Anal. Chem. 1988. 60. 2781-2786
transition time in the submillisecond region can be obtained by this technique. The transition time obtained with a 40-4 preset current is 885 ps. Since the geometry of the gold microwire electrode is uncertain, the calculation of the theoretical value for the transition time is not attempted. The effective scan rate for this chronopotentiogram is about 1100 VIS. Cyclic voltammograms a t similar scan rates were only obtained with an ultramicroelectrode after signal averaging (25). It also shows that the top and bottom portions of the transient are partially removed due to the decay of the curent transient to the preset level in less than one cycle time (125 ps). The cycle time and thus the background overcorrection could be minimized by collecting data at shorter time intervals with smaller potential pulses. We also note that the shape of the transients is sharper than described by eq 2. Similar behavior was predicted in potentiometric stripping analysis (26) and CCC with aq ultramicroelectrode (27). In conclusion, the very low background achieved by sampling the current on the pulse and by the use of a dynamic background correction is essential for the characterization of chronopotentiograms in the millisecond level. The analytical utility of SPPC is clearly demonstrated by the fast anodic stripping analysis of ppb-level Cd(I1) and Pb(I1) in aqueous solution. It appears that an accurate measurement of a submillisecond transition time may require a faster data acquisition frequency.
LITERATURE CITED (1) Ungane, J. J. J . Electroanal. Chem. 1980, 1 , 379. (2) Relnmuth, W. H. Anal. Chem. 1981, 3 3 , 485-487. (3) Anson. F. C. Anal. Chem. 1981, 3 3 , 1123-1124. (4) Laky, R. W.; McIntyre, J. D. E. J . Am. Chem. Soc. 1985, 8 7 ,
3806-38 12.
2781
Shults, W. D.; Haga, F. E.; Mueller, T. R.: Jones, H. C. Anal. Chem. 1985, 3 7 , 1415-1418. Sturrock, P. E.; Hughey, J. L.; Vaudreull, E.; O'Brlen, G. E. J . Electrochem. Soc.1975, 722, 1195-1200. Sturrock, P. E.; Vaudreull. 6. J . Electrochem. Soc. 1975, 722, 1311-1315. Sturrock, P. E.; Gibson, R. H. J . Electrochem. Soc. 1978, 123, 629-631. Kato, Y.; Yamada, A.; Yoshido, N.; Unoura, K.; Tanaka, N. Bull. Chem. Soc. Jpn. 1981, 54, 175-180. Schreiber, M. A.; Last, T. A. Anal. Chem. 1981, 53, 2095-2100. Hussam, A.; Gunaratna, G. Anal. Chem. 1988, 60, 503-507. Hance, G. W.; Kuwana, T. Anal. Chem. 1987. 59, 131-134. Sawyer, D. T.; Roberts, J. L., Jr. Experimental Electrochemistry for Chemlsts; Wlley: New York, 1974; p 77. Aubanel, E. E.; Oldham. K. 6. Byte February 1985, 207-218. Olmstead, M. L.; Nicholson, R. S. J . fhys. Chem. 1988, 72, 1650- 1656. de Vrles, W. T. J . Electroanal. Chem. Interfacial Electrochem. 1988, 77, 31-43. Rodgers, R. S.; Meites. L. J . Electroanal. Chem. Interfacial Electrochem. 1988, 16, 1-11. Bos, P. J . Electroanal. Chem. Interfacial Electrochem. 1972. 3 4 , 475-483. Perone, S. P.; Brumfleld, A. J . Electroanal. Chem. Interfacial Elecbochem. 1987, 13, 124-131. Hulllang, H.; Yagner, D.; Renman. L. Anal. Chlm. Acta 1987, 202, 117-122. Hulllang, H.; Yagner, D.; Renman, L. Anal. Chim. Acta 1987, 202, 123-129. Baranaskl, A. S. Anal. Chem. 1987, 59. 862-666. Kounaves, S. P.; O'Dea, J. J.; Chandrashekar, P.; Osteryoung. J. Anal. Chem. 1987, 59, 386-389. Aoki, K.; Aklmoto, K.; Tokuda, K.; Matsuda, J.; Osteryoung, J. J . E k tmanal. Chem. Intertacial Electrochem. 1985, 182, 281. Howell, J. 0.; Wlghtman, R. M. Anal. Chem. 1984, 56, 524-529. Hussam, A.; Coetzee, J. F. Anal. Chem. 1985, 5 7 , 581-583. Galus, 2.;Schenk, J. 0.; Adams. R. N. J . Ektroanal. Chem. Interfacial Ekctrmhem. 1982, 135, 1.
RECEIVED for review April 11,1988. Accepted September 23, 1988.
Covalent Enzyme Coupling on Cellulose Acetate Membranes for Glucose Sensor Development Robert Sternberg,' Dilbir S. Bindra? George S. Wilson,*Jand Daniel R. Th6venot*J Laboratoire de Biodectrochimie et d'Analyse du Milieu, U.F.R. de Sciences et Technologie, Universitt? Paris-Val de Marne, Avenue du GGnGral de Gaulle, 94010 Creteil Cedex, France, and Department of Chemistry, university of Kansas, Lawrence, Kansas 66045
Methods for hnmoMilzing glucose oxldase (GOx) on cellulose acetate (CA) membranes are compared. The optlmai method Involves covalent coupllng of bovlne serum albumin (BSA) to CA membrane 8nd a subsequent reaction of the membrane with GOx, whlch has prevlousiy been actlvated wHh an excess of p-benzoquinone. This coupling procedure Is fairly reproduclbie and allows the preparatlon of thin membranes (5-20 pm) showing high surface activities (1-3 U/cm2) whkh are s t a b over a perlod of 1-3 months. Electrochemkai and radloiabeilng experiments show that enzyme inactivation as a resuit of lmmobliization is negiiglbie. A good correlation between surface activity of membranes and their GOx load is observed.
INTRODUCTION The performance of an enzyme electrode is ultimately dependent on the ability of ita enzymatic membrane to sustain *Author to whom corres ondence should be directed. Universit6 Paris-Val Marne. University of Kansas.
&
0003-2700/8S/0360-2781$01.50/0
and protect the enzyme. Among the available methods of enzyme immobilization, four methods are currently used: adsorption followed by reticulation with bifunctional reagents such as glutaraldehyde ( I ) , covalent coupling between enzyme and activated support (2-5) or activated enzyme and support (6-8), and reversible immunological coupling (9). Those involving covalent coupling to solid supports are of great interest since they generally yield the best activity stabilities (10). Nevertheless two difficulties may be encountered low levels of activatable or activated surface groups on the support and denaturation of enzyme if covalent coupling is accomplished through functional groups of the enzyme which are essential to its catalytic activity. Highly active and stable membranes may be prepared by acyl azide activation of reconstituted collagen films ( 2 , l l ) . However such membranes have been found to be too thick and too fragile, especially at 37 "C, to be recommended for in vivo applications of enzyme electrodes. As cellulose acetate (CA) membranes of different thickness and permeability may easily be prepared by film casting or coating and because they exhibit significant permselectivity toward anions (12),we have studied their ability to support enzyme and be used for an in vivo implantable glucose sensor. 0 1988 American Chemical Society
2782
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
Flgure 1. Schematic dlagram of rotating membrane electrode: (a) rotating disk electrode shaft and electrical contact, (b) KeLF body, (c) threaded collar, (d) membrane support cap, (e) platinum electrode, and (f) membrane.
The literature is abundant with reports of glucose sensors and many of these are used routinely for in vitro clinical determinations. Despite a history of more than 25 years (131, no completely reliable implantable sensor has yet been developed (14).For such applications a miniaturized sensor is needed and the fabrication of such devices through multilayer film deposition is a major problem. I t is quite evident that miniaturized sensors will yield high sensitivity and stability only if the enzyme layer and the associated polymer layers can be deposited in a reproducible fashion. Commercially available membranes are not suitable for this purpose. The enzyme can be immobilized and protective polymer films deposited by dip coating (15). Because of the low success rate (
