Constant potential amperometric detection at a copper-based

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Srinivasah. S.,Will, F. S., Eds.; The Electrochemical Society: Princeton, NJ, 1977;p 861. (34) Watanabe, M.; Nagano, S.; Sanui, K.; Ogata, N. Polym. J . 1986, 78,

809. (35)Pinkerton, M. J.: LeMest. Y.: Zhana. - H.;Watanabe. M.; Murray, R. W. J . Am. Chem. Soc., in press.

(36) Zalipsky, S.: Gilon. C.; Zilkha, H. Eur. Poly” J . lS83, 79, 1177. (37) Ewlng, A. 0.;Dayton, M. A.; Wightman, R. M. Anal. Chem 1981. 53,

(41) Aoki, K.; Honda, K.; Tokuda. K.; Metsuda, H. J . Electroanel. Chem. Interfacial Electrochem. 1985, 182, 267. (42) Aoki. K.; Tokuda, K. J . phvs. Chem. 1987,237, 163. (43) Morris, R . B.; Franta, D. J.; White. H. S. J . Phys. Chem. 1987, 9 7 , 3559. (44) Petek, M.; Neal, T. E.; Murray, R. W. Anal. Chem. 1971, 43, 1069. (45) Seibold, J. D.: Scott, E. R.; White, H. S. J . Electroanal. Chem. Interfacial Electrochem. 1989. 264, 281.

~2 ._

(38)Dayton, M. A.; Brown, J. C.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1980,52,946. (39)Shoup, D.; Szabo, A. J . Electroanal. Chem. Interfacial Electrochem. 1982, 140, 237. (40)Aoki, K.; Aklmoto, K.; Tokuda, K.; Matsuda, H.; Osteryoung, J. J . Electroanal. Chem. Interfacial Electrochem. 1984, 171, 219.

RECEIVED for review September 12,1989. Accepted December 21,1989. This research was supported in part by grants from The Department of Energy and The National Science Foundation.

Constant Potential Amperometric Detection at a Copper-Based Electrode: Electrode Formation and Operation Peifang Luo, Sunil V. Prabhu,’ and Richard P. Baldwin*

Department of Chemistry, University of Louisville, Louisville, Kentucky 40292

Copper-based chemically modmed electrodes (CMEs) prevlouSry used for the amperometrk detection of carbohydrate compounds can be prepared via several dlfferent chemical and electrochemleal procedures lncludlng exposure of glassy carbon to a CuCi, sdutlon gaiuanlcally with a Cu counter electrode arrd plating of metallic Cu by cathodic depositlon. In the former case, the mechanism most likely involves the formatlon of a coatlng of sllghtly soluble CuCl by a spontaneous redox reactlon between Cu2+and Cu metal. All of the Cu CMEs exhlblt sknUar cycllc voltammetric and constant potentlal amperometrlc response toward carbohydrates and offer detection Hmns at the pkomole level In chromatography applications.

We have recently described the application of a Cu-based chemically modified electrode (CME) for the amperometric detection of carbohydrates and related compounds following separation by anion-exchange chromatography (1-3). This CME is of particular interest for two principal reasons. First, it offers detection capabilities in the nanomoleto-picomole range for simple mono- and oligosaccharides as well as related amino sugars, alditols, and aldonic, uronic, and aldaric acids. And second, it enables the detection to be carried out under constant potential conditions and thus is compatible with the simple constant-potential electrochemical detection units already in common use among chromatographers. Previous reports have focused on evaluating the analytical performance of the Cu CME and on applying it in specific chromatographic schemes for the separation and analysis of various carbohydrate compounds. To this point, however, comparatively little has been determined concerning the structure of the CME and its mechanism of operation. In this communication, we describe experiments that serve to provide more information concerning both of these questions.

* Author to whom correspondence should be addressed. ‘Current address: Merck 81 Co., P.O. Box 2000, Mail Code RSOL-123, Rahway, NJ 07065.

EXPERIMENTAL SECTION Reagents. Carbohydrates and related compounds, purchased from Sigma or Fisher Chemical Cos., were used as received without further purification. Stock solutions (1.0 mM) were prepared in deionized water and were brought to the desired concentration and pH by dilution with mobile phase just prior to use. Mobile phases used for flow injection experiments were prepared from carbonate-free NaOH, and all solutions were degassed with N2 before use. Electrodes. CMEs were prepared from Models MF-2012 and MF-1000 glassy carbon electrodes obtained from Bioanalytical Systems (West Lafayette, IN). The various modification procedures employed are described in detail in the results section. The metallic copper electrode used for comparative experiments was made by sealing a 2-mm-diameter Cu wire in a glass tube; it was polished with alumina and held at -1.3 V vs Ag/AgCl for 3 min in 0.50 M NaOH before voltammetric scans were initiated. In most cases, the electrodes were cycled briefly from 0.0 to +0.6 V in 0.15 M NaOH before use in flow detection systems. This usually served to decrease background currents and the length of time required for the background to become stable. Apparatus. Cyclic voltammetry (CV) was performed with a Pine Instrument Co. (Grove City, PA) Model RDE4 potentiostat with an Ag/AgC1(3.0 M NaCl) reference electrode and a platinum wire auxiliary electrode. Flow injection experiments were carried out with a Beckman Model llOB pump, an SSI (State College, PA) Model LP-21 pulse dampener, a Rheodyne (Berkeley, CA) Model 7125 injector with a 20-wL sample loop, and a Bioanalytical Systems Model LC-4B amperometric detector. All experiments were performed at room temperature.

RESULTS In the initial report ( I ) , the Cu CME was prepared simply by immersing a commercially available glassy carbon electrode assembly into an aqueous CuC12 solution. If, after several minutes of soaking, the electrode was removed from the copper solution and rinsed with water, a light-colored deposit that gradually changed to green could be seen to have formed spontaneously on the glassy carbon. Subsequent X-ray fluorescence measurements made on the modified electrode surface at this point indicated that the principal constituents of the deposit were Cu and C1; scanning electron microscopy revealed that much of the film was in the form of hexagonally

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ANALYTICAL CHEMISTRY, VOL. 62, NO. 7, APRIL 1, 1990

753

Table I. Alternative Methods for Forming Cu CME modification approach A B C

medium

E , for glucose'

probable surface species

procedure

0.050 M CuClz 100 pA reduction current was applied for 1-3 min 0.050 M C U ( N O ~200 ) ~ pA reduction current was applied for 1-3 min 0.050 M Cu(NO& a droplet of Cu(N03)*was placed onto GCE and allowed to air-dry; a drop of 0.15 M NaOH was placed onto the resulting film and allowed to air-dry

(V vs Ag/AgCl)

CuCl Cu metal Cu(OH),/CuO

+0.50 +0.48 +0.50

'CVs were obtained for 0.0050 M glucose in 0.15 M NaOH. shaped crystalline structures, 1-2 pm in diameter, seemingly embedded into and across the glassy carbon surface (2). When subsequently used in voltammetric and flow injection experiments, this electrode, referred to as a Cu CME, gave large currents between +0.4 and +0.6 V vs Ag/AgCl for the oxidation of numerous carbohydrates and related compounds. The principal requirement for the carbohydrate oxidation to be observed was that extremely basic conditions, with hydroxide concentrations of a t least M, had to be maintained. It was subsequently discovered that the procedure described above was successful in efficiently forming an active deposit on the glassy carbon surface only in those instances where the entire cell assembly, including the metallic electrical contacts, was submerged in the CuC1, solution. Furthermore, the rate of the deposition process could be greatly enhanced by deliberately attaching a short piece of freshly polished copper wire to the built-in electrical contact during exposure to the CuCl, solution. Under these conditions, the surfaces of both the glassy carbon electrode and the copper wire lost their initial shiny appearance and took on a dull white cast within 30 s. After the modification process was allowed to continue for 1 min and the electrode was removed from the solution and rinsed with deionized water, the glassy carbon gradually took on the same green color noted above. Most important, the performance of this electrode in CV and flow injection analysis of carbohydrate compounds was qualitatively the same as that of the Cu CME prepared by the original procedure. Subsequently, several other variations in the CME preparation procedure, summarized in Table I, were investigated and found to produce electrodes possessing essentially the same activity toward carbohydrates. The variations studied included constant current reductive deposition from both CuCl, and C U ( N O ~solutions )~ and evaporative deposition of a C U ( N O ~film. ) ~ In addition, a metallic copper electrode, formed by sealing an ordinary Cu wire into a glass tube, was also considered. Each of these approaches produced electrodes that exhibited similar steady-state CVs in both blank and glucose-containing solutions. Shown, for example, in Figure 1 are voltammograms run in 0.50 M NaOH for both the plain copper wire electrode and a CME formed by electrodeposition from CuC1, (i.e., method A from Table I). In both cases, the CVs, which match those previously reported for metallic Cu electrodes in strongly alkaline solution (4-7), show evidence of all three Cu oxidation states. Specifically, the anodic waves labeled A, B, and C correspond to the formation of Cu(I), Cu(11), and Cu(II1) species, respectively, while cathodic peaks D and E represent the re-formation of Cu(1) and Cu(0). (Note that here, as in the previous studies ( 4 - 9 , wave C could be seen in CV only for NaOH concentrations of 0.2 M or higher.) The principal difference exhibited by electrodes prepared by the different procedures involved the current-voltage behavior seen on their very first CV scans. In particular, the principal anodic activity (Le., B') for the CMEs prepared from CuCl, solution was shifted to more positive potentials on the initial scan. The precise appearance of these initial oxidation pro-

T

500pA

1

1

0.80

.

1

0.40

1

1

0.00

1

1

-0.40

1

1

-0.80

1

1

-1.20

E / V vs Ag/AgCl

Flgure 1. Cyclic voltammograms of (a) copper wire electrode and (b) Cu CME in blank solution: electrolyte, 0.50 M NaOH; scan rate, 20 mVls; first cycle (-), second cycle (- - -).

cesses depended on the specific hydroxide concentration employed and the thickness of the film deposited onto the CME surface. For the 0.5 M NaOH conditions in Figure 1,at least two distinct oxidations were observed while, a t lower basicity, the potentials of the waves shifted somewhat, occasionally to such an extent that nearly complete overlap occurred. But, in all cases, these waves disappeared on the second and all later scans; and the CV for the CME exactly matched that for the plain copper electrode. Interestingly, physical exposure of the Cu CME to strongly alkaline solutions changed the CME film to yellow. Subsequent scanning of the electrode potential to values more positive than the carbohydrate oxidation caused the electrode surface to take on a black or dark gray appearance. An additional effect of scanning to extremely positive potentials was a rapid deterioration of the CME surface that was reflected by decreased currents for all the waves in the succeeding CVs. The primary effect of glucose addition on the CV behavior was also identical for both the Cu CME and the plain Cu electrode. As shown in Figure 2 for the CME, this consisted of a marked decrease in peaks B and E and the development of a new anodic wave at +0.50 V. It is worthwhile noting that this glucose-related wave, which was the one used for carbohydrate detection both here and in our earlier studies did not occur at a potential very close to either Cu(I1) or Cu(II1)

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ANALYTICAL CHEMISTRY, VOL. 62, NO. 7, APRIL 1, 1990

CU(S) + cu2+

w 1

,

1

0.40

0.80

1

1

0.00

1

,

1

-0.40

1

-0.80

1

1

-1.20

€I' V vs Ag/AgCI

Figure 2. Cyclic voltammograms of (a) copper wire electrode and (b) CuCME in glucose-containingsolution: electrolyte, 0.50 M NaOH; scan rate, 20 mV/s; glucose concentrations, 0.0 M (-), 0.0020 M (- - -), 0.010 M (. -), and 0.030 M (- - -).

-

formation (waves B and C in Figure 1) but was very reminiscent of the wave seen for the initial CV scan in the blank (wave B'). The fact that electrodes formed via all the different procedures exhibited this same wave at +0.50 V and thereby gave useful carbohydrate detection capabilities demonstrates clearly that the presence of C1-, though helpful for CME formation according to our initial approach (1-3), was not essential to fabricate Cu-based electrodes capable of oxidizing carbohydrate compounds in an analytically useful fashion.

DISCUSSION While several aspects of carbohydrate oxidation at the Cu CME remain to be resolved, the experiments described above allow some the important characteristics to be explained-in particular, the electrode itself and its likely mode of formation. Perhaps most important in this regard is the observation that formation of an active CME surface requires participation not only of Cu2+ but also of metallic Cu, either from the cell assembly's electrical contacts or from intentionally added Cu wire. This suggested to us the involvement of a galvanic Cu+-forming redox reaction as a likely element in the formation of the CME. As the solubility of cuprous chloride is rather low (81, generation of a cuprous ion under the 0.1 M C1- conditions in effect might easily lead to the deposition of CuCl on the glassy carbon surface. This possibility is supported indirectly by several additional factors. First, X-ray fluorescence analysis, described earlier (21,indicated that the freshly formed CME surface contains principally Cu and C1. Second, experiments in which salts, such as C U ( N O ~ were ) ~ , used in place of CuC12 during opencircuit conditions failed to produce an active CME surface. Third, the initial deposition and subsequent reaction of CuCl accounts well for the appearance of the CME observed at various stages in its usage: light-colored initially and then green on exposure to air, CuCl; yellow, after exposure to NaOH, Cu20; and black, after exposure to oxidizing potentials, CuO (8). Finally, measurement of the open-circuit potential during CME formation yielded a value consistent with that calculated for CuCl formation according to the reaction

+ 2c1-

-

2CuCl(s)

The measured potential during CME formation was 260 (f10) mV, while the calculated value ([Cu2+]= 0.050 M, [Cl-] = 0.10 M, K , for CuCl = 1.7 X lo-') was 243 mV. Our conclusion, based on all these observations, is that the deposition of a CuCl film, according to the above reaction, represents the most likely explanation for the formation and initial state of the Cu CME as used previously for carbohydrate detection (1-3). It seems equally clear that, once formed and then subjected to other chemical and electrochemical conditions, the CuCl deposit is readily converted to a Culoxide or hydroxide system similar to that known to occur under highly basic conditions for metallic Cu (4-7). Thus,the freshly deposited CuCl film on its initial CV scan exhibited unique current-voltage properties that subsequently disappeared and were replaced by those characteristic of Cu metal electrodes. Most important, it is apparent that it is the latter, long-lived surface structure that is responsible for the CME's long-term activity toward carbohydrates. The actual mechanism of the carbohydrate oxidation still remains difficult to specify with certainty. Previously, we have suggested an electrocatalytic mechanism for carbohydrate oxidation which might be initiated by a Cu(II1) surface species ( I , 2). An alternative possibility, suggested by the CV behavior in Figure 2, is that the active surface state might be a Cu(I1) species whose formation is shifted from -0.05 V in blank NaOH solution to +0.5 V in the presence of the carbohydrate analyte. It is of interest that Van Effen and Evans reported that simple aldehydes undergo oxidation at both Cu(I1) and Cu(II1) oxide surfaces in 1.0 M NaOH (9). Furthermore, Cu(I1) complexes have previously been used as postcolumn additives for the electrochemical detection of reducing sugars (10). In that approach, the Cu species reacts with the carbohydrate and becomes reduced to the cuprous state which can be monitored amperometrically by reoxidation to Cu2+. However, in contrast to the current work, only reducing sugars gave such a response, and the response occurred at much lower potentials, in the vicinity of the solution-phase Cu(I)/Cu(II) redox couple a t 0.0 V vs AgJAgCl, than with the Cu CME. Thus, it appears that, even if the Cu(I1) state is the important one for the CME, some factor such as adsorption or complexation of the carbohydrate analyte must be acting to shift the cuprous oxidation to the unusually positive potentials seen, for example, in Figure 2. A third possibility, suggested by the work of Frei et al. (11,12),is that complexation of Cu(I1) by the carbohydrates acts to solubilize the oxide/hydroxide layer present in basic solutions and thereby increase the current observed for copper oxidation. This phenomenon, however, was reported by Frei for amino acids as analytes-but only for much less basic conditions and a t much lower potential than in the current work. Finally, there is a particularly important question concerning the best form of Cu electrode to recommend for analytical applications-e.g., metallic copper or the Cu CME formed by any of the several procedures employed. Although any of the Cu electrodes studied here could be used in principle, we found that the Cu CME formed galvanically by attachment of a clean copper wire to the electrode assembly and immersion into 0.05 M CuC12 seemed to exhibit m@y superior performance in terms of background noise levels, detection limits, and ease of operation. The Cu CME formed by this technique exhibited detection limits at the pmol level and provided sufficient stability and reproducibility than 100 glucose injections carried out over a 1.5-h period gave a relative standard deviation of less than 1%.

LITERATURE CITED ( 1 ) Prabhu, S.V.; Baldwin, R. P. Anal. Chem. 1969, 6 1 , 852-856. (2) Prabhu, S. V.; Baldwin, R. P. Anal. Chem. 1969, 61, 2258-2263.

Anal. Chem. 1990,62,755-759 (3) Prabhu. S. V.; Baldwin, R. P. J . Chromatogr., in press. (4) Miller, E. J . €/ecffochem. Soc. 1969, 776, 1675-1680. (5) AW El Haleem, S. M.; Ateya, E. G. J . Electroanel. Chem. Interfacial Electrochem. 1981, 777, 309-319. (6) Pyun, C.-H.; Park, S A . J . Electrochem. SOC. 1986, 733, 2024-2030. (7) Drogowska, M.; Brossard, L.; Menard, H. Corrosion (Houston) 1987, 43, 549-552. (8) The Merck Index, 9th ed.; Merck & Co.: Rahway, NJ, 1976; pp 345-347. (9) Van Effen, R. M.; Evans, D. H. J . ElecffoamI. Chem. Interfacia/€/ectrochem. 1978, 703, 383-399.

755

(10) Watanabe, N.; Inoue, M. Anal. Chem. 1983. 55, 1016-1019. (11) Kok, W. Th.; Hanekamp, H. E.; Bos, P.; Frel, R. W. Anel. Chim. Acte 1982, 742, 31-45. (12) Kok, W. Th.; Brinkman, V. A. Th.; Frei, R. W. J . Chromatogr. 1983, 256, 17-26.

RECEIVED for review November 13, 1989. Accepted January 8, 1990. This work was supported by the National Science Foundation through EPSCoR Grant 86-10671-01 and by the University of Louisville College of Arts and Sciences.

Fiber-optic Glucose Sensor with Electrochemical Generation of Indicator Reagent Hari Gunasingham,* Chin-Huat Tan, and Jimmy K. L. Seow Department of Chemistry, National University of Singapore, Kent Ridge, Singapore 0511

An electrochemicallygenerated tetrathiafuivaieneredox m e diator provides a convenient and reversible indicator reagent for a glucose fiber-optic sensor. The fabrication and characterization of this sensor based on a thin-layer cell conflguration are described. The performance of the fiber-optic sensor is compared with that of an amperometric enzyme electrode in flow-Injection and steady-state analyses. The upper limit of the linear range in the flow-injection mode is about 12 mM, whereas in the steady-state mode it is about 3 mM. The sensor Is less prone to interfering species and dissolved oxygen. It can be used continuously for 2 days without signHicant loss of activity.

INTRODUCTION Although the efficacy of fiber-optic sensors has been demonstrated in many analytical applications (1-3),they do suffer from a number of practical limitations that have prevented their more widespread use. One significant limitation is the difficulty of finding suitable, long-lasting, and reversible indicator reagents. Most colorimetric and fluorometric reagent systems are inherently irreversible because the analytical reaction invariably involves the formation of a tightly bound complex or an irreversible adduct. The requirement of a suitable reversible reagent systems is a particular limitation when developing enzyme-based fiber-optic sensors. Consequently there have been only a few practical examples. There have been essentially three approaches to the development of such sensors: (i) The fiber-optic sensor directly measures enzymatic generation of a spectrophotometrically detectable product (4, 5). (ii) The product of the enzymatic reaction is detected through a secondary reaction resulting in a change in the optical properties of the indicator reagent. For example, a fiber-optic sensor for glucose has been developed where the principle of measurement is the change in the fluorescence of an oxygen-sensitive dye where fluorescence is quenched by oxygen (6). Another example is also a sensor for glucose determination based on chemiluminescence generated by reaction of peroxyoxalate with hydrogen peroxide formed in the enzymatic reaction (7). 0003-2700/90/0362-0755$02.50/0

(iii) A dehydrogenase enzyme is immobilized a t the end of a fiber-optic probe with fluorometric detection of consumed. or generated nicotinamide adenine dinucleotide for measurement of lactate or pyruvate (8). Here a fresh supply of nicotinamide adenine dinucleotide has to be provided in the external sample solution. Another route to developing an enzyme-based fiber-optic sensor that has not been hitherto explored is to make use of a redox mediator dye as the indicator reagent. Such dyes have been well developed in conventional enzymatic analysis (9) and dry chemistry systems (IO). The fundamental problem in regard to their application to fiber-optic sensors, however, is the fact that most redox dyes are irreversible. Consequently, it is necessary to provide a large reagent reservoir or provide a means of renewing the reagent. The subject of this paper concerns the fabrication and characterization of a reversible enzyme-based fiber-optic sensor for glucose. Reversibility is afforded through the electrochemical regeneration of an optically active redox mediator. In this work, use is made of tetrathiafulvalene, which is a good redox mediator for glucose oxidase and also possesses desirable optical properties (11). The usefulness of the glucose fiberoptic sensor is demonstrated in steady-state and flow-injection analysis.

EXPERIMENTAL SECTION Chemicals and Solutions. Tetrathiafulvalene(TTF)(>99%) was obtained from Fluka Chemie AG, Buchs/Switzerland. Glucose oxidase (GOX) (EC 1.1.3.4 Type I1 from Aspergillus niger),bovine serum albumin (BSA) (fractionV 9f3-99%albumin), glutaraldehyde, (GLA) (25% aqueous), and D-(+)-glUCOSewere from Sigma Chemical Co. (St. Louis, MO). All other reagents used were of AnalaR grade. All aqueous solutions were made up in 0.1 M pH 7.4 sodium phosphate buffer solution containing 0.01% (w/v) sodium azide prepared with Millipore Milli Q grade water. Glucose solutions were allowed to mutarotate overnight before use. Construction of Enzyme Electrode. Working electrodes were made from disks cut from 3 mm diameter platinum (Johnson Matthey, England) or glassy carbon (Tokai,Japan) rods. Electrical contact was made with a copper wire connected to one side of the disk with silver-loaded epoxy resin. The disk was then press fit into a Teflon holder and sealed with epoxy resin so that only a circular plane was exposed. Electrodes were polished with abrasive paper and then mirror polished with 0.3-fim diamond paste (K-5000, Kyoto, Japan). Prior to use, electrodes were 0 1990 American Chemical Society