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Detection of metal ions using ion-channel sensor based on self-assembled monolayer of thioctic acid
Talanta 61 (2003) 837 /848 www.elsevier.com/locate/talanta
Detection of metal ions using ion-channel sensor based on selfassembled monolayer of thioctic acid Ruphino Zugle, James Kambo-Dorsa, Victor Patrick Yao Gadzekpo * Department of chemistry, University of Cape Coast, Cape Coast, Ghana Received 16 April 2003; received in revised form 3 June 2003; accepted 3 June 2003
Abstract Gold electrodes were chemically modified with thioctic acid monolayer designed to mimic biological ion-channel membranes. The technique was then used in the determination of alkali, alkaline earth, thallium(I), and lanthanum metal cations as analytes. Cyclic voltammograms (CV) of [Fe(CN)6]3 an electroactive marker, were measured in the presence of the various types of analyte cations. In the absence of the analyte cation, electrostatic repulsion between the marker anions and the carboxylate groups of the receptor monolayer hindered the approach of the marker anion to the electrode surface and hence hindered its reduction. The modified electrodes responded well to the metal cations except the alkali metal cations. The sensors could detect the trivalent cation La3 at concentrations as low as 10 8 M. The response of the sensor to the metal cations increase in the order alkali metalB/alkaline earth metal B/lanthanum metal. As compared to divalent ions, the trivalent ion, La3 can be discriminated in the ratio 1:100. This makes it possible to determine the trivalent ion in a sample matrix containing monovalent and divalent cations. Thallium(I) ion showed marked deviation in its response as compared to monovalent ions of the alkali metals. The ion-channel sensor based on self-assembled monolayer of thioctic acid therefore offers a potential alternative technique for the selective determination of metal ions. # 2003 Published by Elsevier B.V. Keywords: Ion-channel; Self-assembled monolayer; Thioctic acid
1. Introduction Ion-channel is a very general system in biological activities. For instance, in the conversion of intracellular signals via hormones, transfer of information in nerve system, ions and molecules are transported across biological membranes by
* Corresponding author. Fax: /233-42-34612. E-mail address: [email protected] (V.P.Y. Gadzekpo). 0039-9140/03/$ - see front matter # 2003 Published by Elsevier B.V. doi:10.1016/S0039-9140(03)00377-1
receptor channel proteins. The unique feature of ion-channels in biological cell membranes is a selective recognition of substrate and the following amplification of its information. The selective binding of an ion-specific channel allows the permeation of a great amount of ions across the membrane following an electrochemical gradient. It was therefore natural to take advantage of this principle as a new philosophy to build chemical sensors based on artificial receptors. In other words, electrode surfaces can be chemically mod-
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ified with artificial receptors to mimic ion-channel in bio-membranes, thus enhancing the selectivity of such electrodes as often required in electrochemistry. Since the introduction of ion-channel or ionchannel mimetic sensor in 1987 [1], analyte-gated receptor modified electrodes have been developed for analytes ranging from hydrogen ions [2 /5], metal cations [1,6 /9], nucleotides [10 /13], inorganic ions [14], antibodies [15,16], dopamine [17], etc. The variety of receptors used in the modification of the electrodes is equally wide, including phosphate esters, various hydrogen bonding receptors, antibiotics, oligopeptides, DNA, dendrimers, cyclodextrin, calixarenes and antigenic groups. Chemisorption of alkanethiols onto gold, copper or silver metal surfaces to form self-assembled monolayers (SAMs), offer a unique strategy for constructing well-defined channels on electrode surfaces using receptors with controlled chemical features to mimic bio-receptor functions [18 /21]. For instance, adding hydrophilic head group to an alkanethiol SAMs has been proposed not only to improve the quality of packing of the monolayer [22], but also to provide basis for chemical modification of the surface to achieve selectivity and sensitivity. Several applications based on the properties of the terminal groups of monolayer have been reported [3,23,24]. Cheng and Brajtertoth reported an ion-channel effect at a thioctic acid SAM on gold electrodes, functioning in a pHdependent manner [3]. In the case of thioctic acid, the disulphide group interacts with the gold surface. The hydrophobic alkyl chains interact with each other providing the ion-channels, while the carboxyl (/COOH) head groups favourably interact with the aqueous solution phase. The assembly monolayer based electrode responded to pH and the charge on the monolayer was used to control the response to species in solution. CVs of [Fe(CN)6]3 and [Ru(NH3)6]3, electroactive markers, at different pH values with thioctic acid SAM modified electrode were examined by Cheng and Brajter-toth [3]. They reported that the reduction peak of the CVs of [Ru(NH3)6]3, at pH 1.5 and [Fe(CN)6]3 at pH 7.4 and 9.1 are completely suppressed. They explained that at pH
1.5, the carboxylic acid group (COOH) of the thioctic acid is protonated and therefore allows the negatively charged [Fe(CN)6]3 to permeate the monolayer and be reduced, while the positively charged [Ru(NH3)6]3 is repulsed from the monolayer. On the other hand, at pH 7.4 and 9.1, the carboxylic acid group is negatively charged by deprotonation and thereby preventing [Fe(CN)6]3 to permeate the monolayer and be reduced. Therefore, by the principle of ion-channel sensing, positively charged analyte can be added to allow [Fe(CN)6]3 or prevent [Ru(NH3)6]3, to permeate the monolayer and be reduced at pH 7.4 and 9.1. This reasoning has been applied in the detection of protamine, a polycation, with selectivity of about thousand times over polybrene, another polycation [25]. The detection of protamine, a cation, with ion-channel sensor serves as an encouragement to us to wonder whether ionchannel mimetic sensor based on SAM of thioctic acid can be used as an alternative but relatively cheaper technique for the detection of metal cations. In this paper, the use of thioctic acid as a receptor in an ion-channel mimetic sensor for the detection of metal ions of group IA, IIA, Lanthanum ion La3 and thallous ion Tl of group IIIA is reported. It was envisaged that cations with higher charge would have the advantage of being much more strongly bound to the receptor monolayer and/or electrostatically exerting more influence on the access of the marker to the electrode surface. In other words, cations with higher charge are expected to be more sensitively and selectively detected.
2. Experimental 2.1. Reagents Potassium ferricyanide (99%), potassium nitrate (99%), calcium nitrate tetrahydrate (98%), thallous nitrate (98%), magnesium nitrate hexahydrate (99%), barium nitrate (98%), lanthanum chloride (26.6% w/v), sodium sulphate anhydrous (99%), potassium hydroxide (85%), sodium nitrate (98%), di-potassium hydrogen phosphate (98%), potas-
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sium di-hydrogen phosphate (98%) were all laboratory reagent grade chemicals from BDH chemicals Ltd., Poole, UK. Thioctic acid (98%) was from Wako, Japan. 0.3 and 0.05 mM alumina slurry (alpha micropolish alumina No. 2 and gamma micropolish alumina No. 3, respectively) were from Buehler, Lake Bluff, IL, USA. Buffer tablets of pH 4.0 and 9.2 were from BDH chemicals Ltd., Poole, UK. All solutions for the analysis were prepared using doubly distilled water from Sibata WS-12 type distillation plant. 2.2. Self-assembly of thioctic acid monolayer on gold electrodes Gold electrodes were polished first with 0.3 mM alumina slurry on a felt pad for 10 min. A second polishing was done using 0.05 mM alumina slurry for another 10 min on a different felt pad. The gold electrodes were then rinsed several times with doubly distilled water. The polished electrodes were then dipped in 0.5 M KOH solution and then electrocleaned by scanning the potential between 0 and /1.4 V using polarographic analyzer, p-1100 Yanaco, Japan in cyclic voltammetry mode. The potential window was scanned repeatedly until the CVs obtained did not change shape, indicating a perfectly clean gold surface. The electrodes were then rinsed again several times with doubly distilled water and lastly with anhydrous ethanol. The electrodes were then immersed in a 0.1% thioctic acid solution in ethanol for at least 24 h. The modified electrodes were then rinsed several times with water and stored in distilled water at temperature below 258 in a refrigerator until use. Four gold electrodes were modified and used for the analysis.
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was used as the auxiliary electrode. A potential range of 700 mV, ranging from /500 to /200 mV was used for all analysis. Each sample solution contained 6 mM potassium hexacyanoferrate(III) as the electroactive marker and 0.1 M potassium nitrate as the supporting electrolyte. The pH of each sample solutions was adjusted to 7.4 using 2 NaOH in the presence of either H2 PO 4 =HPO4 ; borax or ammonia buffer. CVs of the sample solutions containing the electroactive marker were then taken and interpreted. Before taking the voltammograms, nitrogen gas was passed through each sample solution for 15 min to remove any oxygen present. 2.4. Electrode regeneration The removal of the metal ion from the monolayer surface after detection was done by dipping the sensor into a solution of 0.1 M Na2SO4 (pH adjusted to 1.5 with aliquots of H2SO4). The metal ions were removed from the sensor by placing it in a test tube containing the sulphate solution, pH 1.5, and shaking it for 10 min. Removal of the metal from the monolayer surface was ascertained by running a CV of [Fe(CN)6]3 , the marker ion in a solution containing no metal ion. The reduction current of the marker was found to be negligibly small, showing removal of the metal ion from the monolayer surface, though potassium and sodium ions from the background electrolyte and buffer, respectively were present. However, for the removal of La3 and Tl from the monolayer surface, the surface regeneration period had to be extended to 15 min and even in cases of higher concentration of the metal ions, a longer period was required to again achieve negligible reduction current of the marker.
2.3. Electrochemical measurement All electrochemical measurements were carried out using a polarographic analyzer, p-1100, Yanaco, Japan connected to an X /Y graphical recorder. A three-electrode electrochemical cell was used, with the chemically modified gold electrode as the working electrode; A Ag/AgCl electrode Type SR-P2A from Yanaco, Japan was used as the reference electrode and a platinum wire
3. Results and discussion 3.1. Response behaviour of sensor In preliminary experiments, attempts were made to find out whether the receptor (thioctic acid) would assemble on the gold electrode. It was also to find out whether the metal ions would have the
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anticipated effect of opening the channels for the permeation of the marker [Fe(CN)6]3 ions as observed with the phosphate ester receptor [9]. Fig. 1 shows the response behaviour of the marker on the bare gold electrode and on the modified gold electrode. As shown in the figure, faradaic current flowed when the bare gold electrode was used (a). This can be explained that the bare gold electrode surface did not offer any significant resistance to the approach of the marker ions. Hence, the redox process occurred and current observed. However, on the modified electrode, the faradaic current was scarcely observed in the potential range of /0.5 / /0.2 V versus Ag/AgCl reference electrode. This can be attributed to the electrostatic repulsion between the negatively charged monolayer surface and the marker, [Fe(CN)6]3 . Thus the approach of the marker to the gold surface for the redox process to occur was hindered. Fig. 2 shows the CVs of 6 mM [Fe(CN)6]3 in aqueous solutions containing 1 /10 4 M of the metal ions La3, Ba2, Ca2, and Mg2 As shown in Fig. 2, the addition of the metal ions greatly enhanced the suppressed CV response of the [Fe(CN)6]3 /[Fe(CN)6]4 redox reaction. Another feature that can be observed in the figure is that the different ions have different abilities in
enhancing the suppressed current even though they have the same concentrations. A possible molecular mechanism for the ion responsive change in the voltammograms is as shown in Fig. 3. Interaction of the carboxylate group (COO ) of the monolayer with the metal ions decreases the negative charge density of the monolayer surface, which leads to the decrease in the electrostatic repulsion between the anionic marker, [Fe(CN)6]3 and the carboxylate monolayer head groups on the gold. The packing density of the monolayer in this case is loose and the electroactive marker can permeate into the monolayer. The effect of conformational change of the monolayer induced by the addition of the ions is also an important factor contributing to the change in the voltammograms. Though an important factor, its contribution to the change in the voltammograms was not assessed in this work. 3.2. Response of sensor to calcium ion Calcium binding proteins are typically rich in aspartate (asp) and glutamate (glu), both of which have carboxylate groups as side chains and hence act as anionic ligands for calcium ions [26]. Also because ion-channel-mimetic sensor based on
Fig. 1. CVs of 6 mM K3[Fe(CN)6] in 0.1 M KNO3 aqueous solution, pH 7.4, with: (a) bare gold electrode; (b) gold electrode modified with thioctic acid. Scan rate 100 mVs 1.
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Fig. 2. CVs of 6 mM K3[Fe(CN)6] in 0.1 M KNO3 aqueous solution, pH 7.4 containing 1 /10 4 M of: (a) Mg2 ; (b) Ca2 ; (c) Ba2 ; (d) La3 . Scan rate is 100 mVs 1.
SAM of thioctic acid has been reported [3], hence the response of the sensor to calcium ions is investigated before determining its selectivities versus other cations. The response of the sensor to various concentrations of calcium ions is shown in Fig. 4. In solution without calcium ion (a), the current of the reduction of the marker within the potential window of /0.5 / /0.2 V versus Ag/AgCl reference electrode was negligibly small and the marker reduction irreversible. As the concentration of the calcium ions was increased, a corresponding increase in the redox current was observed as shown from CVs (b) to (j). No sharp peaks occurred at lower calcium ion concentrations. At higher concentrations however, redox peaks appeared and the CVs became reversible, as the calcium ion concentration was further increased. At the highest calcium concentration of 0.1 M, (j), the CV became completely reversible. The redox currents were however lower than those observed on the reversible CV obtained with the bare gold electrode. These results suggest that in the absence of calcium, electrostatic repulsion between the anionic marker species [Fe(CN)6]3 and the negatively charged deprotonated carboxylate head groups on the electrode surface prevent the access of the marker to the electrode surface. Binding of cal-
cium ions to the carboxylate groups reduces the excess negative charge density on the monolayer and facilitates the access of the marker to the electrode. Another interesting observation on the CV response of the sensor to the calcium ion concentration is that the peak potentials tend towards more positive values as the calcium ion concentration increases. This can be understood by considering the electrode kinetics of the marker in the presence of the analyte (Ca2). Binding of the cation, Ca2, to the monolayer alters the profile of the electric potential drop across the electrode interface and thereby changes both the local concentration of the electroactive marker at the electrode surface and the electron transfer rate constant [27]. The CVs show that with increasing calcium ion concentration in the subphase solution, the apparent electron transfer rate constant for any given applied potential of the rising portion of the [Fe(CN)6]3 reduction peak increases. As a result, the potential of the [Fe(CN)6]3 reduction peaks shifts to higher potentials. Large potential difference between the cathodic and anodic peaks is observed at low calcium ion concentration. This is primarily due to slow electrode kinetics of the marker, as the ion-
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Fig. 3. Schematic representation of the ion-channel opening of the monolayer in the presence of the metal ion (Mn ).
channels are not completely opened at low calcium ion concentration. 3.3. Response of sensor to monovalent cations Monovalent cations of group IA, (Na and K ) did not show any appreciable ion-channel opening at the modified thioctic acid gold electrode. Even when 0.1 M KNO3 was used as the supporting electrolyte, no appreciable redox current was observed when no other cation was
present. Quite surprisingly, the ion-gate response of the sensor to thallium(I) ion was interesting. With the four modified electrodes used all showed ion-channel opening in the presence of thallium(I) ions as analyte. In order to be convinced with this, a simple investigation was conducted on the response behaviour with thallium(I) ions. As shown on Fig. 5, CV (a) was obtained using a blank solution containing 0.1 M KNO3 supporting electrolyte, 6 mM K3[Fe(CN)6] at pH 7.4. Freshly prepared 0.1 M TlNO3 solution (5 ml) was spiked into the blank solution and CV (b) obtained. As indicated on the voltammogram, redox current was obtained implying that there was an interaction between thallium(I) ions and the carboxylate head groups of the monolayer leading to a reduction of the negative charge intensity. Thus permeation of the monolayer by marker ions was possible. This unexpected behaviour of thallium(I) ions could not just be merely accidental. A possible explanation could be inferred from the following. When oxygen, nitrogen, and sulphur bonded to organic group are also bonded to thallium(I), the Tl /X bond appears to be more covalent than the bond to alkali metal ions in similar compounds. The thallium compounds appear to be polymeric rather than ionic [28]. As a consequence, when thallium(I) ions interact with the carboxylate head group, multiple bonding occurs and the packing density of the monolayer is greatly altered leading to opening of the channels. The fact that the sensor responded to thallium(I) ions, and on the contrary, the monolayer system of the sensor is not ideal for the detection of thallium(III) ions due to the hydrolysis of its ions at pH 7.4, suggests that the sensor based on thioctic acid monolayer would be useful in the studies of the speciation of thallium in solution. 3.4. Variation of peak potentials with analyte concentration The variation of peak potentials with the logarithm of the cation concentration is shown in Fig. 6. In each case, the peak potentials for the reduction of [Fe(CN)6]3 moves towards more positive
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Fig. 4. CVs of 6 mM K3[Fe(CN)6] obtained with gold electrode modified with thioctic acid monolayer. Scan rate 100 mVs 1, background electrolyte 0.1 M KNO3. Ca2 concentration (in M): (a) 0.0; (b) 1 /10 5; (c) 5 /10 5; (d) 1 /10 4; (e) 5 /10 4; (f) 1 /10 3; (g) 5 /10 3; (h) 1 /10 2; (i) 5 /10 2; (j) 1 /10 1.
peak potentials as the cation concentration is increased. This as explained in the case of calcium ions is due to changes in the profile of the electric potential drop across the electrode interface as the cation binds to the receptor monolayer [27]. Consequently, the local concentration of the electroactive species [Fe(CN)6]3 changes bringing
about changes in the electron transfer rate constant [27]. The trivalent cation La3 is observed to have caused a response of the sensor at low concentration far better than the divalent cations, Ba2, Ca2 and Mg2. This confirms the assertion that higher charged species would have the advantage of being much more strongly bound to
2 Fig. 5. CVs of 6 mM K3[Fe(CN)6] obtained with gold electrode modified with thioctic acid. pH of solution 7.4 with H2 PO 4 =HPO4 buffer: (a) no cation present (blank solution); (b) after freshly prepared solution of Tl ion was spiked into the blank solution.
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Fig. 6. Dependence of peak potentials in CV for 6 mM [Fe(CN)6]3 containing different cation of varying concentrations.
the receptor monolayer and as a consequence electrostatically exert much more influence on the access of the marker ions to the electrode surface. The monovalent cation Tl shows remarkably more positive peak potentials at concentrations of 10 2 and 10 1 M than the divalent cations Ba2, Ca2 and Mg2. This could probably be due to the reasons given in Section 3.2 for the unexpected behaviour of thallium(I). Fig. 7 shows sample CVs containing 10 1 and 10 2 M Ba2 and Tl . As shown, in the CVs the peak potentials corresponding to Tl are more
towards the left (more positive end) than those of Ba2 are. 3.5. Variation of reduction current at fixed potential with concentration of metal ion The response of the sensor to the cations can also be quantified by the current at a potential in the rising portion of the reduction peak of the marker ion. In this region, the marker reduction is still controlled purely by the electrode kinetics and is not affected yet by diffusion in the bulk of the
Fig. 7. CVs of 6 mM K3[Fe(CN)6] in 0.1 M KNO3 aqueous solution, pH 7.4 containing: (a) 10 1 M Tl ; (b) 10 1 M Ba2 ; (c) 10 2 M Tl ; (d) 10 2 M Ba2 .
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sample solution. This alternative method was found appropriate since most of the reduction peaks were too broad for the peak potentials to be located precisely. The reduction currents of the marker were thus measured at an applied potential of /0.11956 V against Ag/AgCl reference electrode. The choice of the applied potential was arbitrary and was only selected to optimise the sensor responses. The voltammetric response of the sensor towards Tl , Mg2, Ca2, Ba2 and La3 as , measured reduction currents of the marker at / 0.11956 V as a function of the cation concentration is shown in Fig. 8. The trivalent ion, La3 is found again to be most sensitive just as in the case of peak potentials. In the plot, Ba2 shows a little bit better response than Mg2 and Ca2 that are almost difficult to distinguish. The plot shows the series La3 / Ba2 /Ca2 :/Mg2 /Tl . The highest sensitivity towards La3 and the least towards Tl can be attributed to differences in charges. La3 is expected to be much more bound to the receptor than Tl . Thus La3 has better ion-channel opening effect than Tl . For the alkaline earth metals, the series cannot be attributed to differences in charges. The order of the effect of these ions is also inconsistent with the stability of the coordination of carboxyl in which Mg2 ion is known to show higher stability than the other ions [26]. The stability of coordina-
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tion decreases in the order Mg2 /Ca2 /Ba2 based on simple electrostatic considerations. Similar results were obtained by Takehara et al. [29],when they used g-L-glutamyl-L-cystein (GluCys) and L-cysteinyl-glycine (Cys-Gly) monolayers that have been modified on gold electrodes. GluCys contains two carboxyl terminal groups while Cys-Gly contains only one terminal carboxyl group. They stated that although no clear explanation could be given, the following factors may account for the discrepancy between the ionchannel response and the complex stability. As compared with the molecule in the bulk of the solution, the conformation changes due to coordination with M2 ion is expected to be restricted in the case of surface confined molecule. However, the observed order Mg2 :/Ca2 B/ Ba2 is approximately consistent with the order of the water exchange rate between the inner hydration shells [30]. Under conditions of restricted conformation change, the slower dehydration of the inner shell may bring about the lower coordination stability of the terminal carboxyl unit. The slight differences in the ion-channel effect of Mg2 and Ca2 may be due to the fact that thioctic acid, the receptor has only one carboxyl unit and thus monodentate. Moreover, Mg2 and Ca2, with atomic radii 65 and 99 picometers, respectively are next to each other and posses comparable extent of hydration, therefore, making
Fig. 8. Reduction current for 6 mM [Fe(CN)6]3 solution at 0.11956 V (versus Ag/AgCl) containing different cations at varying concentrations.
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it difficult for the monodentate receptor to clearly distinguish between them.
the monolayer to the marker than lower charge species.
3.6. Variation of reduction peak areas with concentration of metal ion
3.7. Selectivity
The responses of a typical sensor in separate solutions of thallium(I), magnesium, calcium, barium and lanthanum are as shown in Fig. 9. Shown are the peak areas (cathodic peak areas) for the [Fe(CN)6]3 reduction, obtained manually. The trends in Sections 3.4 and 3.5 can also be seen in the Fig. 9. There is large, intermediate and small responses corresponding to trivalent, divalent and monovalent cations. A feature that is common to the curves, except that of thallium(I) is that above a certain critical concentration of the cations, the reduction peak areas levels off. In the case of La3, leveling off the curve occurs above a concentration of 5 / 10 7 M. While for Mg2, Ca2 and Ba2, it occurs above concentration of 5 /10 4 M. This is attributed to the fact that the receptor monolayer becomes saturated with these ions at higher concentrations. Though it will not be quite appropriate to quantify the preference of the sensor to the metal ions, the results from the peak areas have also illustrated qualitatively that higher charge species have much more influence on the permeability of
The selectivity factors for the various cations using the sensor based on SAM of thioctic acid on gold electrode were determined in separate solution. The method used is similar to the match potential method used in obtaining selectivity coefficients in ion-selective electrodes (ISEs) [31,32] in that a peak potential was fixed and the concentration of any analyte ion, in separate solution, that gave the same peak potential was taken for selectivity determination. From Fig. 6, the concentration of the cation which gave the same peak potential (68.8 mV) as obtained in the case of solution containing 1 / 10 4 M of La3 ions or 1 /10 2 M Ca2 ions were determined. The selectivity factors were then obtained as the ratios of those concentrations and 1 /10 2 M Ca2. The thus obtained selectivity factors are La3, 100; Ba2, 2; Ca2, 1; Mg2, 0.54 and Tl , 0.70. The selectivity ratios obtained above support the general trend that trivalent cations are most selectively sensed than the divalent and monovalent cations. As already explained, because of the high charge of the trivalent ion, La3, it is much more bound to the receptor and thus exerts greater
Fig. 9. Areas of the K3[Fe(CN)6] reduction peaks as a function of the logarithm of the metal ion concentration.
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influence on the permeability of marker. The trend in the selectivity of the alkaline earth metals, Ba2, 2; Ca2, 1 and Mg2, 0.54 is in accordance to the ease with which these hydrophilic cations can undergo partial dehydration that is required for the formation of the interfacial complexes at the surface of the monolayer. The ease of dehydration is in the order Ba2 /Ca2 /Mg2. Thus Ba2 is expected to be much more bound to the monolayer and exert greater influence on the permeation of the marker.
3.8. Reproducibility and lifetime All the four modified electrodes prepared responded to the presence of metal ions. In the case of lanthanum ion, some of the sensors responded to concentrations as low as 10 8 M. The general observation about the four sensors was that at low concentration of the cation, the voltammograms were not quite reproducible. They only became reproducible after three to four scans. Thus all the voltammograms were taken after the fourth scan. The non-reproducible nature of the voltammograms at low concentration of the cation could be due to slow equilibration of the metal ions between the aqueous phase and the hydrophobic organic monolayer. The electrodes used showed generally the same sequence of selectivity for the metal ions. However, the magnitude of the response in some cases differed. The lifetime of the sensors was quite long. For instance, one sensor was used for over 3 months, taking over hundred voltammograms. However, in cases where ammonia buffer was used, the sensor easily lost its ion-channel opening and closing ability and even in the absence of the analyte cation, redox current flowed. This situation seems incomprehensible and the sensor became functional only after putting it in concentrated sulphuric acid for about 2 h and then reassemble the monolayer onto the gold surface. Thus ammonia buffer was avoided as much as possible in the work. Another factor that affected the life span of the sensors was the magnitude of the applied potential. The monolayer broke down at higher applied
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potentials. Thus a working potential window of / 0.5 / /0.2 V was used throughout the work. 3.9. Conclusion The gold electrode chemically modified with thioctic acid monolayer and designed to mimic ion-channel membrane allows the detection of metal ions with [Fe(CN)6]3 as the electroactive marker. The detection is based on the attachment of the positively charged metal ion onto the negatively charged monolayer surface at pH of 7.4. For the trivalent ion La3, detection at concentration as low as 10 8 M was observed with some of the sensors. As compared to La3, divalent cations, for instance Ca2, are discriminated in the ratio 1:100. Such selectivity makes these sensors attractive for the detection of the trivalent ion in the presence of large background of divalent and monovalent ions. The trivalent ion La3, with its high charge, has the advantage of being strongly bound to the receptor monolayer and/or electrostatically exert more influence on the access of the marker to the electrode surface. With these characteristics, a gold electrode chemically modified with a monolayer of thioctic acid to mimic gating at bio-membranes has the possibility of being a sensing device for metal ions especially the trivalent ion (La3 ion).
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[22] C.D. Miller, P. Cuendet, M. Gra¨tzel, J. Phys. Chem. 95 (1991) 877. [23] N. Nakashima, T. Taguchi, Y. Takad, K. Fujio, M. Kunitake, O. Manabe, J. Chem. Commun. (1991) 232. [24] S. Steinberg, Y. Tor, E. Sabatani, I. Rubinstein, J. Am. Chem. Soc. 113 (1992) 5179. [25] V.P.Y. Gadzekpo, K.P. Xiao, H. Aoki, P. Bu¨hlmann, Y. Umezawa, Anal. Chem. 71 (1999) 5109 /5115. [26] D.F. Shriver, P.W. Atkins, C.H. Langford, Inorganic Chemistry, Oxford University Press, Oxford, 1990, p. 610. [27] Q. Cheng, A. Brajter-Toth, Anal. Chem. 67 (1995) 2767. [28] F.A. Cotton, G. Wilkinson, Advanced Inorganic Chemistry, 2nd ed., Interscience Publishers, John Wiley and sons, New York, 1967. [29] H.T. Tien, R.H. Barish, L.-Q. Gu, A.L. Ottova, Anal. Sci. 14 (1998) 3. [30] H. Diebler, M. Eigen, G. Ilgenfritz, G. Maas, R. Winkler, Pure Appl. Chem. 20 (1969) 93. [31] V.P.Y. Gadzekpo, G.D. Christian, Anal. Chim. Acta 164 (1984) 279. [32] Y. Umezawa, P. Bu¨hlmann, K. Umezawa, K. Tohda, S. Amemiya, Pure Appl. Chem. 72 (2000) 10.
Detection of metal ions using ion-channel sensor based on selfassembled monolayer of thioctic acid Ruphino Zugle, James Kambo-Dorsa, Victor Patrick Yao Gadzekpo * Department of chemistry, University of Cape Coast, Cape Coast, Ghana Received 16 April 2003; received in revised form 3 June 2003; accepted 3 June 2003
Abstract Gold electrodes were chemically modified with thioctic acid monolayer designed to mimic biological ion-channel membranes. The technique was then used in the determination of alkali, alkaline earth, thallium(I), and lanthanum metal cations as analytes. Cyclic voltammograms (CV) of [Fe(CN)6]3 an electroactive marker, were measured in the presence of the various types of analyte cations. In the absence of the analyte cation, electrostatic repulsion between the marker anions and the carboxylate groups of the receptor monolayer hindered the approach of the marker anion to the electrode surface and hence hindered its reduction. The modified electrodes responded well to the metal cations except the alkali metal cations. The sensors could detect the trivalent cation La3 at concentrations as low as 10 8 M. The response of the sensor to the metal cations increase in the order alkali metalB/alkaline earth metal B/lanthanum metal. As compared to divalent ions, the trivalent ion, La3 can be discriminated in the ratio 1:100. This makes it possible to determine the trivalent ion in a sample matrix containing monovalent and divalent cations. Thallium(I) ion showed marked deviation in its response as compared to monovalent ions of the alkali metals. The ion-channel sensor based on self-assembled monolayer of thioctic acid therefore offers a potential alternative technique for the selective determination of metal ions. # 2003 Published by Elsevier B.V. Keywords: Ion-channel; Self-assembled monolayer; Thioctic acid
1. Introduction Ion-channel is a very general system in biological activities. For instance, in the conversion of intracellular signals via hormones, transfer of information in nerve system, ions and molecules are transported across biological membranes by
* Corresponding author. Fax: /233-42-34612. E-mail address: [email protected] (V.P.Y. Gadzekpo). 0039-9140/03/$ - see front matter # 2003 Published by Elsevier B.V. doi:10.1016/S0039-9140(03)00377-1
receptor channel proteins. The unique feature of ion-channels in biological cell membranes is a selective recognition of substrate and the following amplification of its information. The selective binding of an ion-specific channel allows the permeation of a great amount of ions across the membrane following an electrochemical gradient. It was therefore natural to take advantage of this principle as a new philosophy to build chemical sensors based on artificial receptors. In other words, electrode surfaces can be chemically mod-
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ified with artificial receptors to mimic ion-channel in bio-membranes, thus enhancing the selectivity of such electrodes as often required in electrochemistry. Since the introduction of ion-channel or ionchannel mimetic sensor in 1987 [1], analyte-gated receptor modified electrodes have been developed for analytes ranging from hydrogen ions [2 /5], metal cations [1,6 /9], nucleotides [10 /13], inorganic ions [14], antibodies [15,16], dopamine [17], etc. The variety of receptors used in the modification of the electrodes is equally wide, including phosphate esters, various hydrogen bonding receptors, antibiotics, oligopeptides, DNA, dendrimers, cyclodextrin, calixarenes and antigenic groups. Chemisorption of alkanethiols onto gold, copper or silver metal surfaces to form self-assembled monolayers (SAMs), offer a unique strategy for constructing well-defined channels on electrode surfaces using receptors with controlled chemical features to mimic bio-receptor functions [18 /21]. For instance, adding hydrophilic head group to an alkanethiol SAMs has been proposed not only to improve the quality of packing of the monolayer [22], but also to provide basis for chemical modification of the surface to achieve selectivity and sensitivity. Several applications based on the properties of the terminal groups of monolayer have been reported [3,23,24]. Cheng and Brajtertoth reported an ion-channel effect at a thioctic acid SAM on gold electrodes, functioning in a pHdependent manner [3]. In the case of thioctic acid, the disulphide group interacts with the gold surface. The hydrophobic alkyl chains interact with each other providing the ion-channels, while the carboxyl (/COOH) head groups favourably interact with the aqueous solution phase. The assembly monolayer based electrode responded to pH and the charge on the monolayer was used to control the response to species in solution. CVs of [Fe(CN)6]3 and [Ru(NH3)6]3, electroactive markers, at different pH values with thioctic acid SAM modified electrode were examined by Cheng and Brajter-toth [3]. They reported that the reduction peak of the CVs of [Ru(NH3)6]3, at pH 1.5 and [Fe(CN)6]3 at pH 7.4 and 9.1 are completely suppressed. They explained that at pH
1.5, the carboxylic acid group (COOH) of the thioctic acid is protonated and therefore allows the negatively charged [Fe(CN)6]3 to permeate the monolayer and be reduced, while the positively charged [Ru(NH3)6]3 is repulsed from the monolayer. On the other hand, at pH 7.4 and 9.1, the carboxylic acid group is negatively charged by deprotonation and thereby preventing [Fe(CN)6]3 to permeate the monolayer and be reduced. Therefore, by the principle of ion-channel sensing, positively charged analyte can be added to allow [Fe(CN)6]3 or prevent [Ru(NH3)6]3, to permeate the monolayer and be reduced at pH 7.4 and 9.1. This reasoning has been applied in the detection of protamine, a polycation, with selectivity of about thousand times over polybrene, another polycation [25]. The detection of protamine, a cation, with ion-channel sensor serves as an encouragement to us to wonder whether ionchannel mimetic sensor based on SAM of thioctic acid can be used as an alternative but relatively cheaper technique for the detection of metal cations. In this paper, the use of thioctic acid as a receptor in an ion-channel mimetic sensor for the detection of metal ions of group IA, IIA, Lanthanum ion La3 and thallous ion Tl of group IIIA is reported. It was envisaged that cations with higher charge would have the advantage of being much more strongly bound to the receptor monolayer and/or electrostatically exerting more influence on the access of the marker to the electrode surface. In other words, cations with higher charge are expected to be more sensitively and selectively detected.
2. Experimental 2.1. Reagents Potassium ferricyanide (99%), potassium nitrate (99%), calcium nitrate tetrahydrate (98%), thallous nitrate (98%), magnesium nitrate hexahydrate (99%), barium nitrate (98%), lanthanum chloride (26.6% w/v), sodium sulphate anhydrous (99%), potassium hydroxide (85%), sodium nitrate (98%), di-potassium hydrogen phosphate (98%), potas-
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sium di-hydrogen phosphate (98%) were all laboratory reagent grade chemicals from BDH chemicals Ltd., Poole, UK. Thioctic acid (98%) was from Wako, Japan. 0.3 and 0.05 mM alumina slurry (alpha micropolish alumina No. 2 and gamma micropolish alumina No. 3, respectively) were from Buehler, Lake Bluff, IL, USA. Buffer tablets of pH 4.0 and 9.2 were from BDH chemicals Ltd., Poole, UK. All solutions for the analysis were prepared using doubly distilled water from Sibata WS-12 type distillation plant. 2.2. Self-assembly of thioctic acid monolayer on gold electrodes Gold electrodes were polished first with 0.3 mM alumina slurry on a felt pad for 10 min. A second polishing was done using 0.05 mM alumina slurry for another 10 min on a different felt pad. The gold electrodes were then rinsed several times with doubly distilled water. The polished electrodes were then dipped in 0.5 M KOH solution and then electrocleaned by scanning the potential between 0 and /1.4 V using polarographic analyzer, p-1100 Yanaco, Japan in cyclic voltammetry mode. The potential window was scanned repeatedly until the CVs obtained did not change shape, indicating a perfectly clean gold surface. The electrodes were then rinsed again several times with doubly distilled water and lastly with anhydrous ethanol. The electrodes were then immersed in a 0.1% thioctic acid solution in ethanol for at least 24 h. The modified electrodes were then rinsed several times with water and stored in distilled water at temperature below 258 in a refrigerator until use. Four gold electrodes were modified and used for the analysis.
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was used as the auxiliary electrode. A potential range of 700 mV, ranging from /500 to /200 mV was used for all analysis. Each sample solution contained 6 mM potassium hexacyanoferrate(III) as the electroactive marker and 0.1 M potassium nitrate as the supporting electrolyte. The pH of each sample solutions was adjusted to 7.4 using 2 NaOH in the presence of either H2 PO 4 =HPO4 ; borax or ammonia buffer. CVs of the sample solutions containing the electroactive marker were then taken and interpreted. Before taking the voltammograms, nitrogen gas was passed through each sample solution for 15 min to remove any oxygen present. 2.4. Electrode regeneration The removal of the metal ion from the monolayer surface after detection was done by dipping the sensor into a solution of 0.1 M Na2SO4 (pH adjusted to 1.5 with aliquots of H2SO4). The metal ions were removed from the sensor by placing it in a test tube containing the sulphate solution, pH 1.5, and shaking it for 10 min. Removal of the metal from the monolayer surface was ascertained by running a CV of [Fe(CN)6]3 , the marker ion in a solution containing no metal ion. The reduction current of the marker was found to be negligibly small, showing removal of the metal ion from the monolayer surface, though potassium and sodium ions from the background electrolyte and buffer, respectively were present. However, for the removal of La3 and Tl from the monolayer surface, the surface regeneration period had to be extended to 15 min and even in cases of higher concentration of the metal ions, a longer period was required to again achieve negligible reduction current of the marker.
2.3. Electrochemical measurement All electrochemical measurements were carried out using a polarographic analyzer, p-1100, Yanaco, Japan connected to an X /Y graphical recorder. A three-electrode electrochemical cell was used, with the chemically modified gold electrode as the working electrode; A Ag/AgCl electrode Type SR-P2A from Yanaco, Japan was used as the reference electrode and a platinum wire
3. Results and discussion 3.1. Response behaviour of sensor In preliminary experiments, attempts were made to find out whether the receptor (thioctic acid) would assemble on the gold electrode. It was also to find out whether the metal ions would have the
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anticipated effect of opening the channels for the permeation of the marker [Fe(CN)6]3 ions as observed with the phosphate ester receptor [9]. Fig. 1 shows the response behaviour of the marker on the bare gold electrode and on the modified gold electrode. As shown in the figure, faradaic current flowed when the bare gold electrode was used (a). This can be explained that the bare gold electrode surface did not offer any significant resistance to the approach of the marker ions. Hence, the redox process occurred and current observed. However, on the modified electrode, the faradaic current was scarcely observed in the potential range of /0.5 / /0.2 V versus Ag/AgCl reference electrode. This can be attributed to the electrostatic repulsion between the negatively charged monolayer surface and the marker, [Fe(CN)6]3 . Thus the approach of the marker to the gold surface for the redox process to occur was hindered. Fig. 2 shows the CVs of 6 mM [Fe(CN)6]3 in aqueous solutions containing 1 /10 4 M of the metal ions La3, Ba2, Ca2, and Mg2 As shown in Fig. 2, the addition of the metal ions greatly enhanced the suppressed CV response of the [Fe(CN)6]3 /[Fe(CN)6]4 redox reaction. Another feature that can be observed in the figure is that the different ions have different abilities in
enhancing the suppressed current even though they have the same concentrations. A possible molecular mechanism for the ion responsive change in the voltammograms is as shown in Fig. 3. Interaction of the carboxylate group (COO ) of the monolayer with the metal ions decreases the negative charge density of the monolayer surface, which leads to the decrease in the electrostatic repulsion between the anionic marker, [Fe(CN)6]3 and the carboxylate monolayer head groups on the gold. The packing density of the monolayer in this case is loose and the electroactive marker can permeate into the monolayer. The effect of conformational change of the monolayer induced by the addition of the ions is also an important factor contributing to the change in the voltammograms. Though an important factor, its contribution to the change in the voltammograms was not assessed in this work. 3.2. Response of sensor to calcium ion Calcium binding proteins are typically rich in aspartate (asp) and glutamate (glu), both of which have carboxylate groups as side chains and hence act as anionic ligands for calcium ions [26]. Also because ion-channel-mimetic sensor based on
Fig. 1. CVs of 6 mM K3[Fe(CN)6] in 0.1 M KNO3 aqueous solution, pH 7.4, with: (a) bare gold electrode; (b) gold electrode modified with thioctic acid. Scan rate 100 mVs 1.
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Fig. 2. CVs of 6 mM K3[Fe(CN)6] in 0.1 M KNO3 aqueous solution, pH 7.4 containing 1 /10 4 M of: (a) Mg2 ; (b) Ca2 ; (c) Ba2 ; (d) La3 . Scan rate is 100 mVs 1.
SAM of thioctic acid has been reported [3], hence the response of the sensor to calcium ions is investigated before determining its selectivities versus other cations. The response of the sensor to various concentrations of calcium ions is shown in Fig. 4. In solution without calcium ion (a), the current of the reduction of the marker within the potential window of /0.5 / /0.2 V versus Ag/AgCl reference electrode was negligibly small and the marker reduction irreversible. As the concentration of the calcium ions was increased, a corresponding increase in the redox current was observed as shown from CVs (b) to (j). No sharp peaks occurred at lower calcium ion concentrations. At higher concentrations however, redox peaks appeared and the CVs became reversible, as the calcium ion concentration was further increased. At the highest calcium concentration of 0.1 M, (j), the CV became completely reversible. The redox currents were however lower than those observed on the reversible CV obtained with the bare gold electrode. These results suggest that in the absence of calcium, electrostatic repulsion between the anionic marker species [Fe(CN)6]3 and the negatively charged deprotonated carboxylate head groups on the electrode surface prevent the access of the marker to the electrode surface. Binding of cal-
cium ions to the carboxylate groups reduces the excess negative charge density on the monolayer and facilitates the access of the marker to the electrode. Another interesting observation on the CV response of the sensor to the calcium ion concentration is that the peak potentials tend towards more positive values as the calcium ion concentration increases. This can be understood by considering the electrode kinetics of the marker in the presence of the analyte (Ca2). Binding of the cation, Ca2, to the monolayer alters the profile of the electric potential drop across the electrode interface and thereby changes both the local concentration of the electroactive marker at the electrode surface and the electron transfer rate constant [27]. The CVs show that with increasing calcium ion concentration in the subphase solution, the apparent electron transfer rate constant for any given applied potential of the rising portion of the [Fe(CN)6]3 reduction peak increases. As a result, the potential of the [Fe(CN)6]3 reduction peaks shifts to higher potentials. Large potential difference between the cathodic and anodic peaks is observed at low calcium ion concentration. This is primarily due to slow electrode kinetics of the marker, as the ion-
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Fig. 3. Schematic representation of the ion-channel opening of the monolayer in the presence of the metal ion (Mn ).
channels are not completely opened at low calcium ion concentration. 3.3. Response of sensor to monovalent cations Monovalent cations of group IA, (Na and K ) did not show any appreciable ion-channel opening at the modified thioctic acid gold electrode. Even when 0.1 M KNO3 was used as the supporting electrolyte, no appreciable redox current was observed when no other cation was
present. Quite surprisingly, the ion-gate response of the sensor to thallium(I) ion was interesting. With the four modified electrodes used all showed ion-channel opening in the presence of thallium(I) ions as analyte. In order to be convinced with this, a simple investigation was conducted on the response behaviour with thallium(I) ions. As shown on Fig. 5, CV (a) was obtained using a blank solution containing 0.1 M KNO3 supporting electrolyte, 6 mM K3[Fe(CN)6] at pH 7.4. Freshly prepared 0.1 M TlNO3 solution (5 ml) was spiked into the blank solution and CV (b) obtained. As indicated on the voltammogram, redox current was obtained implying that there was an interaction between thallium(I) ions and the carboxylate head groups of the monolayer leading to a reduction of the negative charge intensity. Thus permeation of the monolayer by marker ions was possible. This unexpected behaviour of thallium(I) ions could not just be merely accidental. A possible explanation could be inferred from the following. When oxygen, nitrogen, and sulphur bonded to organic group are also bonded to thallium(I), the Tl /X bond appears to be more covalent than the bond to alkali metal ions in similar compounds. The thallium compounds appear to be polymeric rather than ionic [28]. As a consequence, when thallium(I) ions interact with the carboxylate head group, multiple bonding occurs and the packing density of the monolayer is greatly altered leading to opening of the channels. The fact that the sensor responded to thallium(I) ions, and on the contrary, the monolayer system of the sensor is not ideal for the detection of thallium(III) ions due to the hydrolysis of its ions at pH 7.4, suggests that the sensor based on thioctic acid monolayer would be useful in the studies of the speciation of thallium in solution. 3.4. Variation of peak potentials with analyte concentration The variation of peak potentials with the logarithm of the cation concentration is shown in Fig. 6. In each case, the peak potentials for the reduction of [Fe(CN)6]3 moves towards more positive
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Fig. 4. CVs of 6 mM K3[Fe(CN)6] obtained with gold electrode modified with thioctic acid monolayer. Scan rate 100 mVs 1, background electrolyte 0.1 M KNO3. Ca2 concentration (in M): (a) 0.0; (b) 1 /10 5; (c) 5 /10 5; (d) 1 /10 4; (e) 5 /10 4; (f) 1 /10 3; (g) 5 /10 3; (h) 1 /10 2; (i) 5 /10 2; (j) 1 /10 1.
peak potentials as the cation concentration is increased. This as explained in the case of calcium ions is due to changes in the profile of the electric potential drop across the electrode interface as the cation binds to the receptor monolayer [27]. Consequently, the local concentration of the electroactive species [Fe(CN)6]3 changes bringing
about changes in the electron transfer rate constant [27]. The trivalent cation La3 is observed to have caused a response of the sensor at low concentration far better than the divalent cations, Ba2, Ca2 and Mg2. This confirms the assertion that higher charged species would have the advantage of being much more strongly bound to
2 Fig. 5. CVs of 6 mM K3[Fe(CN)6] obtained with gold electrode modified with thioctic acid. pH of solution 7.4 with H2 PO 4 =HPO4 buffer: (a) no cation present (blank solution); (b) after freshly prepared solution of Tl ion was spiked into the blank solution.
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Fig. 6. Dependence of peak potentials in CV for 6 mM [Fe(CN)6]3 containing different cation of varying concentrations.
the receptor monolayer and as a consequence electrostatically exert much more influence on the access of the marker ions to the electrode surface. The monovalent cation Tl shows remarkably more positive peak potentials at concentrations of 10 2 and 10 1 M than the divalent cations Ba2, Ca2 and Mg2. This could probably be due to the reasons given in Section 3.2 for the unexpected behaviour of thallium(I). Fig. 7 shows sample CVs containing 10 1 and 10 2 M Ba2 and Tl . As shown, in the CVs the peak potentials corresponding to Tl are more
towards the left (more positive end) than those of Ba2 are. 3.5. Variation of reduction current at fixed potential with concentration of metal ion The response of the sensor to the cations can also be quantified by the current at a potential in the rising portion of the reduction peak of the marker ion. In this region, the marker reduction is still controlled purely by the electrode kinetics and is not affected yet by diffusion in the bulk of the
Fig. 7. CVs of 6 mM K3[Fe(CN)6] in 0.1 M KNO3 aqueous solution, pH 7.4 containing: (a) 10 1 M Tl ; (b) 10 1 M Ba2 ; (c) 10 2 M Tl ; (d) 10 2 M Ba2 .
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sample solution. This alternative method was found appropriate since most of the reduction peaks were too broad for the peak potentials to be located precisely. The reduction currents of the marker were thus measured at an applied potential of /0.11956 V against Ag/AgCl reference electrode. The choice of the applied potential was arbitrary and was only selected to optimise the sensor responses. The voltammetric response of the sensor towards Tl , Mg2, Ca2, Ba2 and La3 as , measured reduction currents of the marker at / 0.11956 V as a function of the cation concentration is shown in Fig. 8. The trivalent ion, La3 is found again to be most sensitive just as in the case of peak potentials. In the plot, Ba2 shows a little bit better response than Mg2 and Ca2 that are almost difficult to distinguish. The plot shows the series La3 / Ba2 /Ca2 :/Mg2 /Tl . The highest sensitivity towards La3 and the least towards Tl can be attributed to differences in charges. La3 is expected to be much more bound to the receptor than Tl . Thus La3 has better ion-channel opening effect than Tl . For the alkaline earth metals, the series cannot be attributed to differences in charges. The order of the effect of these ions is also inconsistent with the stability of the coordination of carboxyl in which Mg2 ion is known to show higher stability than the other ions [26]. The stability of coordina-
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tion decreases in the order Mg2 /Ca2 /Ba2 based on simple electrostatic considerations. Similar results were obtained by Takehara et al. [29],when they used g-L-glutamyl-L-cystein (GluCys) and L-cysteinyl-glycine (Cys-Gly) monolayers that have been modified on gold electrodes. GluCys contains two carboxyl terminal groups while Cys-Gly contains only one terminal carboxyl group. They stated that although no clear explanation could be given, the following factors may account for the discrepancy between the ionchannel response and the complex stability. As compared with the molecule in the bulk of the solution, the conformation changes due to coordination with M2 ion is expected to be restricted in the case of surface confined molecule. However, the observed order Mg2 :/Ca2 B/ Ba2 is approximately consistent with the order of the water exchange rate between the inner hydration shells [30]. Under conditions of restricted conformation change, the slower dehydration of the inner shell may bring about the lower coordination stability of the terminal carboxyl unit. The slight differences in the ion-channel effect of Mg2 and Ca2 may be due to the fact that thioctic acid, the receptor has only one carboxyl unit and thus monodentate. Moreover, Mg2 and Ca2, with atomic radii 65 and 99 picometers, respectively are next to each other and posses comparable extent of hydration, therefore, making
Fig. 8. Reduction current for 6 mM [Fe(CN)6]3 solution at 0.11956 V (versus Ag/AgCl) containing different cations at varying concentrations.
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it difficult for the monodentate receptor to clearly distinguish between them.
the monolayer to the marker than lower charge species.
3.6. Variation of reduction peak areas with concentration of metal ion
3.7. Selectivity
The responses of a typical sensor in separate solutions of thallium(I), magnesium, calcium, barium and lanthanum are as shown in Fig. 9. Shown are the peak areas (cathodic peak areas) for the [Fe(CN)6]3 reduction, obtained manually. The trends in Sections 3.4 and 3.5 can also be seen in the Fig. 9. There is large, intermediate and small responses corresponding to trivalent, divalent and monovalent cations. A feature that is common to the curves, except that of thallium(I) is that above a certain critical concentration of the cations, the reduction peak areas levels off. In the case of La3, leveling off the curve occurs above a concentration of 5 / 10 7 M. While for Mg2, Ca2 and Ba2, it occurs above concentration of 5 /10 4 M. This is attributed to the fact that the receptor monolayer becomes saturated with these ions at higher concentrations. Though it will not be quite appropriate to quantify the preference of the sensor to the metal ions, the results from the peak areas have also illustrated qualitatively that higher charge species have much more influence on the permeability of
The selectivity factors for the various cations using the sensor based on SAM of thioctic acid on gold electrode were determined in separate solution. The method used is similar to the match potential method used in obtaining selectivity coefficients in ion-selective electrodes (ISEs) [31,32] in that a peak potential was fixed and the concentration of any analyte ion, in separate solution, that gave the same peak potential was taken for selectivity determination. From Fig. 6, the concentration of the cation which gave the same peak potential (68.8 mV) as obtained in the case of solution containing 1 / 10 4 M of La3 ions or 1 /10 2 M Ca2 ions were determined. The selectivity factors were then obtained as the ratios of those concentrations and 1 /10 2 M Ca2. The thus obtained selectivity factors are La3, 100; Ba2, 2; Ca2, 1; Mg2, 0.54 and Tl , 0.70. The selectivity ratios obtained above support the general trend that trivalent cations are most selectively sensed than the divalent and monovalent cations. As already explained, because of the high charge of the trivalent ion, La3, it is much more bound to the receptor and thus exerts greater
Fig. 9. Areas of the K3[Fe(CN)6] reduction peaks as a function of the logarithm of the metal ion concentration.
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influence on the permeability of marker. The trend in the selectivity of the alkaline earth metals, Ba2, 2; Ca2, 1 and Mg2, 0.54 is in accordance to the ease with which these hydrophilic cations can undergo partial dehydration that is required for the formation of the interfacial complexes at the surface of the monolayer. The ease of dehydration is in the order Ba2 /Ca2 /Mg2. Thus Ba2 is expected to be much more bound to the monolayer and exert greater influence on the permeation of the marker.
3.8. Reproducibility and lifetime All the four modified electrodes prepared responded to the presence of metal ions. In the case of lanthanum ion, some of the sensors responded to concentrations as low as 10 8 M. The general observation about the four sensors was that at low concentration of the cation, the voltammograms were not quite reproducible. They only became reproducible after three to four scans. Thus all the voltammograms were taken after the fourth scan. The non-reproducible nature of the voltammograms at low concentration of the cation could be due to slow equilibration of the metal ions between the aqueous phase and the hydrophobic organic monolayer. The electrodes used showed generally the same sequence of selectivity for the metal ions. However, the magnitude of the response in some cases differed. The lifetime of the sensors was quite long. For instance, one sensor was used for over 3 months, taking over hundred voltammograms. However, in cases where ammonia buffer was used, the sensor easily lost its ion-channel opening and closing ability and even in the absence of the analyte cation, redox current flowed. This situation seems incomprehensible and the sensor became functional only after putting it in concentrated sulphuric acid for about 2 h and then reassemble the monolayer onto the gold surface. Thus ammonia buffer was avoided as much as possible in the work. Another factor that affected the life span of the sensors was the magnitude of the applied potential. The monolayer broke down at higher applied
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potentials. Thus a working potential window of / 0.5 / /0.2 V was used throughout the work. 3.9. Conclusion The gold electrode chemically modified with thioctic acid monolayer and designed to mimic ion-channel membrane allows the detection of metal ions with [Fe(CN)6]3 as the electroactive marker. The detection is based on the attachment of the positively charged metal ion onto the negatively charged monolayer surface at pH of 7.4. For the trivalent ion La3, detection at concentration as low as 10 8 M was observed with some of the sensors. As compared to La3, divalent cations, for instance Ca2, are discriminated in the ratio 1:100. Such selectivity makes these sensors attractive for the detection of the trivalent ion in the presence of large background of divalent and monovalent ions. The trivalent ion La3, with its high charge, has the advantage of being strongly bound to the receptor monolayer and/or electrostatically exert more influence on the access of the marker to the electrode surface. With these characteristics, a gold electrode chemically modified with a monolayer of thioctic acid to mimic gating at bio-membranes has the possibility of being a sensing device for metal ions especially the trivalent ion (La3 ion).
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