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A M . Chem. 1002, 64, 646-651
and on the surface concentration of octadecylviologen mediator.
CONCLUSIONS We demonstrated in this report that enzyme immobilization in amphiphilic bilayer assemblies is a viable strategy not only for hydrophilic enzymes such as glucose oxidase but also for more hydrophobic enzymes which traditionally have been more difficult to work with. Immobilization of D. gigas hydrogenase in a bilayer assembly consisting of OTS and C18MV2+molecules involves hydrophobic interactions which lead to the binding of 3 pmol/cm2 of the enzyme in the plane of the bilayer. This corresponds to 50% of the full monolayer coverage. The enzyme immobilization is independent of pH in the range 4.0-9.0. It is strongly inhibited by 0.01 M C1ions and is associated with partial desorption of C18MV2+, suggesting competing interaction on the OTS surface. In spite of the fact that some uncertainties remain concerning mechanistic details of the enzyme interactions with the OTS/ C18MV2+bilayer, this scheme constitutes a general approach of enzyme immobilization at electrodes. It is apparent that success of this approach depends, in general, on self-segregation phenomenon, which is ubiquitous in biological systems, and on the well-known amphiphilic character of bilayer assemblies. This provides enzyme molecules with a possibility of “selecting” their own milieu most compatible with their structure. We are hopeful that the strategy developed here opens a possibility of extending such schemes to integral membrane enzymes in order to construct electrode-enzyme systems useful in analytical measurements. We also showed that efficient coupling of the enzymatic activity to the electrode surface can be accomplished via mobile electron-transfer mediator, C18MV2+,and that the resulting electrocatalytic system functions under both potentiometric and steady-state voltammetric conditions. Rapid potentiometric response of the enzymemodified electrode was found to be independent of the level of enzyme activity and the surface concentration of the electron-transfer mediator. These features make this and other analogous enzymatic systems attractive candidates in the development of selective potentiometric sensors.
ACKNOWLEDGMENT M.M. thanks the US. National Science Foundation for partial support of this collaborative research under Grants CHE-8807846 and CHE-9108378. REFERENCES (1) Updike, S. J.; Hicks, G. P. Netwe 1987, 214, 986. (2) Naparstek, A.; Romette, J. L.; Kemevez, J. P.; Thomas, D. Netwe 1974, 249, 490. (3) Bodilbn. C.; Bowgeds, J. P.; Thomas, D. J . Am. Chem. Soc. 1960, 102, 4231. (4) Cess, A. E. G.; Davis, G.; Francis, Q. D.; Hill, H. A. 0.; Aston, W. J.; Higpins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56, 667. (5) Foulds, N. C.; Lowe, C. R. J . Chem. Soc.,Farady Trans. 1988, 8 2 , 1256. (6) Pandey, P. C. J . Chem. Soc.. Faraday Tfans. 1988, 64, 2259. (7) Sheolln, M.; Huaiguo, X.; Btdong, a. J . Ekicfroenal. Chem. Interfedel Ekictrochem. 1991, 304, 7. (8) W n l , Y.; Heiler, A. J . phys. Chem. 1987, 91, 1285. (9) Heller, A. Acc. Chem. Res. 1890, 2 3 , 128. (10) BodW, C.; Mejda, M. J . Am. Chem. Soc. 1990, 112, 1795. (11) MI#er. C. J.; Mepa, M. J . Electroenal. Chem. IntwfechlElecirochcwn. 1988, 207, 49. (12) Miller, C. J.; Wldrig, C. A.; Charych. D. H.; Majda, M. J . phys. Chem. 1988, 92, 1928. (13) 008s. C. A.: Miller, C. J.; Mapa, M. J . phys. Chem. 1988, 92, 1937. (14) Hatchlkian, E. C.; Bruschl, M.; Legaii, J. Blochem. skphvs. Res. Commun. 1970. 82. 451. (15) VoordOuw. Q.; k%, N. K.; Legal, J.; C h i , E.; Peck, H. D.: Przybyle, A. E. J . Becter(0l. 1989. 171. 2804. (16) MajcJa, M. I n Klnethx and Cak@k In M i c r o h e t Systems; ~ Qratzel, M., Kalyanasunduram, K., Ed.; Surfactant Science Serbs Vol. 3 8 M. Dekker: New York, 1991; Chapter 8, p 227. (17) a s s , C. A.; Mapa, M. J . Ekictroanal. Chem. Interfadel Ekicb.0chem. 1991, 300, 377. (18) Plleni. M. P.; Braun, A. M.; Qratzel, M. Umtochem. Photobkl. 1980, 31. 423. (19) Itaya, K.; Sugawara, S.; Aral, K.; Sako, S. J . Chem. Eng. Jpn. 1984, 17, 514. (20) Furneaux, R. C.; Rigby, W. R.; Davldson, A. P. Netvo 1989, 337, 147. (21) LeCiall, J.; Ljungdehi, P. 0.; M a , I.; Peck, H. D.; Xavler, A. V.; Mowa, J. J. 0.; Teixera, M.; Huynh, B. H.; DerVattanlan, D. V. Elochem. Biophys. Res. Commun. 1982, 106, 610. (22) Femender, V. M.; Aguirre, R.; Hatchkian, E. C. 6 M m . Bbphys. Acta 1984, 790, 1. (23) Llssolo, T.; Puhrln, S.; Thomas, D. J . W .Chem. 1984, 259, 11725. (24) Megs, R. M.; Bourdlllon, C. J . Bkl. Chem. 1986, 280, 14701. 1972, 39, (25) Trasattl, S. J . Electroanel. Chem. I n t e r f e c l e l E k t ” . 163.
RECEIVED for review July 22,1991. Accepted November 27, 1991.
A Hole Can Serve as a Microelectrode Keith B.Oldham Trent University, Peterborough, Canada K9J 7B8
Imperfect mlcrooktrode fabrkatlon may lead to an undetected cowtrlctlonwparatlng the electrode proper from the bulk rdutlon. The “lagoon” that k thereby fonnd has vdtammotrk comoquencos that are examlnd In thk artlde. A mathomatical m0d.l reverb mtk effect of tho lagoon on the dHhmIon4lmtt.d deadystate current but shows that ole& t m t r a d e r rate constants may be ml8calCulat.d by several 0rd.n of magnitude. The orlfke wparathg the lagoon from the bulk ekctrdyte acqulros some of tho 1eatur.r of a ”ekckod.. Steady4ate effects kmtlclal to analytkal voltammotry may ro8ult from the preaence of lagoons.
INTRODUCTION Though still in their experimental infancy, the applications of microelectrode voltammetry, and especially steady-state 0003-2700/92/0364-0646$03.00/0
microelectrode voltammetry, to chemical analysis and to quantify the kinetics of fast electron-transfer reactions are well understood. These topics have been the subject of numerous publications, all but the most recent of which are summarized in four compendia.’+ It has been shown5 that the steady-state voltammetric current I flowing to a microelectrodeof any size or shape obeys the universal equation
Here Idif is the value that I adopts when the reaction rate is controlled solely by the rate of diffusion of the reactant to the electrode; Ikin is the value that I would have if the reaction rate were controlled solely by the kinetics of the electrontransfer reaction; and h is a heterogeneity function whose 0 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 6, MARCH 15, 1992
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2r, Flgure 1. Microelectrode con8tructed by covdng a conductor wlth an insulating layer, kavlng a small aperture.
Flgure 2. Lagoon formed betweem the aperture and the conductor.
significance need not concem us here, except to note that it cannot be negative. E is the eledrode potential measured with respect to the “reversible halfwave potential” and other symbole are defied in Appendix A (note that the signs adopted correspond to the electrode reaction being an oxidation). The kinetic information, if any, present in the steady-state current Z is contained exclusively in the Zb term of eq 1. It is evident, therefore, that reliable kinetic data may be extracted from experimental measurements of the steady-state current only if Z h is smaller than (or, at worst, comparable with) Idif.The full expressions for Idit and Zb, namely
Zdif = vnFDcbfi (2) (where Y is a number, the accessibility fador: reflecting the microelectrode’s shape) and Zb = nFcbkAexp{(l - a)nFE/RTJ (3) show that Zb is proportional to A, the electrode area, whereas Zdir is proportional to d A . Hence, decreasing the size of the microelectrode improves the chances of determining kinetic data. This consideration is the prime motivation behind the search for methods of making ever smaller electrodes. One way of fabricating a microelectrode is to almost cover a conductor with an insulating coating, leaving a tiny uncovered portion, as illustrated in Figure 1. For example, Penner et al! constructed microelectrodes by almost totally coating etched samples of platinum-iridium wire with molten glass. These electrodes were so small that they could not be inspected, or even detected, microscopically, their existence and size being inferred from their voltammetric properties. Thus,the effective radius rd of such a structure was found by treating it as an inlaid disk microelectrode and applying the Saito equation’
2a Figwe a. Model of a lagooned microelectrode.
rameters (k and CY in eq 3);this involves considering the interplay between Zb and Ida.Later in the article some desirable features of lagooned microelectrodes will be described.
TEE MODEL Figure 3 shows the model adopted to facilitate theoretical study of a solution/orifke/lagoon/condudor microelectrode. The coating is treated as an infinitesimally thick insulating sheet perforated by a circular hole of radius rh. The lagoon has the shape of an oblate hemispheroid (with major and minor semiaxes a and b) and is symmetrically positioned behind the hole. The three characteristic lengths, rh, a, and b, are not mutually independent but are correlated by the Pythagorean relationship a2 = b2 rE (5) Further to delineate the geomet$ of the oblate hemispheroidal lagoon, we may note that its volume is 2ua2b/3,and that it makes contact over an area
+
A = ,a2
with the condudor, over an area *b2 with the insulatingsheet, and across an orifice of area q2 with the extemd solution. One may erect a cylindrical coordinate system (r,z, 6) with ita origin at the center of the hole. The z-axis,r = 0, is perpendicular to the sheet with increasing positive values of z corresponding to progress into the electrolyte solution. Our model has total rotational symmetry about the z-axis,so that the B coordinate plays only a minor role in what follows. The oblate hemispheroidal interface between the conductor and the solution may be assigned cylindrical coordinates (R,2, 0) where
Z = - b d m which is the v = 4 / d u version of eq 2. Incomplete adheaion between the condudor and the mating is a possible danger with this fabrication method. The conductor would then not be in contact with the orifice in the insulatingcoating. Instead a “lagoon” could exist between the ofice and the eledrode iteelf, aa illustrated in Figure 2. Such a lagoon might form during the fabrication process itself, perhaps around a submicroscopic pit in the condudor’s surface, or it could arise from a subsequent local breakdown of the coating/conductor seal. One purpose of the present article is to model an electrode with features similar to those in Figure 2, and so predict the voltammetric consequences of lagooning. There are two, largely unrelated, consequences which will be considered separately. The f i t is the effect on Zm, for there is certainly no reason to expect the Saito equation to be obeyed by a lagooned microelectrode. The second consequence is on the use of steady-state voltammetry to measure the kinetic pa-
*ab2 b +ucosh rh U
OlRla
-blZIO
(7)
Ol0
