AMI. Chem. l a w , 65, 523-528
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Amperometric Sensors for Peroxide, Choline, and Acetylcholine Based on Electron Transfer between Horseradish Peroxidase and a Redox Polymer Michael G. Garguilo, Nhan Huynh, Andrew Proctor, and Adrian C. Michael’ Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Amperometrlc sensors have been developed for hydrogen peroxide, choline, and acetylcholine by immobllizatlon of hofseradlrh peroxidase, (HRP), chollne oxidase, and acetylcholinesterase In a cross-linked redox polymer deporlted on glassy carbon electrodes. Peroxide sensors, prepared by knmobllltatlon of HRP alone, gave detection Hmits of 10 nM and a Ilnear response up to ca. 1 mM. Colmmoblllzatlon of HRP and glucose oxidase was used to establish the feadblllty of highly efficient blenzyme sensors at low substrate levels. Replacing glucose oxidase with choline oxidase produced wluon with submlcromolar detection llmits and a llnear rwponso up to 0.8 mM. Addition of acetylcholinesterase to the sensors generated a relatively small rerponse to acetylcMImthat demonstrates the fmadblltty of trlenzyme sensors. At low substrate concentratlons, no lo88 In sendtlvlty during a l d a y experiment was observed. The response times of these sensors are all le88 than 30 8 with 2-8 response times achieved In some cases.
INTRODUCTION Amperometric sensors based on enzymes continue to be a topic of interest due to their considerable potential for sensitivity and selectivity.’ Sensors that are compact and self-contained can be constructed by immobilizing enzymes in polymer films attached to solid electrodes. These are particularly well suited for miniaturization for in vivo use. In sensors based on oxidases, the analytical signal is obtained by amperometrically monitoring the formation of an enzymatic product such as hydrogen peroxide.2-4 consumption of a cosubstrate such as dioxygen,4+ or the recycling of a socalled artificial cosubstrate which mediates electron transfer between the oxidase and the electrode.74 Recently, a strategy for combiningthe role of immobilization and electron-transfer mediation into a single material, i.e. redox polymers, has been demonstrated.10-13 In these materials, the mediator redox ~~
(1) Gorton, L.; Csbregi, E.; Domlnguez, E.; EmnBus, J.; JonssonPettersson, G.; Marko-Varga, G.; Persson, B. Anal. Chin.Acta 1991,250, 2rn-248. - - - - .-. (2) Galiatsatos, C.; Ikariyama, Y .;Mark, J. E.; Heineman, W. R. Biosens. Bioelect. 1990,5, 47-61. (3) Fortier, G.;BBliveau,R.;Leblond, E.;BBlanaer,D. A n d L e t t . 1990, 23,1607-1619. (4) Villarta. R. H.: Cunnineham. D. D.: Guilbault. G. G. Talanta 1991. 38,‘44-55. ‘ (5) Tse, P. H. S.;Gough, D. A. Anal. Chem. 1987,59, 2339-2344. (6) Gough, D. A.; Lucisano, J. Y.; Tse, P. H. S.Anal. Chem. 1986,57, 2351-2357. I
I
( 7 ) Hale, P. D.; Boguslavsky, L. I.; Karan, H. I.; Lan, H. S.; Okamoto, Y.; Skotheim, T. A. Anal. Chim.Acta 1991,248,155-161. (8) Crumbliss, A. L.; Hill, H. A. 0.;Page, D. 3. J. Electroanal. Chem. Interfacial Electrochem. 1986,206, 327-331. (9) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. 0.;Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chern. 1984,56, 667-671. (10) Hale, P. D.; Lee, H. S.; Okamoto, Y.; Skotheim, T. A. Anal. Lett. 1991,24, 345-356. 0003-2700/93/0365-0523$04.00/0
couple is nondiffusing, so electron transport occurs by electron hopping.14J5 There are several advantages to the use of a nondiffusing mediator; most notably, the mediator is prevented from leaching out of the sensor membrane, thereby eliminating the need for a containment membrane while simultaneously improving the longevity of the sensor. The use of artificial cosubstrates is suitable as long as facile electron transfer with the oxidase of interest can be achieved. This is not always possible as some oxidases, choline oxidase for example, exhibit high cosubstrate specificity. In this paper we report on the preparation of a choline sensor based on a nondiffusing mediator and choline oxidase in which a second enzyme, horseradish peroxidase, is used to link the mediator and the oxidase.16-18 In this way the advantages of the nondiffusingmediator are still realized, albeit by an indirect scheme. The development of a choline sensor is significant in several respects, but we are most interested in choline’s importance as a metabolite of acetylcholine, a neurotransmitter in the central and peripheral nervous systems. In addition to measurement of choline, such a sensor could also provide the basis for measurement of the neurotransmitter directly by incorporation of acetylcholinesterase into the sensor for in situ conversion of acetylcholine to choline. Furthermore, a choline sensor would be useful for the determinationof cholinesterase activity in various body fluids, such as blood and amniotic fluid, which are important diagnostic procedures. Although these enzymes have long been used in analytical devices, such as postcolumn reactors for HPLC,1*21 only a few reports of choline and acetycholine sensors exist.22-26 The sensors we are developing involve the use of as many as three consecutive enzymatic reactions to generate the amperometric response. In these devices the relative conversion efficiency of each enzymaticstep must be considered. To this end, we have also constructedbienzyme glucose sensors (11) Gregg, B. A.; Heller, A. Anal. Chern. 1990,62,258-263. (12) Hale, P. D.; Inagaki, T.; Lee, H. S.; Karan, H. I.; Okamoto, Y.; Skotheim, T. A. Anal. Chim.Acta 1990,228, 31-37. (13) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1989,111, 2357-2358. (14) Dalton, E. F.; Surridge, N. A.; Jernigan, J. C.; Wilbourn, K. 0.; Facci, J. S.; Murray, R. W. Chem. Phys. 1990,141,143-157. (15) Chidsey, C. E. D.; Murray, R. W. Science 1986,231, 25-31. (16) Kulys, J.; Schmid, R. D. Biosens. Bioelect. 1991, 6, 43-48. (17) Tatauma, T.; Watanabe, T. Anal. Chim.Acta 1991,242,85-89.
(18)Kulys, J. J.; LaurinaviEiua, V. S.A.;Pealiakiene,M. V.; GureviEiene, V. V. Anal. Chim.Acta 1983,148, 13-18. (19) Gunaratna. P. C.: Wilson. G. S. Anal. Chem. 1990.62.402-407. (20) Damsma, G.; Westerink, ‘B.H. C.; Horn, A. S. J.’ Neurochern. 1985.45. 1649-1652.
(21) Potter, P. E.; Meek, J. L.; Neff, N. H. J. Neurochem. 1983,41, 188-194. (22) Tamiya, E.; Sugiura, Y.; Navera, E. N.; Mizoshita, S.; Nakajima, K.; Akiyama, A.; Karube, I. Anal. Chim.Acta 1991, 251, 129-134. (23) Kawagoe, J. L.; Niehaus, N. E.; Wightman, R. M. Anal. Chem. 1991,63,2961-2965. (24) Morelis, R. M.; Coulet, P. R. Anal. Chim. Acta 1990,231,27-32. (25) Marty, J. L.; Sode, K.; Karube, I. Anal. Chim. Acta 1989, 228, 49-53. (26) Hale, P. D.; Wightman, R. M. Mol. Cryst. Lip. Cryst. 1988,160, 269-279. 0 1993 Amerlcan Chemical Society
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because, in contrast to choline oxidase, glucose oxidase undergoes electron transfer with a variety of redox mediators. With glucose oxidase and HRP coimmobilized in a redox polymer, glucose can be detected via both a monoenzyme scheme (Scheme I) and a bienzyme scheme (Scheme 11).With
Scheme I
glucose + Med(ox)
-
GOx
Med(red)
gluconolactone
-
Med(ox)
+ Med(red)
+ e-
Scheme I1
glucose
H,O,
+ 0,
-
GOx
gluconolactone
+ 2H' + 2Med(red) Med(ox) + e-
-
HRP
-
2H,O
+ H202 + 2Med(ox)
Med(red)
a single sensor, therefore, it is possible to switch between the two detection schemes simply by adjusting the electrode potential with respect to the E l p of the mediator. At positive potentials, the monoenzyme scheme is invoked, whereas at negative potentials the bienzyme scheme is invoked. Thus, the HRP/GOx sensor is a convenient model system for comparison of mono- and bienzyme sensors. . EXPERIMENTAL SECTION Reagents. Acetylcholinesterase type V-S (electriceel),choline oxidase (Alcaligenesspecies), peroxidase type I1 (horseradish), glucose oxidase type I1 (Aspergillus niger), ferrocenemonocarboxylic acid, 4-methylcatechol, acetylcholine chloride, choline chloride, D-(+)-glucose,and 2-bromoethylamine hydrobromide were obtained from Sigma (St. Louis, MO). Hydrogen peroxide (305% aqueous solution) was obtained from Mallinckrodt (Paris, KY). K20sC16was obtained from Johnson Matthey (Ward Hill, MA). Poly(4-vinylpyridine) (PVP, MW 50 000) and poly(ethylene glycol 400 diglycidyl ether) (PEG) were purchased from Polysciences (Warrington, PA). The above chemicals were used as received. Phosphate-buffered saline (PBS), pH 7.4, was prepared with ultrapure water (Nanopure) and used for all electrochemical experiments and preparation of standard solutions. Glucose solutions were allowed to reach equilibrium between the a- and p-anomers before use (ca. 24 h). The crosslinkable redox polymer (PVP-Os(bpy)&l;bpy = 2,2'-bipyridine) was synthesized according to the published procedure.27 The final chlorideform of the redox polymer was additionally washed with acetone. The Os(bpy)zpyCl(py = pyridine) was similarly prepared by replacing PVP with py. Electrochemistry. Conventional 3-mm-diameter glassy carbon electrodes were polished with 5.0-, LO-, and 0.3-pm particle size alumina (Buehler, Lake Bluff, IL), rinsed thoroughly with ultrapure water between each polishing step, and finallysonicated, rinsed again, and dried in air before use. Microcylinder electrodes, ca. 100 pm in length, were made from 7-pm-diameter carbon fibers (T-300,Union Carbide, New York, NY) sealed with Spurr epoxy (Polysciences)into glass capillary tubes.Zs Cyclic voltammetry and constant potential amperometry were performed with apparatus controlled by an 80386-based personal computer (Twinhead SS-600/25C) with an analog interface (Labmaster DMA, Scientific Solutions, Solon, OH) and software developed in-house. A locally constructed potentiostat interface and a picoammeter (Keithley 427, Cleveland, OH) were used to control the applied potential and amplify the electrode current, respectively. (27) Gregg, B. A.; Heller, A. J . Phys. Chem. 1991,95,5970-5975. (281 Michael, A. C.; Justice, J. B., Jr. Anal. Chem. 1987,59,405-410.
Cyclic voltammetry was performed in stationary, air-equilibrated PBS unless stated otherwise. Amperometric measurements were made in a flow injection system equipped with a pneumatically actuated injection valve (Rheodyne 50/5701, Cotati, CA) which was controlled by the computer via a locally constructed interface. During amperometric experiments, the current offset control of the picoammeter was used to suppress the background current so that high gain could be used while avoiding dynamic range limitations. Plain or modified glassy carbon electrodes were mounted in a wall-jet configuration in a custom-madeflow cell. A flow of 1.2 mL/min through the system was created by gravity feed from an elevated reservoir containing air-equilibrated PBS. All experiments were performed with a saturated calomel reference electrode (SCE, Fisher) and a platinum auxiliary electrode. Electrode Modification. Amperometric sensors were prepared by modifying glassy carbon electrodes with an aqueous mixture of the redox polymer, cross-linker (PEG),and appropriate enzymes (see Results and Discussion for detailed recipes). A 5.0-pL aliquot of the mixture was pipetted onto the electrodes andspreadouttocompletelycoverthecarbonsurface.Thesensors were cured in air for at least 48 h following which they were soaked in ultrapure water for 15 min and redried in air for an additional 75 min before use. The final soaking and redrying improved the general performance of the modified electrodes. Spectroscopy. A Hewlett-Packard 8450 diode array spectrophotometer was used in all UV-vis absorbance measurements. Spectra obtained for [Os(bpy)zClzlCland Os(bpy)&12 compared well to those in the literature.29 UV-vis absorbance was also used to determine the concentration of GOx and HRP in aqueous solutions using molar extinction coefficients of 1.31 X 104 M-l cm-l at 450 nm and 1.03X lo6M-I cm-l at 403 nm,30respectively. Absorbance analysisof GOx waa performed with nitrogen-purged solutions. ESCA spectra were obtained, via a custom built interface to an IBM PC compatible, on a Leybold Heraeus LHS10photoelectron spectrometer using unmonochromatized A1Ka X-rays (1486.6 eV) at a power of 240 W (12 kV, 20 mA). Data were acquired in the constant analyzer pass energy mode (U= 100 eV). Pressure in the analysis chamber was maintained 0.99). The inset in Figure 2 shows the low end of this concentration range. At a concentration of 10 nM H202 a signal to noise ratio of ca. 3 is obtained. This detection limit is about 3 orders of magnitude lower than that which has been previously reported for monoenzyme glucose sensorsbased on the same polymeric mediator.43 The improved detection limit obtained with peroxide seneors implies that the elevated rate constant measured for electron transfer between HRP and the soluble mediator, compared to Gox and the soluble mediator,remains operative in the cross-linked polymer. Mono- and Bienzyme Glucose Sensors. Because both GOx and HRP participate in electron transfer with the redox polymer, these enzymes can be coimmobilized on a single sensor which can be used to detect glucose in two modes, as described by Schemes I and 11. This provides a convenient method for appraising the performance of HRP-based multienzyme sensors. Figure 3 shows the response of a glucose sensor prepared by depositing 1.85 pg of redox polymer, 1.11 pg of cross-linker, 7.27 pg of GOx, and 10.9 pg of HRP onto a glassy carbon electrode. Figure 3a compares the temporal response of the sensor operated in the monenzyme mode (Le. at positive potentials) and the bienzyme mode (i.e. at negative (42) Laidler, K. J. Introduction t o the Chemistry of Enzymes; McGraw-Hill: New York, 1954; p 109. (43) Pishko, M. V.; Michael, A. C.; Heller, A. Anal. Chem. 1991, 63, 2260-2272.
0
10
20 30 40
Glucose (mM)
(a)Comperlson of glucose sensor responseto 5 mMsubstrate injections obtained in monoenzyme (EA= 0.45 V) and blenzyme (EA = 0.0 V) mode. (b) Comparison of glucose callbration cwves obtained in the mono- and bienzyme modes. Figure 3.
potentials) in the flow system during injection of a 10-8bolus of 5 mM glucose. The striking feature of Figure 3a is the substantially faster response of the sensor when operated in the bienzyme mode. This is attributed to the elimination of the relatively slow electron transfer between GOx and the redox polymer when the sensor is operated cathodically (see Scheme 11, which shows that electron transfer between GOx and the mediator is not involved when the sensor is operated in the bienzyme mode). Figure 3b shows glucose calibration curves obtained in the mono- and bienzyme mode (currenta are reported as the absolute value to facilitate direct comparison). Below ca. 5 mM glucose the two curves are identical, demonstrating that the bienzyme detection mode is highly efficient in this concentration range. This behavior is ideal for our planned choline and acetylcholine sensors because the biologically significant concentration range for these substrates is micromolar rather than millimolar. As the glucose concentration increases above 5 mM, the calibration curves in Figure 3b deviate with the bienzyme sensor yielding less signal. Beyond 30 mM glucose the influence of the substrate inhibition of HRP is again observed, with the bienzyme glucose calibration curve showing a negative slope. With the bienzyme sensor the substrate inhibition phenomenon is observed at such high concentrations of primary substrate (Le. glucose in this case) that it is unlikely to ever cause significant difficulty. BienzymeCholineSensors. Due to the high cosubstrate selectivity of ChOx for dioxygen, electron transfer between ChOx and the Os-based redox complex, either in solution or in the polymeric form, does not occur. Thus, monoenzyme choline sensors, analogous to those reported for several other oxidase enzyme substrates, are not feasible. However, coimmobilization of ChOx and HRP allows detection of choline via a sequence of reactions analogous to that shown in Scheme I1 for glucose. In contrast to the glucose sensor, the choline sensor can only be operated in the bienzyme mode. Sensors were mounted in the flow injection system and held at a constant potential of 4.1 V vs SCE. Choline standards were loaded into the sample loop of the injection valve which was then placed in the inject mode for 30 8. Because of the
ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1993 400
I-
-
1.86 pg Polymer
I
527
300
n = 3
2 225 -
C
c
m
L
5
150
u
-
0
5
g
s
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0 ~
0
2
4
6
8
10
I
Chox/HRP W p g )
Flguro 4. Optlmkatlon of blenzyme chdlne sensors at a potentlal of -0.1 V vs SCE. The current measured In response to 30 mM chdlne Isshown as a functionof the reclpeusedfor preparationof the sensors. The ChOXHRP enzyme ratio Is based on welghts of the as recehred sdld ensamples.
variable response time of the choline sensors, calibration data were collected near the end of the 30-8 injection (response time discused further, below). In order to establish an optimum recipe for the choline sensors, a series of sensors were prepared and tested at saturating substrate concentrations, i.e. 30 mM choline. The current measured at 30 mM choline, Zm, using a series of sensors prepared in various ways is shown in Figure 4. All the sensorsshown were prepared with 1.85pg of redox polymer and 1.11pg of cross-linker but varying amounts of ChOx and HRP, as indicated in the figure. The figure shows that, for a particular quantity of immobilized ChOx, increasing the amount of HRP on the sensor increases Z-, but that if too much HRP is used then,,Z decreases. Presumably, with small quantities of HRP there is a low probability that the HzOzproduced by ChOx will be reduced before diffusing out of the sensor. With excessively large quantities of HRP electron transport through the polymer is hindered, which is detrimental to the performance of the sensor. This effect is evidenced by an increase in the peak separation and the onset of hysteresis observed in the slow scan voltammogram of the redox polymer in the presence of excessive quantities of enzyme (not shown). Similar considerations define the optimum quantity of ChOx that should be used. As Figure 4 shows, increasing the quantity of ChOx from 0.93 to 1.85 pg caused an increase in Z-. However, further increases in the quantity of ChOx used produced either similar or smaller Zm, values. Thus, the point in Figure 4 marked by an asterisk appears to define the choline sensor with optimum sensitivity. Figure 5 shows a calibration curve obtained using an o p t i m i i choline sensor. The optimized sensor exhibited a K m of ca. 0.8mM and gave a linear response below 100 pM choline. The signal to noise ratio obtained at 1pM choline was ca. 5. Previous estimates place the concentration of choline in the extracellular fluid of brain tissue at ca. 10 pM.44 As the inset in Figure 5 shows, the optimized choline sensor functions very well in this biologically relevant concentration range. The previous paragraphs were concerned solely with the sensitivity of choline sensors. An additional important operating parameter of any sensor is ita response time. This is especially important for choline sensors intended, eventually, for use in neurochemical analysis, where experiments involving rapid fluctuations in choline concentration are anticipated. Typically, the response time of these sensors is (44)Brehm, R.;Lindmar, R.;Ldffelholz, K.J. Neurochern. 1987,48, 1480-1485.
250
0
500
750
1000
Choline (pM)
Figuro 5. Callbration cwves obtalned wlth an optimized blenzyme &dine sensor set at an applled potentlalof -0.1 V vs SCE. The Inset shows the response to 1-10 pM choline. 0.15
1 1 .OmM Choline
0
15
30
Time
5+
C
1.50
.
0'75
.
0.00
L
(3
-0.75
a
45
(SI
'
.
-1.50
600
300
0
E (rnV vs. SCE) Flguro 6. (a) Response tlme of a blenzyme chollne sensor dwing an lnjadon of 1 mM substrate. Electrode potential is at -0.1 V vs SCE. (b) Cyclic voltammogram (50 mV/s) of the sensor In a.
less than 20 s (10-90% of steady-state signal) but is variable. The response time of the sensor is related to the facility of electron transport through the cross-linked polymer. Figure 6 shows results obtained with a rapidly responding optimized choline sensor, prepared with same recipe used in Figure 4. As Figure 6a shows, this sensor exhibited a response time of about 2 s during an injection of 1.0 mM choline. Figure 6b shows the cyclic voltammogram of the redox polymer obtained at 50 mV/s in the absence of choline. The highly symmetrical voltammogram is characteristic of facile oxidation and reduction of the electroactive polymer. Sensors that exhibit increased hysteresis in their cyclic voltammogram alsoexhibit increased amperometric response times to enzyme substrate. Trienzyme Acetylcholine Sensors. Because acetylche line is converted to choline by the actions of AChE, the possibility of developing a trienzyme acetylcholine sensor exists. Figure 7 shows results obtained with electrodes modified with 3.7 pg of redox polymer, 1.11pg of cross-linker,
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1993
0
10
20
30
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50
Time (s) b
1 .OrnM Ach
0
10
20
30
40
50
Time (s) Fbwo 7. (a) The response of a bienzyme chollne sensor before and after the^ addition of 1 mg/mL AChE to a fresh ACh solution. (b) Same as in (a) with a trienzyme acetylcholine sensor. Electrodes are at a constant potential of -0.1 V vs SCE.
5.55 pg of ChOx, and 2.78 pg of HRP, both with (Figure 7a)
and without (Figure 7b) an additional 0.92 pg of AChE. The figure shows the response obtained in the flow system at an applied potential of -0.1 V vs SCE. In these experiments it was important to control for the spontaneous hydrolysis of ACh to Ch in aqueous pH 7.4 buffer, which occurs to a noticeable extent in a matter of minutes following preparation of solutions. Figure 7a shows the response of a choline sensor to a freshly prepared 1.0 mM ACh solution less than 1-min old. The virtual absence of a response demonstrates that little Ch contamination of very fresh ACh solutions occurs. The fresh ACh solution was analyzed again with the choline sensor following addition to AChE, which quickly produced choline in the sample solution, resulting in the large amplitude response shown in Figure 7a. This verified the viability of the commercial AChE preparation in hand and double checked the performance of the sensor. A similar sequence of experiments with AChE incorporated into the sensor itself produced the results shown in Figure 7b. The measurable response to the very fresh ACh solution demonstrates that the trienzyme approach is operative and that AChE has been successfully incorporated into the cross-linked polymer.
Comparison to the choline response obtained after addition of AChE to the sample solution shows that the ACh sensors we have prepared so far operate at low efficiency. This could either be caused by low activity of the immobilized AChE or inefficient collection of choline by coimmobilized ChOx. Low AChE activity could be a result of inhibition by the redox polymer since pyridinium salts are known AChE inhibitors. Thus, while the PVP-based redox polymer has been very useful for peroxide and choline sensors, it may be less than optimal for immobilization of AChE. Finally, comparison of Figure 7a and 7b shows that incorporation of AChE into the cross-linked polymers has little effect on the sensor's choline response.
CONCLUSIONS The peroxide and choline Sensors prepared so far exhibit suitable sensitivities and response times for application to biological problems. With miniaturization equivalentto that demonstrated for glucose sensors, in vivo applications can also be envisioned. In vivo sensing of peroxide, for example, could shed further light on the involvement of thiscytotoxin in neurodegenerativedisorders, such as Parkinson's disease. Peroxide arising in vivo from adventitious autoxidation processes has long been suspected but remains an open question. Likewise, choline sensors for in vivo use would provide a new tool for investigation of cholinergic systems of the central and peripheral nervous system. Prior to in vivo use, however, well-known sources of interference for amperometric sensors must be addressed. One of these is oxygen. The choline sensors we have made utilize oxygen in the scheme for substrate detection. Thus, they may be sensitive to fluctuations in oxygen tension in their surroundings. In the case of oxygen-sensitive glucose sensors, this was solved with barrier membraneswhich reduce the amount of glucose that reaches the sensor to values below its apparent K,. The apparent Kmsof the sensors reported here already exceed the physiologically relevant concentrations of choline, thus oxygen sensitivity is anticipated to be less of problem in this work. An additional interferant of concern is ascorbate, which is easily oxidizable and present at high concentrations in the central nervous system. The sensors described here, however, are operated at potentials too negative to cause direct oxidation of ascorbate at the carbon electrode, which greatly reduces the capacity of ascorbate to interfere with these sensors. Investigations of the effects of oxygen and ascorbate are in progress.
ACKNOWLEDGMENT This work was financially supported by the University of Pittsburgh. N.H. was a NSF-REU summer student. RECE~VED for review June 22, 1992. Accepted November 13, 1992.
