorbed material, presents the highest reduction current. Moreover, the voltammogram for the cashew/phaeo-b bilayer presents a double wave, which indicates a two-step process. There are many examples in the literature where the most important thing is not the quantity of adsorbed material, but the interactions between the involved materials and the quantity of free sites. The synergistic effect between different materials in the bilayer structure may originate new properties compared with the materials separately. In previous works, it was already observed that multilayer films of different materials with cashew gum improves stability in acid
Fig. 5. AFM images of 10 μm × 10 μm: (A) 15 monolayers of phaeo-b, (B) 15 bilayers of PAH/phaeo-b and (C) 15 bilayers of cashew/phaeo-b.
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Table 1 Cathodic peak potential (Pcp) and current density (Jcp) values obtained from Fig. 7 for the working electrodes used in the H2O2 detection.
Fig. 6. Cyclic voltammograms of three layers of phaeo-b, PAH/phaeo-b and cashew/phaeob on ITO electrodes in acidic medium (HCl).
medium and amplifies the electrical response [48,22]. It was also observed that multilayer films of a Fe-pthalocyanine (FeTsPc) with cashew gum intensify the current values in electrochemical measurements [17]. This result indicates that phaeo-b in the form of multilayers possesses efficient electrocatalytical activity towards H2O2. The phaeo-b activates the electrode surface in order to increase the observed current signal. Table 1 displays the cathodic peak potential and current density extracted from Fig. 7. An overview of the values presented in Table 1 allows us to conclude that the phaeo-b is sensitive to H2O2. This signal was intensified in the bilayer systems, where the phaeo-b immobilized with cashew gum offered the higher cathodic current density (410 μA/cm2). Thus, this system seems to be promising for H2O2 detection. As can be seen in Fig. 7, detection of H2O2 by a plain ITO electrode yields only a small current response, whereas the catalytic current detected by the cashew/phaeo-b multilayer modified ITO increased significantly. To verify whether the current was due to the electrocatalytic capability of H2O2, a control experiment without H2O2 was performed. Fig. 7 shows also the cyclic voltammograms of the pure ITO electrode and the cashew/phaeo-b multilayer modified ITO electrode in the absence of H2O2. One observes that no characteristic peak appears in the cyclic voltammogram recorded with the pure ITO and ITO/cashew/ phaeo-b electrode. These results confirmed that the catalytic current for H2O2 was due to the presence of the cashew/phaeo-b multilayer. Fig. 8A displays a typical current-time plot of ITO/cashew/phaeo-b multilayer modified electrode upon successive step additions of
Fig. 7. Cyclic voltammograms of the clean ITO and LbL films of PAH, cashew gum, phaeo-b, cashew/phaeo-b and PAH/phaeo-b.
Working electrode
Pcp (V)
jcp(μA/cm2)
ITO PAH Cashew gum Phaeo-b PAH/phaeo-b Cashew/phaeo-b
−0.30 −0.30 −0.26 −0.14 −0.16 −0.19
−48 −82 −98 −200 −360 −410
1.1 mmol/L H2O2 under continuous stirring into the HCl (0.1 mol/L) electrolyte. The applied potential of 0.0 V was chosen as the working potential for the amperometric determination of H2O2, where the risk for interfering reactions of other electroactive species in the solution is minimized and also where the background current and noise levels reached their lowest values. A rapid current response could be observed when H2O2 aliquots were added into the stirring acid solution. The maximum steady-state current could be achieved within 12 s, indicating a good H2O2 diffusion process in the modified electrode. The current response increased linearly with the concentration of H2O 2 . Fig. 8B shows the calibration curve between the response current and the concentration of H2O2. The electrode presented a linear range from 3.3 mmol/L to 19.8 mmol/L, governed by the equation: j = 0.481 + 0.0561C, with a correlation coefficient of 0.997. The use of a cashew/phaeo-b electrode as nonenzymatic electrochemical sensor to detect H2O2 has many advantages, such as easy preparation of cashew/phaeo-b multilayer films on the electrode, low cost, suitable electrocatalytic activity and most importantly, it can be operated at 0.0 V potential. Therefore, the approach proposed a new and efficient kind of electrochemical sensor to detect H2O2.
4. Conclusions We produced successful films containing phaeophytin-b, a new biological material extracted from T. subulata Sm., together with natural cashew gum and the polymer PAH using the LbL technique, which is considered a simple technique of low cost and is already well accepted in the literature. The films displayed an electrochemical behavior that varied with the polyelectrolyte that was incorporated in the phaeo-b. Through the voltammetric studies it was possible to identify the oxidation of phaeo-b, and it was observed that the film with three monolayers of phaeo-b showed the highest anodic current peak and a good definition of the anodic peak potential, demonstrating to be a very promising material. The performance of the phaeo-b modified electrode was evaluated for the amperometric detection of H2O2. The ITO electrode modified with three bilayers PAH/phaeo-b displayed the second best result. On the other hand, the cashew/phaeo-b film exhibited the best electrochemical response. The presence of the natural gum was very advantageous, since the redox currents were higher for films containing the cashew gum in comparison to PAH/phaeo-b or phaeo-b films, in this way becoming the most efficient sensor for detection of H2O2. The application of a cashew/phaeo film amperometric sensor presents many advantages, such as low cost, environmental friendliness and suitable electrocatalytic activity at 0.0 V potential. Moreover, the prepared sensor exhibited an effective catalytic response to the reduction of H2O2. The amperometric sensor exhibited a linear response in the range from 3.3 mmol/L to 19.8 mmol/L. Therefore, this electrode provides a novel and promising sensor for the detection of H2O2. The possibility of producing films with phaeophytin-b opens up new prospects for using these natural biological materials in technological applications like electrochemical sensors.
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Fig. 8. (A) Typical current–time response curve for successive addition of 1.1 mmol/L H2O2 for the sensor in 0.1 mol/L HCl at the applied potential of 0.0 V, and (B) calibration curve of H2O2 concentration on the modified electrode ITO/cashew/phaeo-b. The dashed line at (B) serves only as guide.
Acknowledgments This work was supported by CNPq, Nanobiomed Network CAPES (#AUX–PE 705/2009), INCT/INEO (573762/2008-2), INCT/ Nanobiotecnologia. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
[18] [19] [20] [21]
[22]
G. Decher, J.D. Hong, J. Schmitt, Thin Solid Films 210–211 (1992) 831–835. Y. Lvov, H. Haas, G. Decher, H. Möhwald, J. Phys. Chem. 97 (1993) 12835–12841. Y. Lvov, G. Decher, H. Möhwald, Langmuir 9 (1993) 481–486. G. Decher, Science 277 (1997) 1232–1237. M. Ferreira, M.F. Rubner, Macromolecules 28 (1995) 7107–7114. F.N. Crespilho, V. Zucolotto, J.R. Siqueira Jr., C.J.L. Constantino, F.C. Nart, O.N. Oliveira Jr., Environ. Sci. Technol. 39 (2005) 5385–5389. V. Zucolotto, N.M. Barbosa Neto, J.J. Rodrigues Jr., C.J.L. Constantino, S.C. Zílio, C.R. Mendonça, R.F. Aroca, O.N. Oliveira Jr., J. Nanosci. Nanotechnol. 4 (2004) 855–860. G.E. Nunes, A.L. Sehnem, I.H. Bechtold, Liq. Cryst. 39 (2012) 205–210. E.A.O. Farias, N.A. Dionisio, P.V. Quelemes, S.H. Leal, J.M.E. Matos, E.C.S. Filho, I.H. Bechtold, J.R.S.A. Leite, C. Eiras, Mater. Sci. Eng. C 35 (2014) 449–454. F.N. Crespilho, V. Zucolotto, O.N. Oliveira Jr., F.C. Nart, Int. J. Electrochem. Sci. 1 (2006) 194–214. W.B. Stockton, M.F. Rubner, Macromolecules 30 (1997) 2717–2725. J.-i. Anzai, Y. Kobayashi, N. Nakamura, M. Nishimura, T. Hoshi, Langmuir 15 (1999) 221–226. E. Kharlampieva, V. Kozlovskaya, S.A. Sukhishvili, Adv. Mater. 21 (2009) 3053–3065. L. Zhou, M. Chen, L. Tian, Y. Guan, Y. Zhang, ACS Appl. Mater. Interfaces 5 (2013) 3541–3548. M.A. Witt, F. Valenga, M.E.R. Dotto, I.H. Bechtold, O. Felix, A.T.N. Pires, G. Decher, Biointerphases 7 (2012) 64. M.F. Zampa, I.M.S. Araújo, V. Costa, C.H.N. Costa, J.R. Santos Jr., V. Zucolotto, C. Eiras, J.R.S.A. Leite, Nanomedicine 5 (2009) 352–358. I.M.S. Araújo, M.F. Zampa, J.B. Moura, J.R. dos Santos Jr., P. Eaton, V. Zucolotto, L.M.C. Veras, R.C.M. de Paula, J.P.A. Feitosa, J.R.S.A. Leite, C. Eiras, Mater. Sci. Eng. C 32 (2012) 1588–1593. G.A. Towle, R.L. Whistler, New York: Van Nostrand Reinhold 1 (1973) 198–248. D.S. Torquato, M.L. Ferreira, G.C. Sá, E.S. Brito, G.A.S. Pinto, E.H.F. Azevedo, World J. Microbiol. Biotechnol. 20 (2004) 505–507. C.G. Monthe, I.A. Souza, G.M.T. Calazans, Med. Nutr. 19 (2008) 50–52. F.M.F. Monteiro, G.M.M. Silva, J.B.R. da Silva, C.S. Porto, L.B. de Carvalho Jr., J.L.L. Filho, A.M.A. Leão-Carneiro, M.G. Carneiro-Cunha, A.L.F. Porto, Process Biochem. 42 (2007) 884–888. S.B.A. Barros, C.M.d-S. Leite, A.C.F. de Brito, J.R. dos Santos Jr., V. Zucolotto, C. Eiras, Int. J. Anal. Chem. (2012) 1–10.
[23] M.A. Calin, S.V. Parasca, J. Optoelectron. Adv. Mater. 8 (2006) 1173–1179. [24] W.-T. Li, H.-W. Tsao, Y.-Y. Chen, S.-W. Cheng, Y.-C. Hsu, Photochem. Photobiol. Sci. 6 (2007) 1341–1348. [25] E.D. Sternberg, D. Dolphin, Tetrahedron 54 (1998) 4151–4202. [26] K. Sadaoka, S. Kashimura, Y. Saga, Bioorg. Med. Chem. 19 (2011) 3901–3905. [27] A.P. Gerola, A. Santana, P.B. França, T.M. Tsubone, H.P.M. de Oliveira, W. Caetano, E. Kimura, N. Hioka, Photochem. Photobiol. 87 (2011) 884–894. [28] S. Schelbert, S. Aubry, B. Burla, B. Agne, F. Kessler, K. Krupinska, S. Hortensteiner, Plant Cell 21 (2009) 767–785. [29] S.G.B. Filho, M.G. Fernandes, O.S. Chaves, M.C.O. Chaves, F.B. Araruna, C. Eiras, J.R.S.A. Leite, M.F. Agra, R. Braz-Filho, M.F.V. Souza, Quim. Nova 37 (2014) 603–609. [30] D.A. Barbosa, K.N. Silva, M.F. Agra, Rev. Bras. Farmacognosia 17 (2007) 396–413. [31] S.M.O. Costa, J.F. Rodrigues, R.C.M. de Paula, Polímeros Ciênc. Tecnol. 6 (1996) 49–55. [32] V. Zucolotto, M. Ferreira, M.R. Cordeiro, C.J.L. Constantino, W.C. Moreira, O.N. Oliveira Jr., Synth. Met. 137 (2003) 969–970. [33] J.R. Siqueira Jr., L.H.S. Gasparotto, F.N. Crespilho, A.J.F. Carvalho, V. Zucolotto, O.N. Oliveira Jr., J. Phys. Chem. B 110 (2006) 22690–22694. [34] M.F. Zampa, A.C.F. Brito, I.L. Kitagawa, C.J.L. Constantino, O.N. Oliveira Jr., H.N. Cunha, V. Zucolotto, J.R.C. Santos Jr., C. Eiras, Biomacromolecules 8 (2007) 3408–3413. [35] M. Kobayashi, S. Akutsu, D. Fujinuma, H. Furukawa, H. Komatsu, Y. Hotota, Y. Kato, Y. Kuroiwa, T. Watanabe, M. Ohnishi-Kameyama, H. Ono, S. Ohkubo, H. Miyashita, in: Z. Dubinsky (Ed.), Photosynthesis, 2013, pp. 47–90 (Chapter 3). [36] M. Raposo, O.N. Oliveira Jr., Langmuir 18 (2002) 6866–6874. [37] M. Raposo, O.N. Oliveira Jr., Braz. J. Phys. 28 (1998). [38] D.L. Elbert, C.B. Herbert, J.A. Hubbell, Langmuir 15 (1999) 5355–5362. [39] C. Picart, J. Mutterer, L. Richert, Y. Luo, G.D. Prestwich, P. Schaaf, J.C. Voegel, P. Lavalle, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 12531–12535. [40] P. Lavalle, C. Gergely, F.J.G. Cuisinier, G. Decher, P. Schaaf, J.C. Voegel, C. Picart, Macromolecules 35 (2002) 4458–4465. [41] A. Tezcaner, D. Hicks, F. Boulmedais, J. Sahel, P. Schaaf, J.-C. Voegel, P. Lavalle, Biomacromolecules 7 (2006) 86–94. [42] G. Strukul (Ed.), Catalytic oxidations with hydrogen peroxide as oxidant, Introduction and Activation Principles, 9, 1992 (chapter 1). [43] M.C.Y. Chang, A. Pralle, E.Y. Isacoff, C.J. Chang, J. Am. Chem. Soc. 126 (2004) 15392–15393. [44] X. Lu, J. Zhou, W. Lu, Q. Liu, J. Li, Biosens. Bioelectron. 23 (2008) 1236–1243. [45] X. Shu, Y. Chen, H. Yuan, S. Gao, D. Xiao, Anal. Chem. 79 (2007) 3695–3702. [46] D.W. King, W.J. Cooper, S.A. Rusak, B.M. Peake, J.J. Kiddle, D.W. O'Sullivan, M.L. Melamed, C.R. Morgan, S.M. Theberge, Anal. Chem. 79 (2007) 4169–4176. [47] J. Jia, B. Wang, A. Wu, G. Cheng, Z. Li, S. Dong, Anal. Chem. 74 (2002) 2217–2223. [48] C. Eiras, A.C. Santos, M.F. Zampa, A.C.F. Brito, C.J.L. Constantino, V. Zucolotto, J.R. Santos Jr., J. Biomater. Sci. Polym. 21 (2010) 1533–1543.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Nanostructured layer-by-layer films containing phaeophytin-b: Electrochemical characterization for sensing purposes Gisele Elias Nunes Pauli a, Felipe B. Araruna b, Carla Eiras b,c, José Roberto S.A. Leite b, Otemberg Souza Chaves d, Severino Gonçalves Brito Filho d, Maria de Fátima Vanderlei de Souza d, Lucas Natálio Chavero a, Maria Luisa Sartorelli a, Ivan H. Bechtold a,⁎ a
Departamento de Física, Universidade Federal de Santa Catarina, Florianópolis, SC 88040900, Brazil Núcleo de Pesquisa em Biodiversidade e Biotecnologia, BIOTEC, Campus Ministro Reis Velloso, CMRV, Universidade Federal do Piauí, UFPI, Parnaíba, Brazil c Laboratório Interdisciplinar de Materiais Avançados, LIMAV, CCN, UFPI, Teresina, PI 64049-550, Brazil d Programa de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos, Universidade Federal da Paraíba, 58051-970 João Pessoa, Paraíba, Brazil b
a r t i c l e
i n f o
Article history: Received 15 July 2014 Received in revised form 27 September 2014 Accepted 6 November 2014 Available online 7 November 2014 Keywords: Phaeophytin-b LbL technique Electrochemical sensor
a b s t r a c t This paper reports the study and characterization of a new platform for practical applications, where the use of phaeophytin-b (phaeo-b), a compound derived from chlorophyll, was characterized and investigated for sensing purposes. Modified electrodes with nanostructured phaeo-b films were fabricated via the layer-by-layer (LbL) technique, where phaeo-b was assembled with cashew gum, a polysaccharide, or with poly(allylamine) hydrochloride (PAH). The multilayer formation was investigated with UV–Vis spectroscopy by monitoring the absorption band associated to phaeo-b at approximately 410 nm, where distinct molecular interactions between the materials were verified. The morphology of the films was analyzed by atomic force microscopy (AFM). The electrochemical properties through redox behavior of phaeo-b were studied with cyclic voltammetry. The produced films were applied as sensors for hydrogen peroxide (H2O2) detection. In terms of sensing, the cashew/phaeo-b film exhibited the most promising result, with a fast response and broad linear range upon the addition of H2O2. This approach provides a simple and inexpensive method for development of a nonenzymatic electrochemical sensor for H2O2. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The search for new materials that can be modified at the molecular level has been objective of numerous studies worldwide. In this regard, the self-assembly LbL technique, proposed by Decher [1–4], in the 90s, has become an interesting choice. Decher's approach makes use of electrostatic interaction of oppositely charged layers, by adsorbing alternatively anionic and cationic polyelectrolytes on solid supports. The main advantage of this technique is the simplicity of the experimental apparatus used in the manufacture of films. Furthermore, the technique can be used for coating surfaces of any shape and size and with film thickness controlled by the number of adsorbed layers. The potential of this method allows fabrication of nanostructured films from a wide range of materials, including polymers [5], biological molecules [6–8] and advanced ceramics [9]. Concerning the fundamental mechanisms involved in the LbL technique, in most cases adsorption of molecules is governed by electrostatic interactions between species bearing opposite charges, but specific interactions or secondary interactions, hydrogen bonding and covalent interactions between the film ⁎ Corresponding author. E-mail address: [email protected] (I.H. Bechtold).
http://dx.doi.org/10.1016/j.msec.2014.11.022 0928-4931/© 2014 Elsevier B.V. All rights reserved.
components have also been shown to be very important, thus opening new alternatives of molecular architectures [4,10–15]. LbL multilayer films are also a versatile platform for numerous applications including sensors [16,17]. A new possibility for formation of nanostructures involves the use of natural gums. The cashew gum is an exudate obtained from the cashew tree (Anacardium occidentale L.), which is known mainly for its nuts that are used as food ingredients, very abundant in the North-eastern Brazil. It is non-toxic, inexpensive, biodegradable, and hydrophilic, characterized as a heteropolysaccharide complex and when solubilized in water, has an anionic nature [18]. The cashew gum has been used in several studies and has shown therapeutic action, emphasized by its antimicrobial [19] and antitumoral [20] activities, biological healing [21], as well as its application in the development of LbL sensors [17,22]. Phaeo-b is a compound derived from chlorophyll, with biomedical properties that have been studied for use in photodynamic therapy [23–25]. Chlorophyll (chl) is the most abundant and widely distributed green pigment in plants and it plays crucial roles in photosynthesis. Chl molecules have cyclic tetrapyrrole moieties as the photofunctional core, having magnesium (Mg) as the central atom, whereas peripheral substituents of the cyclic tetrapyrroles give their structural diversity [24, 26,27]. The demetallization of chlorophyll originates the phaeophytin,
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where the metal ion (Mg2+) is replaced by hydrogen atoms [27,28]. The number of different chls is not so large in nature, with the chlorophyll-a being the most abundant, followed by chlorophyll-b. The chl-a and chlb differ in the C-7 carbon substituent of the chlorin macrocycle. The chla has a methyl group (−CH3) at the C-7 whereas chl-b contains a formyl group (−CHO) at the same position [26]. The phaeo-b used in this work was isolated from the species Turnera subulata belonging to the family Turneraceae [29]. In Brazil its roots are used to treat amenorrhea [30]. The species of Turneraceae family are widely distributed in tropical and subtropical regions of the world. Here, the LbL technique was used to manufacture nanostructured films of phaeo-b containing cashew gum and PAH as polyelectrolytes. Through appropriate characterization techniques (UV–vis and AFM), the film formation and molecular interactions between the materials were investigated. The electrochemical properties and the redox behavior of phaeo-b immobilized in the LbL films were investigated as well. Based on these results, these systems were applied as modified electrodes for the detection of H2O2. Cyclic voltammetry showed that cashew/phaeo-b multilayer films displayed the best catalytic response to the reduction of H2O2. This modified electrode showed a good response time towards H2O2 and the catalytic current was found to be linear to the addition of H2O2 in a wide concentration range. 2. Experimental The samples of cashew gum were collected from the native cashew forest located in the municipality of Ilha Grande, Piauí State (Northeast region of Brazil) and purified as a sodium salt using the method described by Costa and co-workers [31]. Nodules free of bark were selected, triturated and dissolved in ultrapure water at room temperature to give a 5% (w/v) solution that was successively filtered in gaze. The pH of the solution was adjusted to approximately 7.0 by addition of diluted aqueous NaOH (0.05 mol/L). Then, the clear solution was successively filtered through sintered glass (coarse grade) and the cashew gum was precipitated with ethanol (ratio 40:10 alcohol:cashew). After three purification steps, 0.5 g of cashew gum was solubilized in 100 mL of ultrapure water under stirring for 12 h and filtered through sintered glass under vacuum. The final aqueous solution had a concentration of 1.0 mg/mL and pH 7.3. The phaeo-b was solubilized in chloroform at a concentration of 2 mg in 1 mL resulting in 2.5 mL of stock solution. The final solution used in the LbL film formation was obtained by diluting the stock solution to a final volume of 5.0 mL, with 90% alcohol and 10% water resulting in a final phaeo-b concentration of 0.7 mg/mL. Fig. 1 shows the chemical structure of phaeo-b [29]. LbL films were assembled in a monolayer structure only with phaeob and in a bilayer fashion using phaeo-b conjugated with PAH as polycationic solution or cashew gum as polyanionic solution. Phaeo-b and PAH were used without further purification. The PAH was purchased from Sigma-Aldrich and dissolved in aqueous solutions at a
Fig. 1. Chemical structure of the phaeo-b, adapted from [29].
concentration of 1.0 mg/mL and pH 7–8. Therefore, the films were investigated with three distinct architectures: phaeo-b (monolayers), PAH/phaeo-b and cashew/phaeo-b (bilayers). The multilayer films were obtained by immersing the substrates sequentially into the different solutions for 5 min according to the desired architecture and the number of mono/bilayers. According to many examples in the literature of adsorption processes using similar systems, as metallic phthalocyanines [32–34], an immersion period of 5 min assures that the adsorption saturation is completed. After each deposition step the substrates were rinsed in a washing solution (pH 7–8) and dried under N2 flow. The growth of the multilayers was monitored with UV–vis absorption during the deposition steps. The spectra were obtained for solutions (using a quartz cuvette) and also for the variable number of mono/bilayers deposited onto quartz plates, in order to compare the behavior of the material in solution and immobilized as a film. The absorption measurements were performed with an Ocean Optics USB 4000 spectrophotometer. The morphology of the films was analyzed with AFM, Nanosurf model Easyscan2, in tapping mode at 1.0 Hz scanning rate and 512 × 512 lines. Electrochemical measurements were performed using a METROHM AUTOLAB PGSTAT302N potentiostat and a three-electrode electrochemical cell. A Hg/HgCl/KCl (sat.) (SCE) electrode was chosen as the reference electrode; a 7.0 cm2 platinum foil was selected as the auxiliary electrode; and the LbL films deposited with different architectures onto ITO were employed as the working electrodes. All the experiments were performed in a 50 ml electrolytic solution of 0.1 mol/L HCl at room temperature. Amperometric and cyclic voltammetric measurements were employed for detecting H2O2.
3. Results and discussion A wide variety of experimental methods have been employed to characterize the LbL films. One of the most used is UV–vis spectroscopy since most materials processed by the LbL technique absorb light in this wavelength region. In addition, this spectroscopy provides a direct estimation of the amount of material adsorbed during the multilayer deposition. Through this experimental method, firstly, we show the optical characterization of the solutions used in the film formation. Fig. 2 shows the absorbance spectrum of the phaeo-b in solution, where two absorption bands are clearly observed: the first one located between 400 and 450 nm, which is characteristic of porphyrins and their derivatives, corresponding to the Soret band [27]; the second absorption process is observed between 600 and 700 nm, being favorable for photodynamic therapy [24,27]. This result is similar to that obtained by M. Kobayashi et al. [35]. Unlike phaeo-b, PAH and cashew gum solutions (not shown here) do not absorb in the visible region.
Fig. 2. Absorption spectra of the phaeophytin-b in solution.
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The absorption spectra of the phaeo-b films with different numbers of monolayers are presented in Fig. 3, where the profile is very similar to that obtained for phaeo-b in solution. It is shown that at least up to 20 monolayers of phaeo-b could be successfully deposited. The original mechanism of the LbL method is associated with the spontaneous adsorption of oppositely charged polymer layers, and this successfully explains adsorption mechanisms for some polyelectrolytes. However, for multilayered film growth where only one type of material is used (in the case of phaeo-b monolayers), secondary interactions, namely van der Waals forces and H-bonding [11,14,36], prevail over the electrostatic ones and dominate the film formation. An important aspect of LbL films is the linear increase of the thickness with the number of bilayers, meaning that the same amount of material is adsorbed after each deposition step [37]. On the other hand, Fig. 3B shows the dependence of the optical absorbance at 405 nm for phaeo-b LbL films as a function of the number of monolayers and a non-linear increase is observed. It is well established that a non-linear buildup for the first few layers of adsorption can be associated to substrate effects [37]. Films with exponential growth were described by Elbert et al. [38] for poly(L-lysine)/alginate (PLL/ALG) films. Picart and collaborators [39] associated the exponential growth to the ability of at least one of the polyelectrolytes used in the film construction to diffuse in and out of the films. After that, several other studies showed multilayer films exhibiting this type of growth [17,40,41]. From Fig. 3B, it seems that the amount of adsorbed phaeob increases exponentially in the first few layers, indicating that the amount of adsorbed material is not the same at the initial deposition steps, and the linear behavior occurs only after the eighth or tenth monolayer. The absorbance signal is intense for the film containing phaeo-b monolayers, indicating that the system has a large amount of adsorbed material. Therefore, it is assumed that interactions that occur among phaeo-b molecules have a highly attractive character. Fig. 4 displays the UV–vis spectra of the PAH/phaeo-b and cashew/ phaeo-b bilayer structured films. It is worth to underline that the PAH acts as cationic polyelectrolyte while the cashew gum acts as an anionic one. The aim here was to verify the influence of polyelectrolyte charge for the film formation. The results show that PAH is more efficient for production of multilayer films with phaeo-b, since the spectra of Fig. 4A exhibit higher absorbance values and well-defined bands when compared to those obtained with cashew gum, presented in Fig. 4C. It suggests a stronger interaction between PAH and phaeo-b, which can be related to the cationic character of PAH and reveals an anionic character for the phaeo-b. Fig. 4B and D shows the absorbance at the maximum wavelength as a function of the number of deposited bilayers for PAH/phaeo-b and cashew/phaeo-b, respectively. An opposite behavior in relation to phaeo-b monolayer films was observed for the film with bilayers of PAH/phaeo-b, Fig. 4B. The reason for this can be the strong attractive interaction between the glass substrate and PAH molecules. Similar results were obtained by Nunes and collaborators [8] in a dye system.
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The amount of material adsorbed for the PAH/phaeo-b film is constant with the number of deposited bilayers, leading to a linear increase that characterizes a self-regulated [17] process, where the same amount of opposite charge is adsorbed in each deposition step. For the cashew/ phaeo-b film the linearity is not observed, as both substances do not have opposite charges. In this case, the specific secondary interactions prevail over the electrostatic ones, as was also the case for the monolayer films of phaeo-b. AFM measurements were performed in films with 15 layers to investigate the morphological aspects of the different LbL structured films. The results are presented in Fig. 5. From the AFM analyses, the surface mean roughness (RMS) values were obtained by means of the WSxM5.0 software. The film with 15 monolayers of phaeo-b presents a homogeneous covering of the surface with granular characteristics and good monodispersity of the grain sizes (about 200 nm in diameter), which also reflects the low RMS value (9.0 nm), see Fig. 5A. The PAH/ phaeo-b film with 15 bilayers presented in Fig. 5B also displays a granular morphology, but with larger grain domains probably due to agglomerations of the pheo-b molecules in the PAH matrix (RMS of 27 nm). The film with 15 bilayers of cashew/phaeo-b is very flat, with an RMS value of 6.0 nm, without the characteristic granular structure present in the other films, see Fig. 5C. Actually, it seems that a low quantity of phaeo-b molecules is incorporated in this last structure, which correlates well with the reduced values of absorption. After confirming that it was possible to produce LbL films with phaeo-b, and by using cashew gum and PAH, these systems were studied with cyclic voltammetry in order to characterize the immobilized species and also to verify the application feasibility of these films as electrochemical sensors. Fig. 6 shows the voltammograms (50 mV/s) of the films in acidic medium (HCl), where the working electrode containing three monolayers of phaeo-b demonstrated the best electrochemical response in the potential range evaluated, both for the current density (j) as for the definition of the oxidation process of the phaeo-b (at 1.05 V/SCE). Monolayers of cashew and PAH were tested and these films did not exhibit redox processes (not shown here). On the other hand, the bilayer system containing phaeo-b immobilized with PAH showed a higher current value when compared to the system where the PAH was replaced by cashew gum. These effects seem to be driven by the quantity of phaeo-b adsorbed on the films, in accordance with the UV–vis and AFM analysis. With 4 bilayers, absorbance measurements indicate that both phaeo-b and PAH/phaeo-b films have similar quantities of adsorbed materials. However, the presence of PAH seems to have a blocking effect on the oxidation process of phaeo-b. On the other hand, the low absorbance measured for 4 bilayers of cashew/ phaeo-b correlates well with the low quantity of adsorbed material already indicated by absorption and AFM measurements. By comparing with the behavior of a Ni-pthalocyanine (NiTsPc) [34], it is possible to suggest that the oxidation observed at 1.05 V/SCE is related to the phaeo-b macrocycle.
Fig. 3. (A) Absorption spectra of phaeo-b for different numbers of monolayers. (B) Absorbance at 405 nm as a function of the number of monolayers.
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Fig. 4. Absorption spectra at 410 nm, for different numbers of bilayers of (A) PAH/phaeo-b and (C) cashew/phaeo-b. Absorbance as a function of the number of bilayers of (B) PAH/phaeo-b and (D) cashew/phaeo-b. The dashed line at (B) serves only as guide.
In the sequence, the sensing properties of these systems were investigated, where the detection of H2O2 was evaluated. Hydrogen peroxide is a clear, colorless liquid which is completely miscible with water. Industrially, hydrogen peroxide is used mainly in bleaching processes in the industries of paper, textile and cellulose, water purification and, particularly in Europe, in the manufacture of perborate and percarbonate used in detergents [39]. A modest, approximate 10% of the total world production is estimated to be employed in the manufacture of organic chemicals [42]. The reliable and fast determination of hydrogen peroxide is of considerable interest and of great importance in chemical, biological, medicine, food, clinical, pharmaceutical, industrial, and environmental analyses and many other fields [43–45]. Many techniques have been developed to detect hydrogen peroxide as fluorimetry [43] and chemiluminescence [46], but many of these techniques have obvious drawbacks because they are time-consuming and expensive. Due to their simplicity, electrochemical methods [47] have been extensively employed in hydrogen peroxide determination. In order to know the effect of phaeo-b on the reduction of H2O2, when the first is immobilized in different architectures, either forming mono- or bilayers over ITO, voltammetric responses of the different
electrodes are compared and shown in Fig. 7. One observes that both PAH and cashew films enhance the peroxide reduction current in comparison to the plain ITO film. Normalized curves (not shown) indicate, moreover, that the cashew gum film presents a slight catalytic activity towards H2O2 reduction, since the current maximum is detected at lower values of cathodic potential (−0.26 V/SCE). For the pure phaeob film, on the other hand, the reduction current doubles in comparison to the cashew gum film and the current peak occurs at −0.14 V/SCE, indicating an efficient electrocatalytical activity towards H2O2. The most striking result, however, is observed for the cashew/phaeo-b bilayer, which, despite having the lowest quantity of adsorbed material, presents the highest reduction current. Moreover, the voltammogram for the cashew/phaeo-b bilayer presents a double wave, which indicates a two-step process. There are many examples in the literature where the most important thing is not the quantity of adsorbed material, but the interactions between the involved materials and the quantity of free sites. The synergistic effect between different materials in the bilayer structure may originate new properties compared with the materials separately. In previous works, it was already observed that multilayer films of different materials with cashew gum improves stability in acid
Fig. 5. AFM images of 10 μm × 10 μm: (A) 15 monolayers of phaeo-b, (B) 15 bilayers of PAH/phaeo-b and (C) 15 bilayers of cashew/phaeo-b.
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Table 1 Cathodic peak potential (Pcp) and current density (Jcp) values obtained from Fig. 7 for the working electrodes used in the H2O2 detection.
Fig. 6. Cyclic voltammograms of three layers of phaeo-b, PAH/phaeo-b and cashew/phaeob on ITO electrodes in acidic medium (HCl).
medium and amplifies the electrical response [48,22]. It was also observed that multilayer films of a Fe-pthalocyanine (FeTsPc) with cashew gum intensify the current values in electrochemical measurements [17]. This result indicates that phaeo-b in the form of multilayers possesses efficient electrocatalytical activity towards H2O2. The phaeo-b activates the electrode surface in order to increase the observed current signal. Table 1 displays the cathodic peak potential and current density extracted from Fig. 7. An overview of the values presented in Table 1 allows us to conclude that the phaeo-b is sensitive to H2O2. This signal was intensified in the bilayer systems, where the phaeo-b immobilized with cashew gum offered the higher cathodic current density (410 μA/cm2). Thus, this system seems to be promising for H2O2 detection. As can be seen in Fig. 7, detection of H2O2 by a plain ITO electrode yields only a small current response, whereas the catalytic current detected by the cashew/phaeo-b multilayer modified ITO increased significantly. To verify whether the current was due to the electrocatalytic capability of H2O2, a control experiment without H2O2 was performed. Fig. 7 shows also the cyclic voltammograms of the pure ITO electrode and the cashew/phaeo-b multilayer modified ITO electrode in the absence of H2O2. One observes that no characteristic peak appears in the cyclic voltammogram recorded with the pure ITO and ITO/cashew/ phaeo-b electrode. These results confirmed that the catalytic current for H2O2 was due to the presence of the cashew/phaeo-b multilayer. Fig. 8A displays a typical current-time plot of ITO/cashew/phaeo-b multilayer modified electrode upon successive step additions of
Fig. 7. Cyclic voltammograms of the clean ITO and LbL films of PAH, cashew gum, phaeo-b, cashew/phaeo-b and PAH/phaeo-b.
Working electrode
Pcp (V)
jcp(μA/cm2)
ITO PAH Cashew gum Phaeo-b PAH/phaeo-b Cashew/phaeo-b
−0.30 −0.30 −0.26 −0.14 −0.16 −0.19
−48 −82 −98 −200 −360 −410
1.1 mmol/L H2O2 under continuous stirring into the HCl (0.1 mol/L) electrolyte. The applied potential of 0.0 V was chosen as the working potential for the amperometric determination of H2O2, where the risk for interfering reactions of other electroactive species in the solution is minimized and also where the background current and noise levels reached their lowest values. A rapid current response could be observed when H2O2 aliquots were added into the stirring acid solution. The maximum steady-state current could be achieved within 12 s, indicating a good H2O2 diffusion process in the modified electrode. The current response increased linearly with the concentration of H2O 2 . Fig. 8B shows the calibration curve between the response current and the concentration of H2O2. The electrode presented a linear range from 3.3 mmol/L to 19.8 mmol/L, governed by the equation: j = 0.481 + 0.0561C, with a correlation coefficient of 0.997. The use of a cashew/phaeo-b electrode as nonenzymatic electrochemical sensor to detect H2O2 has many advantages, such as easy preparation of cashew/phaeo-b multilayer films on the electrode, low cost, suitable electrocatalytic activity and most importantly, it can be operated at 0.0 V potential. Therefore, the approach proposed a new and efficient kind of electrochemical sensor to detect H2O2.
4. Conclusions We produced successful films containing phaeophytin-b, a new biological material extracted from T. subulata Sm., together with natural cashew gum and the polymer PAH using the LbL technique, which is considered a simple technique of low cost and is already well accepted in the literature. The films displayed an electrochemical behavior that varied with the polyelectrolyte that was incorporated in the phaeo-b. Through the voltammetric studies it was possible to identify the oxidation of phaeo-b, and it was observed that the film with three monolayers of phaeo-b showed the highest anodic current peak and a good definition of the anodic peak potential, demonstrating to be a very promising material. The performance of the phaeo-b modified electrode was evaluated for the amperometric detection of H2O2. The ITO electrode modified with three bilayers PAH/phaeo-b displayed the second best result. On the other hand, the cashew/phaeo-b film exhibited the best electrochemical response. The presence of the natural gum was very advantageous, since the redox currents were higher for films containing the cashew gum in comparison to PAH/phaeo-b or phaeo-b films, in this way becoming the most efficient sensor for detection of H2O2. The application of a cashew/phaeo film amperometric sensor presents many advantages, such as low cost, environmental friendliness and suitable electrocatalytic activity at 0.0 V potential. Moreover, the prepared sensor exhibited an effective catalytic response to the reduction of H2O2. The amperometric sensor exhibited a linear response in the range from 3.3 mmol/L to 19.8 mmol/L. Therefore, this electrode provides a novel and promising sensor for the detection of H2O2. The possibility of producing films with phaeophytin-b opens up new prospects for using these natural biological materials in technological applications like electrochemical sensors.
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Fig. 8. (A) Typical current–time response curve for successive addition of 1.1 mmol/L H2O2 for the sensor in 0.1 mol/L HCl at the applied potential of 0.0 V, and (B) calibration curve of H2O2 concentration on the modified electrode ITO/cashew/phaeo-b. The dashed line at (B) serves only as guide.
Acknowledgments This work was supported by CNPq, Nanobiomed Network CAPES (#AUX–PE 705/2009), INCT/INEO (573762/2008-2), INCT/ Nanobiotecnologia. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
[18] [19] [20] [21]
[22]
G. Decher, J.D. Hong, J. Schmitt, Thin Solid Films 210–211 (1992) 831–835. Y. Lvov, H. Haas, G. Decher, H. Möhwald, J. Phys. Chem. 97 (1993) 12835–12841. Y. Lvov, G. Decher, H. Möhwald, Langmuir 9 (1993) 481–486. G. Decher, Science 277 (1997) 1232–1237. M. Ferreira, M.F. Rubner, Macromolecules 28 (1995) 7107–7114. F.N. Crespilho, V. Zucolotto, J.R. Siqueira Jr., C.J.L. Constantino, F.C. Nart, O.N. Oliveira Jr., Environ. Sci. Technol. 39 (2005) 5385–5389. V. Zucolotto, N.M. Barbosa Neto, J.J. Rodrigues Jr., C.J.L. Constantino, S.C. Zílio, C.R. Mendonça, R.F. Aroca, O.N. Oliveira Jr., J. Nanosci. Nanotechnol. 4 (2004) 855–860. G.E. Nunes, A.L. Sehnem, I.H. Bechtold, Liq. Cryst. 39 (2012) 205–210. E.A.O. Farias, N.A. Dionisio, P.V. Quelemes, S.H. Leal, J.M.E. Matos, E.C.S. Filho, I.H. Bechtold, J.R.S.A. Leite, C. Eiras, Mater. Sci. Eng. C 35 (2014) 449–454. F.N. Crespilho, V. Zucolotto, O.N. Oliveira Jr., F.C. Nart, Int. J. Electrochem. Sci. 1 (2006) 194–214. W.B. Stockton, M.F. Rubner, Macromolecules 30 (1997) 2717–2725. J.-i. Anzai, Y. Kobayashi, N. Nakamura, M. Nishimura, T. Hoshi, Langmuir 15 (1999) 221–226. E. Kharlampieva, V. Kozlovskaya, S.A. Sukhishvili, Adv. Mater. 21 (2009) 3053–3065. L. Zhou, M. Chen, L. Tian, Y. Guan, Y. Zhang, ACS Appl. Mater. Interfaces 5 (2013) 3541–3548. M.A. Witt, F. Valenga, M.E.R. Dotto, I.H. Bechtold, O. Felix, A.T.N. Pires, G. Decher, Biointerphases 7 (2012) 64. M.F. Zampa, I.M.S. Araújo, V. Costa, C.H.N. Costa, J.R. Santos Jr., V. Zucolotto, C. Eiras, J.R.S.A. Leite, Nanomedicine 5 (2009) 352–358. I.M.S. Araújo, M.F. Zampa, J.B. Moura, J.R. dos Santos Jr., P. Eaton, V. Zucolotto, L.M.C. Veras, R.C.M. de Paula, J.P.A. Feitosa, J.R.S.A. Leite, C. Eiras, Mater. Sci. Eng. C 32 (2012) 1588–1593. G.A. Towle, R.L. Whistler, New York: Van Nostrand Reinhold 1 (1973) 198–248. D.S. Torquato, M.L. Ferreira, G.C. Sá, E.S. Brito, G.A.S. Pinto, E.H.F. Azevedo, World J. Microbiol. Biotechnol. 20 (2004) 505–507. C.G. Monthe, I.A. Souza, G.M.T. Calazans, Med. Nutr. 19 (2008) 50–52. F.M.F. Monteiro, G.M.M. Silva, J.B.R. da Silva, C.S. Porto, L.B. de Carvalho Jr., J.L.L. Filho, A.M.A. Leão-Carneiro, M.G. Carneiro-Cunha, A.L.F. Porto, Process Biochem. 42 (2007) 884–888. S.B.A. Barros, C.M.d-S. Leite, A.C.F. de Brito, J.R. dos Santos Jr., V. Zucolotto, C. Eiras, Int. J. Anal. Chem. (2012) 1–10.
[23] M.A. Calin, S.V. Parasca, J. Optoelectron. Adv. Mater. 8 (2006) 1173–1179. [24] W.-T. Li, H.-W. Tsao, Y.-Y. Chen, S.-W. Cheng, Y.-C. Hsu, Photochem. Photobiol. Sci. 6 (2007) 1341–1348. [25] E.D. Sternberg, D. Dolphin, Tetrahedron 54 (1998) 4151–4202. [26] K. Sadaoka, S. Kashimura, Y. Saga, Bioorg. Med. Chem. 19 (2011) 3901–3905. [27] A.P. Gerola, A. Santana, P.B. França, T.M. Tsubone, H.P.M. de Oliveira, W. Caetano, E. Kimura, N. Hioka, Photochem. Photobiol. 87 (2011) 884–894. [28] S. Schelbert, S. Aubry, B. Burla, B. Agne, F. Kessler, K. Krupinska, S. Hortensteiner, Plant Cell 21 (2009) 767–785. [29] S.G.B. Filho, M.G. Fernandes, O.S. Chaves, M.C.O. Chaves, F.B. Araruna, C. Eiras, J.R.S.A. Leite, M.F. Agra, R. Braz-Filho, M.F.V. Souza, Quim. Nova 37 (2014) 603–609. [30] D.A. Barbosa, K.N. Silva, M.F. Agra, Rev. Bras. Farmacognosia 17 (2007) 396–413. [31] S.M.O. Costa, J.F. Rodrigues, R.C.M. de Paula, Polímeros Ciênc. Tecnol. 6 (1996) 49–55. [32] V. Zucolotto, M. Ferreira, M.R. Cordeiro, C.J.L. Constantino, W.C. Moreira, O.N. Oliveira Jr., Synth. Met. 137 (2003) 969–970. [33] J.R. Siqueira Jr., L.H.S. Gasparotto, F.N. Crespilho, A.J.F. Carvalho, V. Zucolotto, O.N. Oliveira Jr., J. Phys. Chem. B 110 (2006) 22690–22694. [34] M.F. Zampa, A.C.F. Brito, I.L. Kitagawa, C.J.L. Constantino, O.N. Oliveira Jr., H.N. Cunha, V. Zucolotto, J.R.C. Santos Jr., C. Eiras, Biomacromolecules 8 (2007) 3408–3413. [35] M. Kobayashi, S. Akutsu, D. Fujinuma, H. Furukawa, H. Komatsu, Y. Hotota, Y. Kato, Y. Kuroiwa, T. Watanabe, M. Ohnishi-Kameyama, H. Ono, S. Ohkubo, H. Miyashita, in: Z. Dubinsky (Ed.), Photosynthesis, 2013, pp. 47–90 (Chapter 3). [36] M. Raposo, O.N. Oliveira Jr., Langmuir 18 (2002) 6866–6874. [37] M. Raposo, O.N. Oliveira Jr., Braz. J. Phys. 28 (1998). [38] D.L. Elbert, C.B. Herbert, J.A. Hubbell, Langmuir 15 (1999) 5355–5362. [39] C. Picart, J. Mutterer, L. Richert, Y. Luo, G.D. Prestwich, P. Schaaf, J.C. Voegel, P. Lavalle, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 12531–12535. [40] P. Lavalle, C. Gergely, F.J.G. Cuisinier, G. Decher, P. Schaaf, J.C. Voegel, C. Picart, Macromolecules 35 (2002) 4458–4465. [41] A. Tezcaner, D. Hicks, F. Boulmedais, J. Sahel, P. Schaaf, J.-C. Voegel, P. Lavalle, Biomacromolecules 7 (2006) 86–94. [42] G. Strukul (Ed.), Catalytic oxidations with hydrogen peroxide as oxidant, Introduction and Activation Principles, 9, 1992 (chapter 1). [43] M.C.Y. Chang, A. Pralle, E.Y. Isacoff, C.J. Chang, J. Am. Chem. Soc. 126 (2004) 15392–15393. [44] X. Lu, J. Zhou, W. Lu, Q. Liu, J. Li, Biosens. Bioelectron. 23 (2008) 1236–1243. [45] X. Shu, Y. Chen, H. Yuan, S. Gao, D. Xiao, Anal. Chem. 79 (2007) 3695–3702. [46] D.W. King, W.J. Cooper, S.A. Rusak, B.M. Peake, J.J. Kiddle, D.W. O'Sullivan, M.L. Melamed, C.R. Morgan, S.M. Theberge, Anal. Chem. 79 (2007) 4169–4176. [47] J. Jia, B. Wang, A. Wu, G. Cheng, Z. Li, S. Dong, Anal. Chem. 74 (2002) 2217–2223. [48] C. Eiras, A.C. Santos, M.F. Zampa, A.C.F. Brito, C.J.L. Constantino, V. Zucolotto, J.R. Santos Jr., J. Biomater. Sci. Polym. 21 (2010) 1533–1543.
