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Electroanalysis of cationic species at membrane-carbon electrodes modified by polysaccharides. Bioaccumulation at microorganism-modified electrodes
Talanta 51 (2000) 1077 – 1086 www.elsevier.com/locate/talanta
Electroanalysis of cationic species at membrane-carbon electrodes modified by polysaccharides. Bioaccumulation at microorganism-modified electrodes E. Lojou *, P. Bianco Unite´ de Bioe´nerge´tique et Inge´nierie des Prote´ines, I.F.R. 1, C.N.R.S., 13402 Marseille cedex 20, France Received 12 November 1999; received in revised form 14 January 2000; accepted 20 January 2000
Abstract Membrane-carbon electrodes modified with polysaccharides suspensions entrapped between a dialysis membrane and the carbon surface were used for electroanalysis of various cationic species. Cationic complexes of ruthenium and cobalt, metallic cations (Cu2 + , Fe3 + , UO22 + ) as well as methylviologen were considered. By investigating various parameters (concentration of the suspension, pH) binding of the cations by the polysaccharides was demonstrated. Comparison of cations uptake by different kinds of polysaccharides such as alginic acid, polygalacturonic acid, pectin, dextran and agar was performed. This study has been extended to natural biomaterials, alga and lichen, which are known to contain polysaccharides. The interest of the membrane – electrode strategy is described. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Modified-electrode; Polysaccharides; Alga; Lichen
1. Introduction Modified electrodes offer great advantages for the development of voltammetric measurement schemes with enhanced selectivity and sensitivity. A fruitful direction is to use surfaces capable of preferentially accumulating target analytes from solutions [1]. In such an approach, the preconcentration step is followed by voltammetric quantification of the surface bound analyte. Collecting * Corresponding author. Tel.: +33-4-91164404; fax: + 334-91164578. E-mail address: [email protected] (E. Lojou)
analytes can be based on coordination reactions, covalent attachment or electrostatic interaction [1] at a surface carrying an appropriate ligand or ion-exchanger. Another interest of modified electrodes is that the surfaces can be tailored by using a variety of potentially accumulating (incorporating) substances. The extent and the affinity of these substances can be compared and quantified. For example, bioaccumulation at electrodes modified by micro-organisms or biomass has been investigated [2–4]. In the most popular schemes, the preconcentration agent is commonly introduced into the surface as part of an appropriate poly-
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meric film or by mixing with a carbon paste matrix. An alternative can be to use membrane electrodes as described in previous works [5–7]. In this technique, the preconcentrating agent (generally a cation-exchanger) remains entrapped between a dialysis membrane and the electrode surface, provided that its molar mass is higher than the membrane cutoff. In this configuration, cationic species to be ‘incorporated’ (i.e. preconcentrated) are within the bulk solution in the electrochemical cell. They are able to pass across the membrane contrary to large incorporating material which remains imprisoned in the close vicinity of the electrode surface. Information on the capability of clays and humic acid suspensions to accumulate cations was gained using this technique as reported in previous works [7,8]. Polysaccharides are natural macromolecules which play a prominent role in that they act in both a structural function (e.g. as constituents of plant fibers, wood, and cell walls) and as a means of energy storage (via their conversion into glycogen and starch). These macromolecules have a high percentage of alcohol-, keto-, and carboxyl groups that are responsible for their complexing ability. The interaction of polysaccharides with metal cations has been considered in relatively few investigations, and almost solely with copper, lead and cadmium [9 – 12]. This paper describes the incorporating properties of some natural ionic polysaccharides acting as modifier agents of membrane-carbon electrodes towards a series of cations. Polysaccharides investigated here such as alginic, polygalacturonic acids and agar –agar are present in natural aquatic media (seaweed) or edible fruits (e.g. apple, lemon, etc.). Dextran is produced by bacteria growing on a sucrose substrate. The technology based on the use of membrane modified-electrodes can be extended to micro-organisms which are supposed to contain polysaccharides. The predisposition for organic cation uptake by micro-organisms such as algae and lichens from their substratum and from rain- and sea water is well documented. Both types of micro-organisms are widely used as biological monitors of air- and water pollution [13,14]. In this
paper, it is shown that it is possible to detect the bioaccumulation of cationic species by cyclic voltammetry at a membrane biomaterial-modified electrode. We have chosen an alga (Fucus 6esiculosus) and a lichen (E6ernia prunastri ) commonly found in sea water and on oak trunks, respectively. The advantage and performances of such biomaterial-modified electrodes are described and compared to those of other conventional modified electrodes.
2. Experimental
2.1. Materials Stock solutions of polysaccharide were prepared by dispersing commercial materials in deionized water: alginic acid from Macrocystis pyrifera (Sigma) (AA is the abbreviation for alginic acid), polygalacturonic acid from orange (Sigma), pectin from citrus fruit (Sigma), agar (Fluka), dextran sulfate sodium salt from Leuconostoc ssp. (Fluka) and lichenan from Cetraria islandica (ICN). Suspensions were stirred for 1 h, and then stocked at +4°C when not in use. The common alga Fucus 6esiculosus (Fv) was purchased under a fine powder form from a local herb-shop (Marseille). The lichen E6ernia prunastri was collected on oak trunks from Basse Provence (South France). It was dried in an oven and then ground into a fine powder. Weighed amounts of alga or lichen were suspended into water and stirred for 1 h before using. Suspensions were kept at +4°C when not in use. Hexaammineruthenium(III) chloride was purchased from Strem Chemicals. Cobalt(III) sepulchrate trichloride (abbreviation: Co(sep)3 + ) and Tris(2,2’-bipyridine)-ruthenium(II) chloride (abbreviation: Ru(bpy)23 + ) were obtained from Aldrich and Sigma, respectively. All other chemicals were reagent grade. All solutions were prepared from distilled, deionized water. Unless stated otherwise, supporting electrolyte was either 10 mM Tris chloride buffer, pH 7.6, or 0.1 M sodium cacodylate buffer, pH 5.1.
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2.2. Apparatus and procedure An EG&G 273A potentiostat controlled by a Prolinea 4/66 microcomputer with EG&G PARC 270/250 software was used for cyclic voltammetry (CV). A three-electrode system consisting of a Metrohm Ag/AgCl/saturated NaCl reference electrode, a platinum wire auxiliary electrode and the working electrode (see above) was used throughout. Unless otherwise stated, all potentials are referred to the Ag/AgCl/saturated NaCl electrode. All experiments were carried out at room temperature (23°C). Working electrodes were constructed from 4 mm rods of pyrolytic graphite (PG) from Le Carbone Lorraine (Paris) housed in epoxy sheaths and cut with the disk face parallel to the edge plane. First, the electrode surface was polished with 0.05 mm alumina slurry. Then, the membrane electrode was prepared as described in [5–7]. A small volume (3 ml) of polysaccharide or biomaterial suspension was deposited on a piece of the dialysis membrane (Visking MWCO 6000/8000 membrane, 30 mm thick, which does not allow particles with molar mass superior to 6000–8000
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to diffuse). The polished electrode surface was pressed down on the membrane; a rubber ring having a diameter that fitted the electrode snugly was gently pushed around the electrode body so that the entrapped suspension formed a uniform thin layer on the carbon surface. The so-mounted electrode was then transferred to the electrochemical cell containing the bulk solution of interest. In such a configuration, small ions could diffuse through the membrane, but particles in the suspension remained entrapped in the close vicinity of the carbon electrode surface. Membrane electrode fillings were renewed before each measurement. Some experiments had to be performed at mercury-film glassy carbon electrodes [15,16]. After polishing of glassy carbon (GC) surface electrodes (constructed from 3 mm diameter rods of GC from AIMCOR, Tokyo) to a mirror finish, the preformed films were electrodeposited for 2 min at − 1.0 V from 100 mM Hg(NO3)2 in 20 mM KCl solution. The preformed film-electrodes were used immediately, with as little exposure to air as possible, and renewed before each experiment.
3. Results and discussion
3.1. Characterization of the membrane alginic acid-modified electrode
Fig. 1. Steady-state cyclic voltammograms at the membrane PG electrode of 90 mM Ru(NH3)36 + in 10 mM Tris chloride buffer solution, pH 7.6, in the absence of alginic acid (broken line), in the presence of 5 g l − 1 AA filling (solid line). Scan rate: 20 mV s − 1.
3.1.1. Interaction between Ru(NH3) 36 + and alginic acid In a first step, the performances of the membrane AA-modified electrode are explored using well-known Ru(NH3)36 + as a cation model. The ability of alginic acid to incorporate Ru(NH3)36 + cations is illustrated from cyclic voltammograms (Fig. 1) at the membrane-electrode modified as described in the experimental section. The concentration of Ru(NH3)36 + is 90 mM in 10 mM Tris chloride buffer solution, pH 7.6. A concentration of 5 g l − 1 AA-filling is used. Gradual increase in peak currents upon repetitive cycling illustrates the uptake of Ru(NH3)36 + by AA suspension. The steady-state voltammogram in Fig. 1 (solid line) is attained after approximately 10 min. The steadystate peak currents are significantly larger than
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those observed when AA is absent from the membrane filling (broken line). The magnitude of cation uptake is well represented by the ratio of the steady-state CV cathodic peak current at the membrane AA-modified electrode to that measured at the membrane electrode in the absence of AA. This enhancement ratio, Re, is 3.4. It can be concluded that Ru(NH3)36 + cations are bound by alginate anions, as a result of favorable electrostatic interactions. The CV peak potentials are Epc = − 0.17 V, Epa = −0.09 V (i.e. E1/2 = − 0.13 V) in the absence of AA, and Epc = −0.25 V, Epa = − 0.16 V (i.e. E1/2 = − 0.20 V) when using 5 g l − 1 AA-filling. The more negative value of E1/2 in the presence of AA is indicative of a stabilization of Ru(NH3)36 + cations by alginic acid. When the membrane Ru(NH3)36 + -loaded electrode was transferred to a blank solution, the majority of incorporated cations was retained by the film over the first minutes of immersion. The retention capability of the AA suspension was estimated by measuring the retention ratio, ip(15)/ ip(0). It is defined as the ratio of the CV peak current 15 min after transfer to the pure supporting electrolyte solution to that recorded immediately after the transfer process. For example, a retention ratio of 0.8 was obtained after accumulation from 30 mM solution. Different concentrations of AA suspension in the range 0– 10 g l − 1 were prepared. Then, repetitive cyclic voltammograms were recorded to detect both the time necessary for obtaining steady-state currents and the extent of the incorporation process. Poor increases in peak currents were detected for filling values under 1 g l − 1. Virtually no incorporation was observed for 10 g l − 1 filling probably because of too poor diffusion in the jellied AA -suspension. The maximum value in CV peak currents was observed for 5 g l − 1 AA filling. The capability of AA suspension to incorporate Ru(NH3)36 + (from 30 mM solution) has been examined as a function of pH. Measurements were performed in 10 mM Tris chloride buffer solutions adjusted at selected pH with acetic acid, hydrochloride acid or sodium hydroxide. The enhancement ratio Re remains virtually unmodified
within the 5–9 pH range. A low decrease in Re value is observed when pH drops from 5 to 3.
3.1.2. Comparison with other polysaccharides Alginic acid is a natural polymer containing D-mannuronic acid units. The ability of membrane-modified electrode to take up Ru(NH3)36 + from solutions has been investigated for other polysaccharides: polygalacturonic acid, pectin (a polymer of methyl-D-galacturonate), agar (which contains residues of D- and L-galactose), and dextran (which contains backbones of D-glucose units linked predominantly a-D(1-6)). Results are given in Fig. 2 (A) and (B) for comparison. The same profiles are obtained for all the tested polysaccharides. Enhancement ratios increase as the polysaccharide concentrations are raised up as expected from the increased binding capacity of the suspensions. Decreases in Re values are observed at higher modifier concentrations because of the increasing viscosity of the prisoned suspension. In particular, poorly reproducible responses are obtained for pectin concentrations superior to 20 g l − 1. 3.1.3. Incorporation of cationic species: Cu 2 + , UO 22 + , Ru(bpy) 23 + , Co(sep) 3 + , Fe 3 + The binding capacity of alginic acid towards a series of cationic species has been investigated using the membrane AA-modified electrode. CV experiments were performed at the same 110 mM cation concentration using a 5 g l − 1 AA-suspension. Supporting electrolyte was 10 mM Tris chloride buffer, pH 7.6 for Ru(bpy)23 + and Co(sep)3 + . 0.1 M cacodylate buffer, pH 5.1 was used in the case of Cu2 + , UO22 + and Fe3 + to prevent hydroxocomplexes from precipitating. To avoid the presence of metallic copper at the electrode surface, a preformed mercury film (see experimental section) was electrodeposited. The membrane AAmodified mercured GC electrode was used for studying Cu2 + incorporation. The extent of incorporation was evaluated from the enhancement ratio as defined in Section 3.1.1. Incorporations were detected for all the cations investigated, as evidenced from the comparison between the CV curves obtained at the membrane electrode containing/not containing alginic acid. A marked
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shift (of at least 80 mV) in peak potentials towards more negative values occurs when AA is present within the membrane. Indeed, alginic acid stabilizes the oxidized form, thus resulting in negative shift in the potential values. Data in Table 1 compare the enhancement ratios measured for the various cations investigated in this work. It is noticeable that a high level of incorporated cations is attained with Fe3 + . This is not surprising when considering the strong affinity of iron(III) to oxygenated ligands and its triple positive charge. However, another factor to be considered is the unavoidable presence of soluble hydroxocomplexes at pH 5.1. Their extent, structure and charge depend largely on the nature of the metal cation to be incorporated, and on the type of counter anion. In contrast, in the set of experiments performed at pH 7.6, no marked difference can be noted between the other investigated cations. An average value of about 3 is evaluated, though differences in charge and redox potential for these cationic species. Note for example that the reduction/reoxidation of Ru(bpy)23 + /3 + couple is observed in the + 0.9 to + 1.2 V range contrary to very less positive potential values for the other couples.
3.2. Bioaccumulation in natural materials Fig. 2. Dependence of steady-state cathodic CV peak currents on the concentration of polysaccharide within the membrane filling for 90 mM Ru(NH3)36 + in 10 mM Tris chloride buffer solution, pH 7.6; (A) suspension of ( ) alginic acid, () polygalacturonic acid and (B) suspension of ( ) agar, ()dextran, ( ) pectin.
Table 1 Enhancement ratio Re for various cations from steady-state CV experiments at the membrane AA-modified electrode (5 g l−1 AA filling) Cationa Re
Cu2+ 1.9
UO2+ 2 2
Fe3+ 14
Cationb Re
Ru(bpy)2+ 3 3.5
Ru(NH3)3+ 6 3.4
Co(sep)3+ 2.8
a b
110 mM in 0.1 M sodium cacodylate buffer, pH 5.1. 110 mM in 10 mM Tris chloride buffer, pH 7.6.
3.2.1. E6aluation of alga binding capacity The presence of an alga suspension within the membrane electrode results in effective accumulation of cationic species, as shown when studying Ru(NH3)36 + and MV2 + cation models. This is well illustrated in Fig. 3 (A) which compares steady-state cyclic voltammograms obtained at pH 7.6 with the unmodified and membrane alga-modified electrode (25 g l − 1 suspension) for Ru(NH3)36 + cationic species. The peak currents are significantly higher when alga material is present in the membrane electrode. The dependence of the peak currents on the accumulation time was examined. For this experiment, the ‘preconcentration/medium exchange/ voltammetry’ procedure was used. For the preconcentration step, the membrane modified electrode was immersed in a stirred solution containing the cation of interest for a given period of
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time. This step proceeded at open circuit. The electrode was then removed from the preconcentration cell, rinsed with deionized water and placed in the electrochemical cell containing the buffer solution. From Fig. 3 (B), it can be seen that the response rises progressively with the accumulation time before leveling off at time of around 10 min. Such a profile slightly differs from the kinetics of metallic cations uptake previously
Fig. 3. (A) Steady-state cyclic voltammograms at the membrane-PG electrode in 10 mM Tris chloride buffer solution, pH 7.6 of 100 mM Ru(NH3)36 + , in the absence of Fv alga (fine line), in the presence of 25 g l − 1 Fv alga suspension (solid line); Scan rate: 20 mV s − 1. (B) Cathodic peak currents at the alga-modified electrode as a function of the accumulation time; 100 mM Ru(NH3)36 + ; 10 mM Tris chloride buffer solution, pH 7.6; 25 g l − 1 Fv alga suspension; Scan rate: 20 mV s − 1.
described [2]. It represents in fact two concomitant processes, especially in the first minutes of accumulation: transport throughout the membrane material and cation binding with the biological matter. Weaker but effective accumulation was also observed in the case of the toxic herbicide methylviologen. The enhancement ratios are Re = 10.1 and 2.1 for the Ru(NH3)36 + /alga and MV2 + / alga couples, respectively. Further information regarding the bioaccumulation process was obtained by studying the effect of the suspension concentration, pH and ionic strength of the buffer solution. The CV peak heights increase with the concentration of the alga suspension as expected from the increased binding capacity of the membrane electrode. Fig. 4 (A) describes the particular case of MV2 + incorporation. A maximum of cation uptake is reached with 20 g l − 1 alga concentration in the suspension. A strong decrease in peak heights is observed for alga concentrations higher than about 25 g l − 1 because of the jellification of the suspension. Fig. 4 (B) shows the pH-dependence of cation accumulation rate in 5 g l − 1 alga-suspension in the case of MV2 + . The cation binding to the alga suspension increases when pH is raised up from 3.5 to 6, as a consequence of the deprotonation of coordinating groups on the biomaterial surface [17], and Re decreases when pH is raised up from 6 to 9. It must be reminded that a low decrease in Re values is observed within the 3–5 pH range when using membrane AA-modified electrode (see Section 3.1.1). No variation of Re is noted with AA-suspension in the 5–9 pH range. This result suggests that different chemical functions are involved in the cation uptake. The dependence of the enhancement ratio Re for Ru(NH3)36 + incorporation upon ionic strength was examined by addition of sodium chloride in solution. As the ionic strength increases, Re progressively decreases (Fig. 4 (C)). Because ruthenium under the hexammine-complexed form is not able to give anionic complexes, such ionic strength dependence must be attributed to competition of sodium cation for the surface exchange sites (note the sodium level is 4× 103 higher than that of the analyte).
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Fig. 4. (A) Effect of the concentration of Fv alga-suspension on the steady-state CV cathodic peak current for 100 mM MV2 + ; 10 mM Tris chloride buffer solution, pH 7.6; 5 g l − 1 Fv alga suspension; Scan rate: 20 mV s − 1. (B) pH-dependence of the enhancement ratio Re at the membrane Fv alga suspension-modified electrode for 100 mM MV2 + ; concentration of the suspension: 5 g l − 1; 10 mM Tris chloride buffer solution, pH 7.6; Scan rate: 20 mV s − 1. (C) Ionic strength-dependence of the enhancement ratio Re at the membrane Fv alga suspension-modified electrode for 100 mM Ru(NH3)36 + ; concentration of the suspension: 25 g l − 1; 10 mM Tris chloride buffer solution, pH 7.6; Scan rate: 20 mV s − 1.
The ability of the membrane alga-modified electrode to trap other cationic has been investigated in the case of two metal cations of environmental interest, Cu2 + and Fe3 + . The membrane modified electrode was first immersed for a given period of time in the electrochemical cell containing the supporting electrolyte and Cu2 + or Fe3 + cations under stirring. This preliminary step allowed cations to preconcentrate into the biomaterial. After stopping stirring, CV curves were recorded. The same experiments were performed in the absence of biomaterial for comparison. Accumulation of both cations occurs in the algamodified electrode. Steady-state currents were attained upon 10 min of repetitive scanning. Respective enhancement ratios of 2 and 5 were measured for Cu2 + and Fe3 + . The same value has been previously evaluated for Cu2 + at the membrane AA-modified electrode (see Table 1).
The marked affinity of iron (III) for AA as noted above (Table 1) is also observed in the case of the natural alga, thus suggesting the very likely involvement of alginic acid in the cation uptake process.
3.2.2. Comparison with lichen binding capacity Lichens are a group of non-vascular plants composed of fungal and alga species growing in a symbiotic relationship. They were recognized as potential indicators of air pollution as early as the 1860s. They have also been found to act as accumulators of trace metals and radioactive elements [18]. Despite the wide diversity of the basic growth forms, all lichens have a similar internal morphology. The bulk of the lichen’s body is formed from filaments of the fungal partner. At its outer surface, the filaments are packed tightly together to form the cortex. The alga partner cells
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Fig. 5. (A) Steady-state cyclic voltammograms at the membrane-PG electrode in 10 mM Tris chloride buffer solution, pH 7.6 of 100 mM Ru(NH3)36 + , in the absence of lichen (fine line), in the presence of 80 g l − 1 lichen suspension (solid line); Scan rate: 20 mV s − 1. (B) Effect of the concentration of the lichen-suspension on the enhancement ratio ( ) and steady-state CV cathodic peak potential () for 100 mM Ru(NH3)36 + ; 10 mM Tris chloride buffer solution, pH 7.6; Scan rate: 20 mV s − 1.
are distributed just below the cortex in a layer where the fungal filaments are not so dense. Below the alga layer is the medulla, a loosely woven layer of fungal filaments. Most lichens have an extracellular matrix which is a gelatinous secretion of polysaccharides such as lichenan and isolichenan. From this general structure, it was of a great interest to consider the binding capacity of lichens, since cations can be accumulated a priori in several parts of this layered fungal and alga species. Fig. 5 (A) illustrates the accumulation of cationic species in the lichen suspension within the membrane electrode. In the case of Ru(NH3)36 + cations an enhancement ratio of 4.8 is reached. When compared to the enhancement ratio observed with alginic acid, this result indicates that lichen exhibits high cation binding capacity. Richardson et al. [19] suggested that a
proportion of the metal binding sites might be located on the extracellular matrix. No incorporation of cationic species has been observed in suspension of lichenan, however. This result agrees with previous data [20] which attest that the removal of all of the extracellular matrix does not decrease the metal-binding capacity. It thus appears that metal binding is primarily an interaction between metal ions and sites located on or within the cell walls of the fungal component. The pH-dependence of the cation accumulation rate in the lichen-suspension is similar to that obtained in the presence of alga: an increase in CV peak heights when pH is raised up from 5 to 7.5, followed by a decrease in the accumulation capacity of the lichen material at basic pH values. This similarity in the pH-dependence of CV peak heights suggests that similar chemical functions
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are involved in cation binding in both alga and lichen. The effect of the lichen concentration on the cation bioaccumulation can be described from the evolution of peak potential values and Re according to a two step process (Fig. 5 (B)). Within the lower lichen concentrations (0 – 20 g l − 1 range), no accumulation of Ru(NH3)36 + species can be observed (Re =1). Complexation of the cationic species by the lichen suspension occurs, however, as demonstrated by the decrease in peak potential from − 185 to −240 mV. Once this complexation equilibrium has been reached, the CV peak heights increase with increasing lichen concentration until a limit of around 100 g l − 1 is attained. At this concentration, the jellification of the suspension is supposed to slow down the accumulation process as already observed with the alga suspension. This peculiar behavior of lichen compared to alga suspension can be attributed to the structure of lichen where the alga layer is hindered by the fungal layer. The interaction between Cu2 + or Fe3 + and the lichen suspension was then studied, following the same experimental procedure as that described for the alga suspension. Concerning Cu2 + , the most noticable feature is the negative shift of the reduction potential. It seems that Cu2 + cations and lichen materials interact via a complexation reaction rather than via an accumulation process. It is not the case for Fe3 + , since increasing voltammetric peak heights upon repetitive cycling are observed with an enhancement ratio of 7. Again a high affinity of Fe(III) for the biomaterial is observed which suggests the involvement of polysaccharides in the cation uptake process.
4. Conclusion Membrane-modified electrodes offer an alternative to carbon-paste electrodes for gaining insights into the interaction of ion-exchanging polymers with cationic species, in particular in the case of polysaccharides as demonstrated in this work. Several advantages of the membrane electrode can be underlined from this and
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previous papers [5–8]. First, the entrapped modifying agent is not able to be released into the solution under investigation (providing it has a sufficiently high enough molar mass). There are no limitations from the presence of diluting agent (paraffin or silicon grease) which can usually result in increased electrode resistance. Because of the thin-layer arrangement of the membrane electrode, the effect of oxygen is minimized upon cathodic cycling. Another major advantage is the easy removal of the membrane electrode, without need of either soaking in acidic medium or treatment with NaCl and EDTA [20]. The application of membrane electrodes modified with biomaterials such as alga or lichen to metal collection is also illustrated in this work. Heavy metals are normal constituents in sea water where they are present in low concentrations. However several metals (mercury, cadmium, etc…) occur in industrial and domestic wastes, so that they can contaminate natural waters. Although they have relatively low solubility (especially at neutral pH), they can reach higher levels in organisms via biomagnification. This point has been well-established in this work from measurements at the membrane electrode in the case of typical cations such as Cu2 + and Fe3 + . A favorite candidate for incorporating in algae, especially in F. 6esiculosus seems to be Fe(III), as previously outlined [21] from measurements on samples from inhabiting coastal areas around Great Britain. In conclusion, this work has illustrated the performances of the membrane electrodes modified with natural substances or biomaterials. Insights in the interactions with metals can be gained by varying the nature of the suspension, thus offering perspectives for enlarged comparisons of the behavior of various natural substances towards cationic species. References [1] J. Wang, Analytical Electrochemistry, VCH, New York, 1994. [2] J. Wang, M.S. Lin, Anal. Chem. 60 (1988) 1545. [3] J. Gardea-Torresdey, D. Darnall, J. Wang, Anal. Chem. 60 (1988) 72. [4] J. Wang, B. Tian, G.R. Rayson, Talanta 39 (1992) 1637.
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[5] J. Haladjian, P. Bianco, F. Nunzi, M. Bruschi, Anal. Chim. Acta 289 (1994) 15. [6] P. Bianco, N. Aghroud, Electroanalysis 9 (1997) 602. [7] M. Pecorari, P. Bianco, Electroanalysis 10 (1998) 181. [8] L. Ghantous, E. Lojou, P. Bianco, Electroanalysis 10 (1998) 1249. [9] E. Reisenhofer, A. Cesaro, F. Delben, G. Manzini, S. Paoletti, Bioelectrochem. Bioenerg. 12 (1984) 455. [10] H.P. van Leeuwen, R. Cleven, J. Buffle, Pure Appl. Chem. 61 (1989) 255. [11] A.M. Nadal, C. Arino, M. Esteban, E. Casassas, Electroanalysis 3 (1991) 309. [12] C. Arino, A.M. Nadal, M. Esteban, E. Casassas, Electroanalysis 4 (1992) 757. [13] J. Garty, M. Kauppi, Environ. Toxic. Chem. 16 (1997) 2404.
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[14] D.H.S. Richardson, Bot. Linnean. Soc. 96 (1988) 31. [15] G.E. Batley, T.M. Florence, J. Electroanal. Chem. 55 (1974) 23. [16] S.C. Petrovic, H.D. Dewald, Anal. Chim. Acta 357 (1997) 33. [17] B. Greene, M.T. Henzl, M. Hosea, D.W. Darnall, Biotechnol. Bioeng. 28 (1986) 764. [18] V. Ahmadjian, The lichen symbiosis, Wiley, New York, NY, 1993. [19] D.H.S. Richardson, E. Nieboer, Cellular Interactions in Symbiosis and Parasitism, Ohio state University Press, Colombus, 1980, p. 75. [20] E. Dempsey, M. Smyth, D.H.S. Richardson, Analyst 117 (1992) 1467. [21] L.C. Ray, J.P. Gaur, H.D. Kumar, Phycology Biol. Rev. 56 (1981) 99.
Electroanalysis of cationic species at membrane-carbon electrodes modified by polysaccharides. Bioaccumulation at microorganism-modified electrodes E. Lojou *, P. Bianco Unite´ de Bioe´nerge´tique et Inge´nierie des Prote´ines, I.F.R. 1, C.N.R.S., 13402 Marseille cedex 20, France Received 12 November 1999; received in revised form 14 January 2000; accepted 20 January 2000
Abstract Membrane-carbon electrodes modified with polysaccharides suspensions entrapped between a dialysis membrane and the carbon surface were used for electroanalysis of various cationic species. Cationic complexes of ruthenium and cobalt, metallic cations (Cu2 + , Fe3 + , UO22 + ) as well as methylviologen were considered. By investigating various parameters (concentration of the suspension, pH) binding of the cations by the polysaccharides was demonstrated. Comparison of cations uptake by different kinds of polysaccharides such as alginic acid, polygalacturonic acid, pectin, dextran and agar was performed. This study has been extended to natural biomaterials, alga and lichen, which are known to contain polysaccharides. The interest of the membrane – electrode strategy is described. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Modified-electrode; Polysaccharides; Alga; Lichen
1. Introduction Modified electrodes offer great advantages for the development of voltammetric measurement schemes with enhanced selectivity and sensitivity. A fruitful direction is to use surfaces capable of preferentially accumulating target analytes from solutions [1]. In such an approach, the preconcentration step is followed by voltammetric quantification of the surface bound analyte. Collecting * Corresponding author. Tel.: +33-4-91164404; fax: + 334-91164578. E-mail address: [email protected] (E. Lojou)
analytes can be based on coordination reactions, covalent attachment or electrostatic interaction [1] at a surface carrying an appropriate ligand or ion-exchanger. Another interest of modified electrodes is that the surfaces can be tailored by using a variety of potentially accumulating (incorporating) substances. The extent and the affinity of these substances can be compared and quantified. For example, bioaccumulation at electrodes modified by micro-organisms or biomass has been investigated [2–4]. In the most popular schemes, the preconcentration agent is commonly introduced into the surface as part of an appropriate poly-
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meric film or by mixing with a carbon paste matrix. An alternative can be to use membrane electrodes as described in previous works [5–7]. In this technique, the preconcentrating agent (generally a cation-exchanger) remains entrapped between a dialysis membrane and the electrode surface, provided that its molar mass is higher than the membrane cutoff. In this configuration, cationic species to be ‘incorporated’ (i.e. preconcentrated) are within the bulk solution in the electrochemical cell. They are able to pass across the membrane contrary to large incorporating material which remains imprisoned in the close vicinity of the electrode surface. Information on the capability of clays and humic acid suspensions to accumulate cations was gained using this technique as reported in previous works [7,8]. Polysaccharides are natural macromolecules which play a prominent role in that they act in both a structural function (e.g. as constituents of plant fibers, wood, and cell walls) and as a means of energy storage (via their conversion into glycogen and starch). These macromolecules have a high percentage of alcohol-, keto-, and carboxyl groups that are responsible for their complexing ability. The interaction of polysaccharides with metal cations has been considered in relatively few investigations, and almost solely with copper, lead and cadmium [9 – 12]. This paper describes the incorporating properties of some natural ionic polysaccharides acting as modifier agents of membrane-carbon electrodes towards a series of cations. Polysaccharides investigated here such as alginic, polygalacturonic acids and agar –agar are present in natural aquatic media (seaweed) or edible fruits (e.g. apple, lemon, etc.). Dextran is produced by bacteria growing on a sucrose substrate. The technology based on the use of membrane modified-electrodes can be extended to micro-organisms which are supposed to contain polysaccharides. The predisposition for organic cation uptake by micro-organisms such as algae and lichens from their substratum and from rain- and sea water is well documented. Both types of micro-organisms are widely used as biological monitors of air- and water pollution [13,14]. In this
paper, it is shown that it is possible to detect the bioaccumulation of cationic species by cyclic voltammetry at a membrane biomaterial-modified electrode. We have chosen an alga (Fucus 6esiculosus) and a lichen (E6ernia prunastri ) commonly found in sea water and on oak trunks, respectively. The advantage and performances of such biomaterial-modified electrodes are described and compared to those of other conventional modified electrodes.
2. Experimental
2.1. Materials Stock solutions of polysaccharide were prepared by dispersing commercial materials in deionized water: alginic acid from Macrocystis pyrifera (Sigma) (AA is the abbreviation for alginic acid), polygalacturonic acid from orange (Sigma), pectin from citrus fruit (Sigma), agar (Fluka), dextran sulfate sodium salt from Leuconostoc ssp. (Fluka) and lichenan from Cetraria islandica (ICN). Suspensions were stirred for 1 h, and then stocked at +4°C when not in use. The common alga Fucus 6esiculosus (Fv) was purchased under a fine powder form from a local herb-shop (Marseille). The lichen E6ernia prunastri was collected on oak trunks from Basse Provence (South France). It was dried in an oven and then ground into a fine powder. Weighed amounts of alga or lichen were suspended into water and stirred for 1 h before using. Suspensions were kept at +4°C when not in use. Hexaammineruthenium(III) chloride was purchased from Strem Chemicals. Cobalt(III) sepulchrate trichloride (abbreviation: Co(sep)3 + ) and Tris(2,2’-bipyridine)-ruthenium(II) chloride (abbreviation: Ru(bpy)23 + ) were obtained from Aldrich and Sigma, respectively. All other chemicals were reagent grade. All solutions were prepared from distilled, deionized water. Unless stated otherwise, supporting electrolyte was either 10 mM Tris chloride buffer, pH 7.6, or 0.1 M sodium cacodylate buffer, pH 5.1.
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2.2. Apparatus and procedure An EG&G 273A potentiostat controlled by a Prolinea 4/66 microcomputer with EG&G PARC 270/250 software was used for cyclic voltammetry (CV). A three-electrode system consisting of a Metrohm Ag/AgCl/saturated NaCl reference electrode, a platinum wire auxiliary electrode and the working electrode (see above) was used throughout. Unless otherwise stated, all potentials are referred to the Ag/AgCl/saturated NaCl electrode. All experiments were carried out at room temperature (23°C). Working electrodes were constructed from 4 mm rods of pyrolytic graphite (PG) from Le Carbone Lorraine (Paris) housed in epoxy sheaths and cut with the disk face parallel to the edge plane. First, the electrode surface was polished with 0.05 mm alumina slurry. Then, the membrane electrode was prepared as described in [5–7]. A small volume (3 ml) of polysaccharide or biomaterial suspension was deposited on a piece of the dialysis membrane (Visking MWCO 6000/8000 membrane, 30 mm thick, which does not allow particles with molar mass superior to 6000–8000
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to diffuse). The polished electrode surface was pressed down on the membrane; a rubber ring having a diameter that fitted the electrode snugly was gently pushed around the electrode body so that the entrapped suspension formed a uniform thin layer on the carbon surface. The so-mounted electrode was then transferred to the electrochemical cell containing the bulk solution of interest. In such a configuration, small ions could diffuse through the membrane, but particles in the suspension remained entrapped in the close vicinity of the carbon electrode surface. Membrane electrode fillings were renewed before each measurement. Some experiments had to be performed at mercury-film glassy carbon electrodes [15,16]. After polishing of glassy carbon (GC) surface electrodes (constructed from 3 mm diameter rods of GC from AIMCOR, Tokyo) to a mirror finish, the preformed films were electrodeposited for 2 min at − 1.0 V from 100 mM Hg(NO3)2 in 20 mM KCl solution. The preformed film-electrodes were used immediately, with as little exposure to air as possible, and renewed before each experiment.
3. Results and discussion
3.1. Characterization of the membrane alginic acid-modified electrode
Fig. 1. Steady-state cyclic voltammograms at the membrane PG electrode of 90 mM Ru(NH3)36 + in 10 mM Tris chloride buffer solution, pH 7.6, in the absence of alginic acid (broken line), in the presence of 5 g l − 1 AA filling (solid line). Scan rate: 20 mV s − 1.
3.1.1. Interaction between Ru(NH3) 36 + and alginic acid In a first step, the performances of the membrane AA-modified electrode are explored using well-known Ru(NH3)36 + as a cation model. The ability of alginic acid to incorporate Ru(NH3)36 + cations is illustrated from cyclic voltammograms (Fig. 1) at the membrane-electrode modified as described in the experimental section. The concentration of Ru(NH3)36 + is 90 mM in 10 mM Tris chloride buffer solution, pH 7.6. A concentration of 5 g l − 1 AA-filling is used. Gradual increase in peak currents upon repetitive cycling illustrates the uptake of Ru(NH3)36 + by AA suspension. The steady-state voltammogram in Fig. 1 (solid line) is attained after approximately 10 min. The steadystate peak currents are significantly larger than
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those observed when AA is absent from the membrane filling (broken line). The magnitude of cation uptake is well represented by the ratio of the steady-state CV cathodic peak current at the membrane AA-modified electrode to that measured at the membrane electrode in the absence of AA. This enhancement ratio, Re, is 3.4. It can be concluded that Ru(NH3)36 + cations are bound by alginate anions, as a result of favorable electrostatic interactions. The CV peak potentials are Epc = − 0.17 V, Epa = −0.09 V (i.e. E1/2 = − 0.13 V) in the absence of AA, and Epc = −0.25 V, Epa = − 0.16 V (i.e. E1/2 = − 0.20 V) when using 5 g l − 1 AA-filling. The more negative value of E1/2 in the presence of AA is indicative of a stabilization of Ru(NH3)36 + cations by alginic acid. When the membrane Ru(NH3)36 + -loaded electrode was transferred to a blank solution, the majority of incorporated cations was retained by the film over the first minutes of immersion. The retention capability of the AA suspension was estimated by measuring the retention ratio, ip(15)/ ip(0). It is defined as the ratio of the CV peak current 15 min after transfer to the pure supporting electrolyte solution to that recorded immediately after the transfer process. For example, a retention ratio of 0.8 was obtained after accumulation from 30 mM solution. Different concentrations of AA suspension in the range 0– 10 g l − 1 were prepared. Then, repetitive cyclic voltammograms were recorded to detect both the time necessary for obtaining steady-state currents and the extent of the incorporation process. Poor increases in peak currents were detected for filling values under 1 g l − 1. Virtually no incorporation was observed for 10 g l − 1 filling probably because of too poor diffusion in the jellied AA -suspension. The maximum value in CV peak currents was observed for 5 g l − 1 AA filling. The capability of AA suspension to incorporate Ru(NH3)36 + (from 30 mM solution) has been examined as a function of pH. Measurements were performed in 10 mM Tris chloride buffer solutions adjusted at selected pH with acetic acid, hydrochloride acid or sodium hydroxide. The enhancement ratio Re remains virtually unmodified
within the 5–9 pH range. A low decrease in Re value is observed when pH drops from 5 to 3.
3.1.2. Comparison with other polysaccharides Alginic acid is a natural polymer containing D-mannuronic acid units. The ability of membrane-modified electrode to take up Ru(NH3)36 + from solutions has been investigated for other polysaccharides: polygalacturonic acid, pectin (a polymer of methyl-D-galacturonate), agar (which contains residues of D- and L-galactose), and dextran (which contains backbones of D-glucose units linked predominantly a-D(1-6)). Results are given in Fig. 2 (A) and (B) for comparison. The same profiles are obtained for all the tested polysaccharides. Enhancement ratios increase as the polysaccharide concentrations are raised up as expected from the increased binding capacity of the suspensions. Decreases in Re values are observed at higher modifier concentrations because of the increasing viscosity of the prisoned suspension. In particular, poorly reproducible responses are obtained for pectin concentrations superior to 20 g l − 1. 3.1.3. Incorporation of cationic species: Cu 2 + , UO 22 + , Ru(bpy) 23 + , Co(sep) 3 + , Fe 3 + The binding capacity of alginic acid towards a series of cationic species has been investigated using the membrane AA-modified electrode. CV experiments were performed at the same 110 mM cation concentration using a 5 g l − 1 AA-suspension. Supporting electrolyte was 10 mM Tris chloride buffer, pH 7.6 for Ru(bpy)23 + and Co(sep)3 + . 0.1 M cacodylate buffer, pH 5.1 was used in the case of Cu2 + , UO22 + and Fe3 + to prevent hydroxocomplexes from precipitating. To avoid the presence of metallic copper at the electrode surface, a preformed mercury film (see experimental section) was electrodeposited. The membrane AAmodified mercured GC electrode was used for studying Cu2 + incorporation. The extent of incorporation was evaluated from the enhancement ratio as defined in Section 3.1.1. Incorporations were detected for all the cations investigated, as evidenced from the comparison between the CV curves obtained at the membrane electrode containing/not containing alginic acid. A marked
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shift (of at least 80 mV) in peak potentials towards more negative values occurs when AA is present within the membrane. Indeed, alginic acid stabilizes the oxidized form, thus resulting in negative shift in the potential values. Data in Table 1 compare the enhancement ratios measured for the various cations investigated in this work. It is noticeable that a high level of incorporated cations is attained with Fe3 + . This is not surprising when considering the strong affinity of iron(III) to oxygenated ligands and its triple positive charge. However, another factor to be considered is the unavoidable presence of soluble hydroxocomplexes at pH 5.1. Their extent, structure and charge depend largely on the nature of the metal cation to be incorporated, and on the type of counter anion. In contrast, in the set of experiments performed at pH 7.6, no marked difference can be noted between the other investigated cations. An average value of about 3 is evaluated, though differences in charge and redox potential for these cationic species. Note for example that the reduction/reoxidation of Ru(bpy)23 + /3 + couple is observed in the + 0.9 to + 1.2 V range contrary to very less positive potential values for the other couples.
3.2. Bioaccumulation in natural materials Fig. 2. Dependence of steady-state cathodic CV peak currents on the concentration of polysaccharide within the membrane filling for 90 mM Ru(NH3)36 + in 10 mM Tris chloride buffer solution, pH 7.6; (A) suspension of ( ) alginic acid, () polygalacturonic acid and (B) suspension of ( ) agar, ()dextran, ( ) pectin.
Table 1 Enhancement ratio Re for various cations from steady-state CV experiments at the membrane AA-modified electrode (5 g l−1 AA filling) Cationa Re
Cu2+ 1.9
UO2+ 2 2
Fe3+ 14
Cationb Re
Ru(bpy)2+ 3 3.5
Ru(NH3)3+ 6 3.4
Co(sep)3+ 2.8
a b
110 mM in 0.1 M sodium cacodylate buffer, pH 5.1. 110 mM in 10 mM Tris chloride buffer, pH 7.6.
3.2.1. E6aluation of alga binding capacity The presence of an alga suspension within the membrane electrode results in effective accumulation of cationic species, as shown when studying Ru(NH3)36 + and MV2 + cation models. This is well illustrated in Fig. 3 (A) which compares steady-state cyclic voltammograms obtained at pH 7.6 with the unmodified and membrane alga-modified electrode (25 g l − 1 suspension) for Ru(NH3)36 + cationic species. The peak currents are significantly higher when alga material is present in the membrane electrode. The dependence of the peak currents on the accumulation time was examined. For this experiment, the ‘preconcentration/medium exchange/ voltammetry’ procedure was used. For the preconcentration step, the membrane modified electrode was immersed in a stirred solution containing the cation of interest for a given period of
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time. This step proceeded at open circuit. The electrode was then removed from the preconcentration cell, rinsed with deionized water and placed in the electrochemical cell containing the buffer solution. From Fig. 3 (B), it can be seen that the response rises progressively with the accumulation time before leveling off at time of around 10 min. Such a profile slightly differs from the kinetics of metallic cations uptake previously
Fig. 3. (A) Steady-state cyclic voltammograms at the membrane-PG electrode in 10 mM Tris chloride buffer solution, pH 7.6 of 100 mM Ru(NH3)36 + , in the absence of Fv alga (fine line), in the presence of 25 g l − 1 Fv alga suspension (solid line); Scan rate: 20 mV s − 1. (B) Cathodic peak currents at the alga-modified electrode as a function of the accumulation time; 100 mM Ru(NH3)36 + ; 10 mM Tris chloride buffer solution, pH 7.6; 25 g l − 1 Fv alga suspension; Scan rate: 20 mV s − 1.
described [2]. It represents in fact two concomitant processes, especially in the first minutes of accumulation: transport throughout the membrane material and cation binding with the biological matter. Weaker but effective accumulation was also observed in the case of the toxic herbicide methylviologen. The enhancement ratios are Re = 10.1 and 2.1 for the Ru(NH3)36 + /alga and MV2 + / alga couples, respectively. Further information regarding the bioaccumulation process was obtained by studying the effect of the suspension concentration, pH and ionic strength of the buffer solution. The CV peak heights increase with the concentration of the alga suspension as expected from the increased binding capacity of the membrane electrode. Fig. 4 (A) describes the particular case of MV2 + incorporation. A maximum of cation uptake is reached with 20 g l − 1 alga concentration in the suspension. A strong decrease in peak heights is observed for alga concentrations higher than about 25 g l − 1 because of the jellification of the suspension. Fig. 4 (B) shows the pH-dependence of cation accumulation rate in 5 g l − 1 alga-suspension in the case of MV2 + . The cation binding to the alga suspension increases when pH is raised up from 3.5 to 6, as a consequence of the deprotonation of coordinating groups on the biomaterial surface [17], and Re decreases when pH is raised up from 6 to 9. It must be reminded that a low decrease in Re values is observed within the 3–5 pH range when using membrane AA-modified electrode (see Section 3.1.1). No variation of Re is noted with AA-suspension in the 5–9 pH range. This result suggests that different chemical functions are involved in the cation uptake. The dependence of the enhancement ratio Re for Ru(NH3)36 + incorporation upon ionic strength was examined by addition of sodium chloride in solution. As the ionic strength increases, Re progressively decreases (Fig. 4 (C)). Because ruthenium under the hexammine-complexed form is not able to give anionic complexes, such ionic strength dependence must be attributed to competition of sodium cation for the surface exchange sites (note the sodium level is 4× 103 higher than that of the analyte).
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Fig. 4. (A) Effect of the concentration of Fv alga-suspension on the steady-state CV cathodic peak current for 100 mM MV2 + ; 10 mM Tris chloride buffer solution, pH 7.6; 5 g l − 1 Fv alga suspension; Scan rate: 20 mV s − 1. (B) pH-dependence of the enhancement ratio Re at the membrane Fv alga suspension-modified electrode for 100 mM MV2 + ; concentration of the suspension: 5 g l − 1; 10 mM Tris chloride buffer solution, pH 7.6; Scan rate: 20 mV s − 1. (C) Ionic strength-dependence of the enhancement ratio Re at the membrane Fv alga suspension-modified electrode for 100 mM Ru(NH3)36 + ; concentration of the suspension: 25 g l − 1; 10 mM Tris chloride buffer solution, pH 7.6; Scan rate: 20 mV s − 1.
The ability of the membrane alga-modified electrode to trap other cationic has been investigated in the case of two metal cations of environmental interest, Cu2 + and Fe3 + . The membrane modified electrode was first immersed for a given period of time in the electrochemical cell containing the supporting electrolyte and Cu2 + or Fe3 + cations under stirring. This preliminary step allowed cations to preconcentrate into the biomaterial. After stopping stirring, CV curves were recorded. The same experiments were performed in the absence of biomaterial for comparison. Accumulation of both cations occurs in the algamodified electrode. Steady-state currents were attained upon 10 min of repetitive scanning. Respective enhancement ratios of 2 and 5 were measured for Cu2 + and Fe3 + . The same value has been previously evaluated for Cu2 + at the membrane AA-modified electrode (see Table 1).
The marked affinity of iron (III) for AA as noted above (Table 1) is also observed in the case of the natural alga, thus suggesting the very likely involvement of alginic acid in the cation uptake process.
3.2.2. Comparison with lichen binding capacity Lichens are a group of non-vascular plants composed of fungal and alga species growing in a symbiotic relationship. They were recognized as potential indicators of air pollution as early as the 1860s. They have also been found to act as accumulators of trace metals and radioactive elements [18]. Despite the wide diversity of the basic growth forms, all lichens have a similar internal morphology. The bulk of the lichen’s body is formed from filaments of the fungal partner. At its outer surface, the filaments are packed tightly together to form the cortex. The alga partner cells
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Fig. 5. (A) Steady-state cyclic voltammograms at the membrane-PG electrode in 10 mM Tris chloride buffer solution, pH 7.6 of 100 mM Ru(NH3)36 + , in the absence of lichen (fine line), in the presence of 80 g l − 1 lichen suspension (solid line); Scan rate: 20 mV s − 1. (B) Effect of the concentration of the lichen-suspension on the enhancement ratio ( ) and steady-state CV cathodic peak potential () for 100 mM Ru(NH3)36 + ; 10 mM Tris chloride buffer solution, pH 7.6; Scan rate: 20 mV s − 1.
are distributed just below the cortex in a layer where the fungal filaments are not so dense. Below the alga layer is the medulla, a loosely woven layer of fungal filaments. Most lichens have an extracellular matrix which is a gelatinous secretion of polysaccharides such as lichenan and isolichenan. From this general structure, it was of a great interest to consider the binding capacity of lichens, since cations can be accumulated a priori in several parts of this layered fungal and alga species. Fig. 5 (A) illustrates the accumulation of cationic species in the lichen suspension within the membrane electrode. In the case of Ru(NH3)36 + cations an enhancement ratio of 4.8 is reached. When compared to the enhancement ratio observed with alginic acid, this result indicates that lichen exhibits high cation binding capacity. Richardson et al. [19] suggested that a
proportion of the metal binding sites might be located on the extracellular matrix. No incorporation of cationic species has been observed in suspension of lichenan, however. This result agrees with previous data [20] which attest that the removal of all of the extracellular matrix does not decrease the metal-binding capacity. It thus appears that metal binding is primarily an interaction between metal ions and sites located on or within the cell walls of the fungal component. The pH-dependence of the cation accumulation rate in the lichen-suspension is similar to that obtained in the presence of alga: an increase in CV peak heights when pH is raised up from 5 to 7.5, followed by a decrease in the accumulation capacity of the lichen material at basic pH values. This similarity in the pH-dependence of CV peak heights suggests that similar chemical functions
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are involved in cation binding in both alga and lichen. The effect of the lichen concentration on the cation bioaccumulation can be described from the evolution of peak potential values and Re according to a two step process (Fig. 5 (B)). Within the lower lichen concentrations (0 – 20 g l − 1 range), no accumulation of Ru(NH3)36 + species can be observed (Re =1). Complexation of the cationic species by the lichen suspension occurs, however, as demonstrated by the decrease in peak potential from − 185 to −240 mV. Once this complexation equilibrium has been reached, the CV peak heights increase with increasing lichen concentration until a limit of around 100 g l − 1 is attained. At this concentration, the jellification of the suspension is supposed to slow down the accumulation process as already observed with the alga suspension. This peculiar behavior of lichen compared to alga suspension can be attributed to the structure of lichen where the alga layer is hindered by the fungal layer. The interaction between Cu2 + or Fe3 + and the lichen suspension was then studied, following the same experimental procedure as that described for the alga suspension. Concerning Cu2 + , the most noticable feature is the negative shift of the reduction potential. It seems that Cu2 + cations and lichen materials interact via a complexation reaction rather than via an accumulation process. It is not the case for Fe3 + , since increasing voltammetric peak heights upon repetitive cycling are observed with an enhancement ratio of 7. Again a high affinity of Fe(III) for the biomaterial is observed which suggests the involvement of polysaccharides in the cation uptake process.
4. Conclusion Membrane-modified electrodes offer an alternative to carbon-paste electrodes for gaining insights into the interaction of ion-exchanging polymers with cationic species, in particular in the case of polysaccharides as demonstrated in this work. Several advantages of the membrane electrode can be underlined from this and
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previous papers [5–8]. First, the entrapped modifying agent is not able to be released into the solution under investigation (providing it has a sufficiently high enough molar mass). There are no limitations from the presence of diluting agent (paraffin or silicon grease) which can usually result in increased electrode resistance. Because of the thin-layer arrangement of the membrane electrode, the effect of oxygen is minimized upon cathodic cycling. Another major advantage is the easy removal of the membrane electrode, without need of either soaking in acidic medium or treatment with NaCl and EDTA [20]. The application of membrane electrodes modified with biomaterials such as alga or lichen to metal collection is also illustrated in this work. Heavy metals are normal constituents in sea water where they are present in low concentrations. However several metals (mercury, cadmium, etc…) occur in industrial and domestic wastes, so that they can contaminate natural waters. Although they have relatively low solubility (especially at neutral pH), they can reach higher levels in organisms via biomagnification. This point has been well-established in this work from measurements at the membrane electrode in the case of typical cations such as Cu2 + and Fe3 + . A favorite candidate for incorporating in algae, especially in F. 6esiculosus seems to be Fe(III), as previously outlined [21] from measurements on samples from inhabiting coastal areas around Great Britain. In conclusion, this work has illustrated the performances of the membrane electrodes modified with natural substances or biomaterials. Insights in the interactions with metals can be gained by varying the nature of the suspension, thus offering perspectives for enlarged comparisons of the behavior of various natural substances towards cationic species. References [1] J. Wang, Analytical Electrochemistry, VCH, New York, 1994. [2] J. Wang, M.S. Lin, Anal. Chem. 60 (1988) 1545. [3] J. Gardea-Torresdey, D. Darnall, J. Wang, Anal. Chem. 60 (1988) 72. [4] J. Wang, B. Tian, G.R. Rayson, Talanta 39 (1992) 1637.
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[5] J. Haladjian, P. Bianco, F. Nunzi, M. Bruschi, Anal. Chim. Acta 289 (1994) 15. [6] P. Bianco, N. Aghroud, Electroanalysis 9 (1997) 602. [7] M. Pecorari, P. Bianco, Electroanalysis 10 (1998) 181. [8] L. Ghantous, E. Lojou, P. Bianco, Electroanalysis 10 (1998) 1249. [9] E. Reisenhofer, A. Cesaro, F. Delben, G. Manzini, S. Paoletti, Bioelectrochem. Bioenerg. 12 (1984) 455. [10] H.P. van Leeuwen, R. Cleven, J. Buffle, Pure Appl. Chem. 61 (1989) 255. [11] A.M. Nadal, C. Arino, M. Esteban, E. Casassas, Electroanalysis 3 (1991) 309. [12] C. Arino, A.M. Nadal, M. Esteban, E. Casassas, Electroanalysis 4 (1992) 757. [13] J. Garty, M. Kauppi, Environ. Toxic. Chem. 16 (1997) 2404.
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[14] D.H.S. Richardson, Bot. Linnean. Soc. 96 (1988) 31. [15] G.E. Batley, T.M. Florence, J. Electroanal. Chem. 55 (1974) 23. [16] S.C. Petrovic, H.D. Dewald, Anal. Chim. Acta 357 (1997) 33. [17] B. Greene, M.T. Henzl, M. Hosea, D.W. Darnall, Biotechnol. Bioeng. 28 (1986) 764. [18] V. Ahmadjian, The lichen symbiosis, Wiley, New York, NY, 1993. [19] D.H.S. Richardson, E. Nieboer, Cellular Interactions in Symbiosis and Parasitism, Ohio state University Press, Colombus, 1980, p. 75. [20] E. Dempsey, M. Smyth, D.H.S. Richardson, Analyst 117 (1992) 1467. [21] L.C. Ray, J.P. Gaur, H.D. Kumar, Phycology Biol. Rev. 56 (1981) 99.