Amino acid analysis using disposable copper nanoparticle plated electrodes Jyh-Myng Zen,* Cheng-Teng Hsu, Annamalai Senthil Kumar, Hueih-Jing Lyuu and Ker-Yun Lin Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan. E-mail: [email protected]; Fax: +886 4 22862547 Received 2nd February 2004, Accepted 10th May 2004 First published as an Advance Article on the web 21st July 2004

A disposable copper nanoparticle-plated screen-printed carbon electrode (designated as Cun-SPE100-nm) provides a new material for the determination of native amino acids. All 20 underivatized amino acids can be sensitively determined at 0.0 V vs. Ag/AgCl in pH 8 phosphate buffer solution. The precisely controlled copper nanoparticles can boost up the CuIIO/CuI2O redox signal on the working surface without any prior pretreatment procedure. The formation of a reversible 1:1 CuIIO–amino acid complex on the Cun-SPE100-nm was proposed to play a key role in the reaction mechanism. Stable detection responses were obtained for all amino acids by flow injection analysis with detection limits (S/N = 3) that lie in the range of 24 nM–2.7 mM. Selected amino acids from six representative chemical natures were separated by HPLC and detected at the Cun-SPE100-nm with promising results.

DOI: 10.1039/b401573h

Introduction L-a-amino acids are the basic building blocks of peptides and proteins as the biosynthetic precursors of many biologically relevant molecules as well as metabolic fuels.1–4 Detection of amino acids is thus very important in proteomics, protein sequencing, gene functionalities, enzyme and hormone characteristics and clinical disorder diagnostics, etc.1,5–10 Traditional analytical methods for amino acids rely on chromoscence or fluorescence based tagging agents for quantification.11–15 In some cases, the reactions either ended with multiple tagging or the compounds themselves reacting with water to form a detrimental fluorescent alcohols. Most reagents are also unable to couple with secondary amino acids (e.g., proline and hydroxyproline). Spectroscopic methods, such as Raman, NMR, electron spray ionization ion mobility and circular dichroism, on the other hand, are expensive and time consuming in sample preparation, operation and quantification for amino acid analysis.16–19 Up to now, inexpensive and direct quantification assays for amino acids by an easy route still remains a challenge in analytical biochemistry. Electroanalysis appears to be a good way to overcome difficulties in amino acid analysis.20–32 The lack of a strong electrochemically active group in most native amino acids, however, has limited earlier developments in electrochemical methods. So far, the most attractive electrode materials with good electrochemical performance for amino acids are non-noble metals like Ni and Cu.22–32 The anodic response at Ni electrodes has been identified as electrocatalysis with electron-transfer mediation involving NiII/III redox species in the surface oxide. Detection of amino acids at Cu electrodes, on the other hand, is more versatile. As illustrated in Scheme 1, apart from the most-known surface-catalyzed oxidation reaction in highly alkaline media (pH 4 13), a complexation mechanism with a relatively feeble current response was observed at low potentials (ca. 20.2 to +0.2 V vs. Ag/AgCl) at pH ˆ groups have 6–11.25,27–29 In the mid-1980’s, both Kok and Stulík studied the complexation process of amino acids at electrochemically pretreated (20.5 V for 5 min and then +0.15 V for 15 min) copper-disk electrodes.25–28 Baldwin and co-workers further extended it to amino acid assays using the complexation route, but failed to get stable responses. Later, they adopted an electrocatalytic approach at an applied potential of ~ 0.5 V vs. Ag/AgCl in 0.1 M NaOH for amino acid analysis.29 Kuhr and co-workers used more complex sinusoidal voltammetry to expand the application of the copper electrode through the electrocatalytic mechanism in 0.1

M NaOH.33 Although numerous methods have been introduced for the analysis of amino acids, all of them show various shortcomings. We report here a low potential (0.0 V vs. Ag/AgCl) and stable electrochemical detection scheme for all native amino acids at physiological pH by a disposable screen-printed carbon electrode modified with copper nanoparticles (designated as Cun-SPE). Our group recently reported an electrochemically deposited normalsized ( > 500 nm) copper-plated screen-printed carbon electrode (Cu-SPE) for H2O2, glucose, dissolved O2 and o-dihydrophenolic compounds (e.g. catechol and dopamine) detection.34–37 A specific five-member CuII-o-quinolate complex formed in the intermediate was proposed for the selective detection of o-diphenols.36 Unfortunately similar to classical Cu-based redox systems in amino acid analysis,29 the CuSPE always deactivates the working surface through fouling. In this work, the Cun-SPE with enriched CuIIO/ CuI2O surface was found stable and sensitive to the amino acid analysis. A new preparation route is introduced for the formation of copper-nanoparticle (Cun) on screen-printed electrode (SPE). Since this CuIIO/CuI2O redox system is so unique for direct amino acid analysis, the details of the complexation characteristic was probed by product analysis with 1H-NMR spectroscopy. Finally, flow

Scheme 1 General routes for amino acid detection at copper-based electrodes.

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injection analysis (FIA) was successfully demonstrated for the sensitive determination of all amino acids. This economical and easy method is expected to have a great impact in biochemical applications.

Experimental Apparatus, electrodes and electrochemical cells Cyclic voltammetric (CV) and chronoamperometric (i–t) experiments were carried out with a CHI 627 electrochemical workstation (CH Instruments, Austin, TX, USA). The three-electrode system consists of a Cun-SPE working electrode (geometric area = 0.2 cm2), an Ag/AgCl reference electrode and a platinum (geometric area = 0.07 cm2) auxiliary electrode. The disposable SPEs were purchased from Zensor R&D (Taichung, Taiwan). The SPEs were characterized with a model system of 3 mM Fe(CN)632 in 0.1 M KCl solution. An average anodic current value of 88.4 ± 2 mA with a RSD value of 2.3% and peak potential separation (DEp) inbetween 175—191 mV at v = 50 mV s21 for 10 sets of SPEs indicated good quality control of the SPEs. The FIA system is consisted of a Cole–Parmer microprocessor pump drive, a Rehodyne model 7125-sample injection valve (20 mL loop) with interconnecting Teflon tube and a Zensor SF-100 thinlayer detecting electrochemical system (specifically designed for SPE). Chromatographic separations were performed using a silicabased HPLC column (Prevail organic acid 5 u, Alltech, 100 3 4.6 mm) with a mobile phase of 0.1 M, pH 8 PBS. Product analysis was carried out by a Varian 400 MHz NMR spectrometer. The electrolysis alanine solution (in pH 8 PBS at 0.0 V vs. Ag/AgCl) from FIA experiments was extracted by ethyl acetate. A rotary vacuum evaporator is later used to remove the solvent and then to the 1H-NMR analysis. The electrode preparation procedure is a modified version of our earlier one.34,36,37 In brief, a Cu layer was electrochemically plated on a clean bare SPE in 200 ppm Cu(NO3)2 + 0.1 M HNO3 at an applied potential of 20.7 V for 300 s ( ~ 0.5 C of charge passed) under photo-irradiation (Perkin Elmer Xenon fiber optic light source). Prior to the experiments the SPE was thoroughly washed with copious amount of water. The light intensity was measured by a TES-1335 light meter (Taiwan). Basic idea for such a preparation is from our previous studies on a Cu-SPE towards photoelectrochemical detection of dissolved oxygen.37 We observed an efficient anodic oxide behavior of the Cu-SPE under photoirradiation and in turn an improved performance.

Results and discussion Electrochemical behavior of amino acids at the Cun-SPE100-nm Fig. 1A shows the typical SEM pictures of the Cu-SPE and CunSPE under different preparation conditions. It is obvious that the new photo-irradiation procedure results to relatively fine Cu particles on the SPE (Fig. 1B). The Cu particle size can be easily controlled by adjusting the light intensity as ~ 500 nm (without illumination), ~ 400 nm (by illumination at 49 klx) and ~ 100 nm (by illumination at 399 klx), respectively. The CV behaviors of the Cu-SPE and the Cun-SPEs in pH 8 PBS were first compared. A much higher peak current response at the Cun-SPE100-nm than those at Cun-SPE400-nm and Cu-SPE indicates higher surface area due to smaller particle size. Strong light-irradiation can even attenuate the Cu particles with small grooves on the outer surface to further increases the surface area on Cun-SPE100-nm. Conceptional presentation in Fig. 1(i)b clearly depicts the increased surface area effect of the Cun-SPE100-nm. For all three electrodes, reduction peaks at ~ 20.20 V (C1, CuIIO ? CuI2O) and 20.25 V (C2, Cu2IO ? Cu0) with an anodic shoulder at E1/2 = 20.07 V vs. Ag/AgCl (A1&A2) were observed.34–37 Surface concentration of CuII (GCu(II) = Q/ nFA) on the Cun-SPE can be calculated by integration the C1 redox peak area (i.e., Q) based on the cyclic voltammogram obtained at a scan rate of 2 mV s21. A value of GCu(II) = 13 nmol cm22 was observed for the Cun-SPE100-nm at pH 8 PBS. The CuIIO/CuI2O enriched Cun-SPE100-nm surface is unique for the stable and selective complexation mechanism for amino acid detection as will be discussed later. It is noteworthy that the CuIIO formed at copper electrode usually considered as a passive material for electrochemical applications, but it is quite useful for the CuIIO-amino acid complexation over the Cu2IO oxidation state. The CV responses of the Cun-SPE100-nm with amino acids were next studied. As shown in Fig. 2A, the addition of alanine (background electrolyte dotted trace; alanine addition solid trace) causes an obvious change in the CV. This is also true for all the 20 amino acids (data not shown). The C1 response found to markedly increased at the expense of the C2 peak together with increase in the anodic peak at 20.05 V in the presence of amino acids. Most important of all, as shown in Fig. 2B, virtually no difference in CV was observed with either aniline (with –NH2) or acetic acid (with –COOH), indicating that this enhancement in anodic current is an

Chemicals and reagents Amino acids were bought from ICN Biomedicals Inc. (Aurora, OH, USA). All the other compounds used in this work were of ACScertified reagent grade. A 200 ppm CuII solution in 0.1 M HNO3 was used for the platting experiments. Aqueous solutions were prepared using deionized water (18 MW cm21). Unless otherwise mentioned, a pH 8 phosphate buffer solution (PBS) of I = 0.1 M was used as the base electrolyte. More dilute standards were prepared by suitable dilution with the same PBS.

Procedures After the Cun-SPE100-nm preparation step, the strip was washed thoroughly with deionized water before subsequent static experiments in pH 8 PBS. Note that, unlike the requirement in classical copper-disk electrode, no extra pretrements were performed.25–28 For FIA, the Cun-SPE100-nm was equilibrated in pH 8 PBS carrier solution at 0.0 V until the current became constant. Noramly it takes ~ 250 s for the stabilization of the Cun-SPE. The quantification of all amino acids samples were achieved by measuring the anodic current from chronoamperometric curve at an applied potential of 0.0 V. All experiments were performed at room temperature of 25 ± 2°C. 842

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Fig. 1 (A) SEM morphological view and typical CV (in pH 8 PBS) of CuSPE and Cun-SPE prepared without (iii) and with 49 (ii) & 399 (ia & ib) klx of light illumination. (B) Conceptional representation of the proposed plating method for the Cun-SPE. CV scan rate (v) = 10 mV s21.

exclusive effect for amino acids. Continuous CV experiments with amino acids also show no change in either peak potential or current for up to 30 min of cycling. Parallel experiments with a normal CuSPE, on the other hand, showed > 80% decrease in the CV signal for just a few minutes of scans. This fouling behavior is consistent with the classical Cu-based redox system as reported earlier.29 As mentioned earlier, two types of electrochemical mechanisms were observed for amino acids at a Cu-wire electrode in 0.1 M NaOH (Scheme 1). Normally, amino acids possess a bidentate ligand, where the –COO2 and –N terminals function as the chelating site.38–41 Classical solution phase study indicates a twostep process for the CuII(metal ion)-amino acid complexation as follows: CuII + A2 ) CuA+ (step 1) + A2 ) CuA2 (A2: a-amino acid) (step 2) (1) The free CuII first gets complexed with an A2 ligand to form a 1:1 complex of CuA+ and then further couples to another A2 ligand to form a 1:2 complex of CuA2. The complexation rate constant of 1–4 3 109 M21s21 for step 1 is relatively faster than the constant of 2 3 108 M21s21 for step 2.38–41 The specific increase in the C1 redox peak in the presence of amino acids provides first evidence for the formation of the CuIIO-amino acid complexes in the present system (Fig. 2A). The fact that parallel CV experiments with aniline and acetic acid do not show any alteration in the electrochemical characteristics verifies the selective complexation of CuIIO-amino acid at the Cun-SPE100-nm (Fig. 2B). The scan rate (v) effect on the CV behavior of alanine at the CunSPE100-nm was further studied. Double logarithmic plots of ipa vs. v result in a slope of ~ 1 in blank electrolyte and ~ 0.5 in the presence of alanine. This suggests a change of charge transfer mechanism from adsorption-controlled to diffusion-controlled as the copperalanine complex starts to form. Furthermore, the plot of current

Fig. 2 Typical CV responses of the Cun-SPE100-nm in the absence (dotted line) and presence (solid line) of (A) 2 mM alanine and (B) 2 mM aniline + 2 mM acetic acid in pH 8 PBS at a scan rate of 10 mV s21.

function [if = (ipacomplex – ipablank)/v1/2] vs. v1/2 shows an inverse trend indicating a diagnostic criteria of the EC kinetics.42,43 A possible reaction pathway at the Cun-SPE100-nm is therefore the electrogenerated CuIIO in couple with the CuIIO-alanine complex formation. 1H-NMR

study and reaction mechanism

In order to verify the complexation mechanism, product analysis by 1H-NMR was carried out for alanine in this study. As reported earlier, R–CN and/or R–CHO final products were identified for amino acid oxidation through the electrocatalytic process.24 Hence, if the alanine oxidation is under the electrocatalysis pathway, a CH3CN and/or CH3CHO NMR signal should be observed. No change in the NMR spectra would otherwise indicate the complexation pathway. Virtually identical 1H-NMR (D2O) spectra of alanine before and after electrolysis on the Cun-SPE100-nm indicates a pure complexation pathway (either CuA+ or CuA2) for the present system. Scheme 2 sketches the possible complexation mechanism on the Cun-SPE100-nm. In step 1, the bi-dentate amino acid ligand is first chelated with Cun, followed by reversible reduction of CuIIO ? Cu2IO. As soon as the reduced Cu2IO is regenerated back to CuIIO, the same cycle can be repeated again. The geometric structure of the chelation on Cun is essential since CuIIO is not as free on the electrode surface as CuII metal ion in aqueous solution. The Cu(II)A2 structure must somehow be sterically hindered at the CunSPE surface (step 2). Ultimately, by knowing the reaction order (m) of the overall reaction one can easily differentiate the nature of reaction pathway on the Cun-SPE100-nm. The FIA responses under optimized hydrodynamic conditions can be taken as the steadystate kinetics current, and a double logarithmic plot of peak current vs. [amino acid] was made. A slope value of 0.99 ± 0.2 was obtained for all amino acids, it is thus concluded that first order reaction mechanism with the formation of reversible 1:1 CuIIOamino acid complex (step 1) takes place on the Cun-SPE100-nm interface. It is likely to occur on the cupric oxide (CuIIO) surface and hence the interaction is not as strong as that of CuII ion in aqueous solution. The reaction mechanism implies a weak adsorption of amino acid on the Cun during the complexation process, where the CuIIO is electro-generated on the surface. The adsorbed amino acid can be easily desorbed as the CuIIO layer is reduced into CuI2O. Interestingly, these adsorption/desorption characteristics shows

Scheme 2 Reaction mechanism for the copper(II)-amino acid complexation at the Cun-SPE100-nm. Analyst, 2004, 129, 841–845

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little damage to the underlying Cun surface as characterized from CV, FIA and H1-NMR studies.

Analytical applications The determination of all the 20 amino acids using the CunSPE100-nm by FIA was evaluated. By taking 50 mM alanine as a standard with pH 8 PBS as carrier solution, the effect of hydrodynamic flow rate (Hf) was first optimized. As can be seen in Fig. 3, the current responses are almost unaltered in the range of 100 mL to 1 mL. The retention time and peak area, however, are strongly influenced by Hf. An increase in peak area of about five times at Hf = 100 mL min21 (10.3 mC) than that at Hf = 1 mL min21 (1.9 mC) was observed. Obviously, this has something to do with the complexation rate constant and the same trend was also observed for other amino acids like serine and aspartic acid. In compromise, an Hf of 300 mL min21 was taken as optimal for further analysis. Fig. 4 shows the typical FIA response of alanine under the optimized Hf. The calibration plot is linear up to 500 mM with sensitivity and regression coefficient of 7.29 nA mM21 and 0.9992, respectively. Eleven continuous injections of 5 mM alanine resulted in a small RSD of 1.26% indicating good reproducibility of the electrode. The detection limit (DL, S/N = 3) was 24 nM. Analytical experiments were then extended into all amino acids. The results

obtained are summarized in Table 1. The calibration plots are linear in the window of 5–500 mM with DL in the range of 24 nM–2.7 mM as a result of different side chain substitution of amino acids.24 The analytical results of Cun-SPE are better than those of the conventional copper disk electrode with ~ one order lower in DL.26 The increase in CuIIO-amino acid complex formation at CunSPE100-nm also results in a higher current sensitivity over the conventional copper disk electrode. Additonal physico-chemical characterization together with optimized modification conditions are necessary to further improve the DL. Overall, the advantage of this approach is easy preparation, simple electrochemical operation and no prior electrochemical pretreatment procedures. The durability was checked with a fresh-prepared Cun-SPE100-nm by continuous injection (n = 10) for up to 4 h (which is the period normally required in amino acid separation studies). Unlike the fouling off electrochemical behaviour as per Baldwin and coworkers,29 no marked alteration (RSD = 4.1%) in the FIA signals of alanine, glutamine and iso-leucine were noticed. Finally, ion-chromatographic method combined with CunSPE100-nm based electrochemical detection was demonstrated for the determination of amino acids in neutral phosphate medium. Fig. 5 shows the typical HPLC separation results for amino acids chosen each one from the six representative chemical natures of amino acids.1 All these results strongly validate the usefulness of the present approach for further real sample analysis.

Conclusions A new copper-nanoparticle-modified screen-printed carbon electrode prepared by a photo-electrochemical deposition method served as an efficient, stable and appreciably sensitive sensing system without any electrode pretreatments for the detection of all 20 amino acids by flow injection analysis at a low working potential in pH 8 PBS. The complexation kinetics operated on the CunSPE100-nm was identified as a reversible pathway by 1H-NMR, unlike the formation of RCN or RCHO products in the conventional electrocatalytic approach at copper metal electrodes. The kinetic model shows that the copper-amino acid complexation at the CunSPE100-nm is a first order reaction with the formation of a 1:1 ratio of CuII and amino acids. Stable FIA responses were obtained at 0.0 V vs. Ag/AgCl for 20 amino acids with good sensitivity. Extended studies with HPLC separation of representative amino acids from Table 1 FIA of 20 a-amino acids at the Cun-SPE100-nma Fig. 3 Typical FIA responses of alanine (50 mM) with different flow rates (Hf) at the Cun-SPE100-nm in pH 8 PBS carrier solution. Insert figure corresponds to the plot of peak area (in mC) against Hf for alanine, aspartic acid and serine.

DL (S/N = 3) Amino acid Ala Arg

Fig. 4 FIA responses of the Cun-SPE100-nm at increasing concentration of alanine. 844

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Linear range/mM

R

Sensitivity/ nA mM21

/nM

5—500 0.9990 7.29 24 5—300 0.9960 5.97 587 5—500 0.9894 5.05 695 Asn 5—300 0.9998 11.68 94 5—500 0.9901 9.78 112 Asp 5—500 0.9951 5.98 2693 Cys 10—300 0.9955 6.41 875 Glu 5—500 0.9901 4.90 624 Gln 5—500 0.9962 7.40 221 Gly 5—500 0.9996 2.83 161 His 5—500 0.9961 12.07 44 Iso-Leu 5—500 0.9976 4.40 297 Leu 5—300 0.9978 7.44 231 Lys 5—500 0.9994 4.30 1890 Met 5—500 0.9929 8.99 102 Phe 5—500 0.9991 7.62 472 Pro 5—300 0.9988 3.70 2545 Ser 5—500 0.9914 7.58 1393 Thr 5—500 0.9954 8.46 429 Trp 5—500 0.9993 6.66 389 Tyr 5—500 0.9999 7.17 548 Val 5—500 0.9992 4.21 574 a FIA detected at 0.0 V vs. Ag/AgCl in pH 8 PBS carrier solution.

/ng 0.04 2.47 2.93 0.28 0.34 7.17 2.12 1.84 0.65 0.21 0.14 0.78 0.61 7.23 0.30 1.56 5.86 2.93 1.02 1.59 1.98 1.34

Fig. 5 Chromatogram of a mixture of amino acids (50 mM) from six representative chemical natures using a silica-based HPLC column with a Cun-SPE100-nm detector.

six different chemical natures also show promising results. The disposable nature of the Cun-SPE100-nm is expected to be useful in amino acid analysis in biological samples.

Acknowledgement The authors gratefully acknowledge financial support from National Science Council of the Republic of China. Helpful 1H-NMR spectroscopy experiments from Dr Bapu Chaudhari and SEM pictures from Dr Hong-Yi Tang are appreciated.

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