Anal. Chem. 1982, 5 4 , 777-782
analytically useful. It would, nevertheless, be more desirable analytically that dopamine be more strongly catalyzed than ascorbic acid (the reverw of Figure 10). Lastly, we call attention to the anomalous difference between the oxidation potentials of dopamine and ascorbic acid and for the redox sites in the iridium metalated film. In an uncomplicated EC electrocatalytic reaction mechanism, the dopamine and ascorbic acid reactions should occur at potentials close to that for the ostensible catalyst wave. Again, uncertainty in the chemical makeup of the film obviates a molecular interpretation of this problem. We should note that one way the film could remain inactive until nearly all of the catalyst sites become oxidized would be a reactivity strongly dependent on the degree of electrostatic cross-linking within the film. Oxidation of the anionic catalyst sites would relieve the electrostatic interactions by lowering the charge on those sites. ACKNOWLEDGMENT Helpful discussions wiith B. P. Sullivan are gratefully acknowledged. ILXTEBATURE CITED Murray, R. W. Acc. Chem. Res. 1980, 13, 135. Oyama, N.; Anson, F. C. Anal. Chem. 1980, 52. 1192. Shigehara, K.; Oyama, N.; Anson, F. C. Inorg. Chem. 1981, 20. 518. Collman, J. P.; Uenlseviclh, P.; Konai, Y.; Marrocco, M.; Koval, C.; Anson, F. C. J. Am. Chem. SOC. 1980, 102,6027. Bettelheirn, A.; Chan, R. J. H.; Kuwana, T. J. flectroanal. Chem. 1980, 110, 93. Degrand, C.; Miller, L. L. J . Am. Chem. SOC. 1980, 102,5728. CaivErt, J. M.; Meyer, T. Y. Inorg. Chem. 1981, 20,27. Abruna, H. D.; Walsh, J. I..; Meyer, T. J.; Murray, R. W. J. Am. Chem. SOC. 1980, 102, 3272. Rocklin, R. D.; Murray, R. W. J. Phys. Chem. 1981, 85, 2104. Ikeda, T.; LeMner, C. R.; Murray, R. W. J , Am. Chem. Soc., In press.
777
(11) Lewls, N. S.;Bocarsly, A. B.; Wrighton, M. S. J. Phys. Chem. 1980, 84, 2033. (12) Andrieux, C. P.; Saveant, J. M. J. E/ectroanal. Chem. 1978, 93, 163. (13) Andrieux, C. P.; Dumas-Bouchiat, J. M.; Saveant, J. M. J. Electroanal. Chem. 1080, 114, 159. (14) Anson, F. C. J. Phys. Chem. 1980, 8 4 , 3336. (15) Daum, P.; Murray, R. W. J. Phys. Chem. 1981, 85,389. (16) Murray, R. W. Phllos. Trans. R . SOC. London, Ser. A 1981, 302, 253. (17) Nowak, R. J.; Schultz, F. A.; UmaAa, M.; Lam, R.; Murray, R. W. Anal. Chem. 1980, 52,315. (18) Daurn, P.; Lenhard, J. R.; Rolison, D.; Murray, R. W. J. Am. Chem. SOC.1980, 102,4649. (19) Facci, J., Unlverslty of North Carolina, unpublished results, 1980. (20) Oyama, N.; Anson, F. C. J. Am. Chem. SOC. 1979, 101, 739. (21) Martln, G.W. Ph.D. Thesis, University of North Carolina, 1977. (22) Bottomley, F.; Clarkson, S. G.; Tong, S. J. Chem. Soc., Dalton Trans. 1074, 2344. (23) Deleplne, M. C.R. Heebd. Seances Acad. Sci. 1911, 152, 1390, 1589. (24) Salmon, D., Ph.D. Thesls, University of North Carolina, 1977. (25) Brown, A. P.; Anson, F. C. Anal. Chem. 1977, 49, 1589. (26) Smith, D. F.; Willman, K.; Kuo, K.; Murray, R. W. J. Electroanal. Chem. 1979, 95,217. (27) Peerce, P. J.; Bard, A. J. J. Elecfroanal. Chem. 1980, 114, 89. (28) Nakahama, S., University of North Carolina, unpublished results, 1980. (29) Evans, J. F.; Kuwana, T.; Henne, M. T.; Royer, G. P. J. Electroanal. Chem. 1977, 80, 409. (30) Bettleheim, A.; Chan, R. J. H.; Kuwana, T. J. Elecfroanal. Chem. 1979, 99, 391. (31) Cheng, H.-Y.; Strope, E.; Adams, R. N. Anal. Chem. 1979, 51,2243. (32) Peerce, P. J.; Bard, A. J. J. Elecfroanal. Chem. 1980, 112, 97. (33) Gough, D. A.; Leypoidt, J. K. Anal. Chem. 1979, 51,439. (34) Kolthoff, I. M.; Ligane, J. J. “Polarography”; Interscience: New York, 1952; Vol. 2, p 729. (35) Bard, A. J.; Faulkner, L. R. “Electrochemical Methods”; Wiley: New York, 1980; p 222. (36) Lane, R. F.; Hubbard, A. T. Anal. Chem. 1976, 48, 1287. (37) Adams, R. N. Anal. Chem. 1878, 48, 1126 A.
RECEIVED for review July 27,1981. Accepted November 30, 1981. This research was supported in part by a grant from the Office of Naval Research.
Optimization of a Tissue-Based Membrane Electrode for Guanine M. A. Arnold and 0. A. Rechnltx” Department of Chemlstty, University of Delaware, Newark, Delaware 1971 1
An optlmlratlon strategy 11s proposed for the development of tlssue-based membrane electrodes where varlables which affect substrate dtffuslon and blocatalytlc actlvlty are consldered. The optlrnlratloii strategy Is Illustrated through the development of a rabblt liver based membrane electrode for guanlne wlth hlgh selectlvlty and other deslrable operatlng characterlstlcs.
Although the empirical development of bioselective membrane electrodes using animal and plant tissue slices is proceeding at a rapid pace ( I ) , the available theoretical treatments (2-4) are not yet capable of predicting electrode properties from fundamental considwations. Indeed, the complexity of tissue-based potentiometric membrane electrodes makes it unlikely that optimization of electrode response characteristics can be achieved on any basis other than trial and error in the immediate future. Yet, tissue-based bioselective membrane electrodes contain certain biochemical and mechanical features in common. As 0003-2700/82/0354-0777$01.25/0
a result, it should be possible to facilitate the future development of such electrodes in a systematic, if empirical, manner with regard to the principal variables of selectivity, sensitivity, response time, electrode life, and operating conditions. This paper presents a possible optimization strategy based upon a consideration of the biochemical processes and membrane phases involved in biocatalytic membrane electrodes and illustrates this approach through the step-by-step optimization of a new potentiometric membrane electrode for guanine which employs rabbit liver tissue as the biocatalyst. Figure 1 shows a schematic representation of the various rate processes which take place at the surface of a biocatalytic membrane electrode. The rate processes which are responsible for the electrode response include the kinetics of substrate diffusion and of the biocatalytic reaction. The optimization strategy which is being proposed involves optimizing the parameters which affect these rate processes, respectively. Variables which are related to the substrate diffusion kinetics include the support membrane characteristics and the thickness of the biocatalytic layer. Parameters which deal with the biocatalytic activity include the effects of pH and activator 0 1982 American Chemical Society
778
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982 BULK SOLUTION
I
BIOCATALYTIC LAYER
I
I I
SOLUTION/ BIOCATALYST INTERFACE
Schematic representation of the diffusion and reaction kinetic processes at biocatalytic membrane electrodes.
Flgure 1.
concentration as well as those factors which control activity stability and biocatalytic selectivity. In order to substantiate the proposed optimization strategy, we have selected to present the development of a novel tissue-based membrane electrode for guanine. For this electrode, a thin slice of rabbit liver tissue is held at the surface of an ammonia gas sensing electrode with a support membrane. The biocatalytic activity shown in eq 1 is utilized for the determination of guanine by measurement of the liberated ammonia. For comparison purposes, an isolated enzyme-based guanine electrode is also considered where the activity of eq 1is provided by the commercially available enzyme, guanase (E.C. 3.5.4.3). 0
guanine
0
xanthine
EXPERIMENTAL SECTION Apparatus. All potentiometric measurementswere made with a Corning Model 12 pH/mV meter in conjunction with a Heath-Schlumberger Model SR-240 potentiometric recorder. Measurements were made in thermostated cells at 25 OC. The Orion Model 95-10 ammonia gas-sensing electrode was used for the construction of all biocatalytic membrane electrodes. Reagents. Analytical grade reagents and deionized-distilled water were used unless otherwise noted for the preparation of all solutions. Guanine, guanosine, adenine, adenosine, adenosine 5’-monophosphate (AMP),guanosine 5’-monophosphate(GMP), inosine 5’-monophosphate (IMP),creatinine, creatine, guanase, and all amino acids were purchased from Sigma Chemical Co., St. Louis, MO. Ultrapure urea was obtained from Schwarz/Mann, Orangeburg, NY. Several rabbit livers were purchased frozen and packed individually in plastic containers from Dutchland LaboratoryAnimals, Denver, PA. The livers were stored at -25 OC until use. Procedures. Rabbit Liver Based Guanine Membrane Electrode Construction. The rabbit liver based guanine membrane electrode was constructed by using the procedure previously reported for other tissue-based membrane electrodes (5,6).In this case, a thin slice of the rabbit liver (approximately 0.5 mm thick) was immobilized at the surface of an ammonia gas sensing electrode by means of a cellophane dialysis membrane (Techicon, type “C”). The rabbit liver slice was sandwiched between two cellophane dialysis membranes and the sandwiched tissue slice was placed on the surface of the gas-permeable membrane of the ammonia gas sensing electrode. The screw cap of the electrode assembly was used to hold the membranes and the tissue slice in place. The electrode system was allowed to condition overnight in a pH 8.0,0.2 M borate buffer which contained 0.02% sodium azide as a tissue preservative. The electrode was stored in this buffer at room temperature between measurements unless otherwise noted. Enzyme-Based Guanine Selective Electrode Construction. The enzyme-basedguanine selective electrode was prepared es-
sentially as previously described (7); however, the enzyme solution was dialyzed overnight vs, 4 L of a 0.1 M borate, pH 8.0 buffer at 4 “C prior to its immobilization. This dialysis step was found to be necessary in order to reduce background ammonia in the commerciallyobtained enzyme solution. After dialysis, 10 fiL of the enzyme solution was sandwiched between the gas-permeable membrane and a cellophane dialysis membrane (Techicon,type “C”). The electrode was conditioned and stored in the working buffer. Guanine Response Curves. Guanine response curves were obtained by monitoring the resulting steady-state potentials from the tissue-basedguanine selective electrode which was immersed in 10 mL of the working buffer to which standard additions of a 0.01 M guanine standard were added. Standard guanine solutions were prepared by adding the appropriate amount of guanine to water and adjusting the solution to a pH of approximately 11.7 with 6 M sodium hydroxide. The standard guanine solutions were kept at room temperature. Determination of the Relative Deaminating Activities. The relative guanine and adenosine deaminating activities of the rabbit liver based guanine electrode were measured by the previously described initial rate method (8). For guanine deaminating activity measurements, the tissue-based guanine electrode was immersed in 3 mL of the buffer being studied and 30 fiL of the 0.01 M guanine standard was added. For the adenosine deaminating measurements the tissue-based guanine electrode was immersed in 10 mL of the appropriate buffer and 100 fiL of a 0.1 M adenosine standard was added. In each case, the resulting production of ammonia was monitored directly with the ammonia gas sensing electrode. RESULTS AND DISCUSSION Evaluation of Electrode Response. Since the response of a biocatalytic membrane electrode is ultimately limited by the response of the internal sensing electrode, a comparison of the relative responses is a convenient method for determining the efficiency of the biocatalytic layer. Moreover, the responses of biocatalytic membrane electrodes which use different types of biocatalysts but which respond to the same substrate can be compared in order to evaluate the relative merits of the biocatalysts. From such comparison studies, several advantages have been realized for the tissue-based systems. The direct comparison of the four classes of biocatalysts (i.e., isolated enzymes, bacterial cells, subcellular fractions, and tissue slices) has been reported for glutamine membrane electrodes (9). This study concluded that the tissue-based membrane electrode is the system of choice based upon stabilization of the biocatalytic activity and ease of electrode construction. A further advantage of tissue-based membrane electrodes has been demonstrated by the rabbit muscle based AMP membrane electrode which provides much greater biocatalytic activity than the comparable enzyme electrode (6). Another possible, but as yet unreported, advantage of tissue-based biocatalytic layers is that a tissue slice might provide a biocatalytic pathway not available with isolated enzymes. Thus, it may be possible to eliminate time-consuming purification procedures by using intact tissue slices. These possibilities have recently been reviewed and discussed with respect to their analytical usefulness (I). Table I compares the typical response characteristics observed for the rabbit liver based guanine electrode and for an unmodified ammonia gas sensing electrode using the recent IUPAC recommendations (IO). Because of the limited solubility of guanine in aqueous solutions (11, 12), guanine response curves are only linear to an upper guanine concenM. From Table I it can be tration of approximately 3 X seen that the response of the tissue-based guanine electrode compares favorably to the response of the internal sensing electrode which suggests that the optimum electrode performance is obtained under the operating conditions defined in Table I.
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982
Table I. Comparison of Response Characteristics for the Tissue-Based Guanine Electrode and the Ammonia Gas Sensing Electrodea guanine electrode
characteristic
ammonia electrode
779
lo!
P . - I
/
'si
v
slope
48.0 mV/decade 57.8 mV/decade linear range 1.26 x 10-51.12 x 10-52.8 X 1 0 - 4 M >1.0 X M limit of detection 6.0 x M 4.0 x M response time 7.5-6 min 3-2 min 35 min 20 min recovery time a Response curves obtained by using a 0.2 M borate, 0.02%NaN,, pH 8.0 buffer at 25 "C.
i.1
F
21
I
02
0.4
I
06 0.8 Thickness, (mml
I
10.0
Flgure 3. Effect of tissue thickness on the response time of the rabbit muscle based AMP electrode: change in AMP concentration: (0)from M. 1.4 to 3.4 X M and (0)from 3.0 to 6.6 X b------l
-a
Flgure 2. General conflguraition of a biocatalytic membrane electrode: (a) outer support membrane); (b) biocatalytic layer; (c) inner support membrane; (d) gas-permeable membrane; (e) internal electrolyte solution; (f) internal combination pH electrode: (9) electrode body.
Recently, an isolated tmzyme-based guanine electrode has been reported where the isolated enzyme, guanase, is coupled to an ammonia gas sensing electrode (7).For the response of this electrode at 27 "C, a slope of 20.0 mV/decade guanine concentration is reported. In our study, we independently obtained a slope of 20.8 mtV/decade under the same conditions and, thus, confirm the poor response of the enzyme based system. The tissue-based guanine electrode, on the other hand, typically yields a slope of 48 mV/decade guanine concentration; this illustrates the superior response of the tissue-based electrode over the enzyme-based system. Biocatalyst Immobilization. Because animal based biocatalytic sections also contain connective tissue, special consideration must be given to the method of biocatalyst immobilization. Methods reported to date include crosslinking with a bovine serum albumin (BSA)-glutaraldehyde matrix (8)and physical retainment using support membranes (5,6); the latter is more commonly used owing to its inherent convenience (9). Figure 2 shows the several membrane layers which are involved in the immobilization of tissue slices at the surface of a gas-sensing potentiometric membrane electrode using support membranes. Typically, a thin slice of the tissue (0.3-0.5 mm) is sandwiched between an inner and an outer support membrane after which the tissue-membrane sandwich is placed on the surface of the internal sensing electrode. The tissue-membrane sandwich is held in place via the screw cap of the electrode assembly as shown in Figure 2. Nylon webbing is commonly employed as the outer membrane since intact tissue permits the use of large pore sizes (37-149 pm). The use of large pore size membranes allows unrestricted diffusion of substrate and product between bulk solution and biocatalytic layer (see Figure 111resulting in optimum response and recovery times. However, some tissue slices do not have the necessary mechanical integrity to permit the use of large pore support membranes. In such cases, a cellophane dialysis support membrane or a BSA-glutaraldehyde cross-linking immobilization can be wed. A cellophane dialysis membrane is also interposed between the biocatalytic layer and the gas-permeable hydrophobic membrane of the internal electrode. This arrangement serves to protect the hydrophobic membrane from lipids, proteins, and other componentsof the biocatalytic material and appears
to prevent contamination and leakage of the electrolyte solution. The thickness of the tissue slice is also an important variable in electrode optimization. With an increase in the tissue thickness, the amount of biocatalyst at the electrode surface is increased, but the advantages of increasing the amount of biocatalytic activity are quickly counterbalanced by increases in the electrode response and recovery times. Figure 3 shows the effect of tissue thickness on the response time of the rabbit muscle based adenosine 5'-monophosphate (AMP) membrane electrode (6). An increase in the electrode response time is observed with an increase in the tissue thickness. This behavior is commonly found with biocatalytic membrane electrodes (13)and sets a practical limit (approximately 0.8 mm) on the thickness of the biocatalytic layer employed. In the case of the rabbit liver electrode described here, tissue thicknesses of 0.5 mrn are employed in conjunction with dialysis support membranes. pH and Activator Concentration. Because of the pH dependencies of both the biocatalytic activity and the gassensing internal electrode, the pH of the working buffer is critical with respect to the performance of biocatalytic membrane electrodes. The effect of pH on the electrode performance can be determined by the initial rate method of measuring relative biocatalytic activities (14). By use of this method to biocatalytic membrane electrodes, the observed rate of product production is a function of the biocatalyst's ability to produce the product and of the internal sensing electrode's ability to measure this product production. As a result, the observed rate is a measure of the relative electrode activity to a particular substrate under the experimental conditions being studied. Generally, the relative electrode activity with respect to pH results in a broad pH profile with the optimum pH typically being that which produces the maximum rate. It will be shown below, however, that for the tissue-based guanine electrode the optimum pH for the electrode activity is not the optimum pH for the overall electrode system due to stability considerations. The effect of activators on the biocatalytic activity is usually determined by using the initial rate method described above. A profile of the relative biocatalytic activity vs. the activator concentration permits evaluation of the optimum activator concentration. The effect of potassium ion concentration on the response of the rabbit muscle based AMP membrane electrode is an example of such activator effects (6). Figure 4 shows a pH profile for the rabbit liver based guanine membrane electrode. From this profile, it can be seen that the maximum electrode activity is available at pH 9.5; whereas, at pH 8.0 only 50% of the maximum activity is
780
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982
Table 11. Stock Tissue Section Storage Requirements and Lifetimes min life-
substrate measd
biocatalyst
glutamine
porcine kidney rabbit muscle rabbit liver mouse small intestine mucosal cells yellow swash
AMP
guanine adenosine
b,i
I.
glutamate I
8.0
I
8.0 OH
storage time, condition months -25 "C -25 "C -25 "C -25"Cin
6
7
I 2
100%
glycerol
+ 4 "C
1
I
10.0
Flgure 4. Effect of pH on the guanine deaminating activity of the rabbit liver based guanine electrode using a 0.2 M borate, 0.02% sodium azide buffer.
available. It will be shown that a more practical electrode performance is obtained a t pH 8.0 due to the pH-dependent stability of the biocatalytic activity. Electrode Lifetime and Biocatalytic Stability. The useful lifetime of a biocatalytic membrane electrode is an important practical consideration for analytical purposes. The electrode lifetime is frequently limited by the stability of the biocatalytic activity being employed. For tissue-based biocatalytic membrane electrodes, special precautions must be taken in order to ensure that a practical useful lifetime is obtained. The use of an antimicrobial agent as a tissue preservative is critical for a successful electrode system. The antimicrobial agent acts to prevent bacterial contamination of the tissue-based biocatalytic layer; such contamination could introduce undesired biocatalytic pathways with resulting loss of selectivity. Two commonly used antimicrobial agents are sodium azide and chlorhexidine diacetate. Sodium azide is a convenient antimicrobial agent in alkaline solution and at a concentration of 0.02% it is very effective in preventing bacterial growth. It should be noted, however, that azide can inhibit certain enzymatic activities (15). Chlorhexidine diacetate, on the other hand, can be used in either acidic or alkaline solutions a t a concentration of 0.002%. For some tissue-based membrane electrodes, a stabilizing agent is also required to stabilize the tissue enzyme or enzymes necessary for the biocatalytic activity. For example, the addition of 40% glycerol to the working buffer of the yellow squash-based glutamate membrane electrode substantially increases the useful lifetime of the electrode by stabilizing the glutamate decarboxylating activity (16). All of the tissue-based membrane electrodes reported to date have required only simple storage conditions, e.g., the electrode is stored by immersion in the appropriate buffer at room temperature. In contrast, enzyme electrodes frequently need to be stored under refrigeration to preserve enzyme stability; as a result, additional temperature reequilibration is required. The overall lifetime of tissue biocatalysts depends not only upon the thin slice actually immobilized at the electrode but also upon that of the stock tissue from which slices are taken. Table I1 summarizes the storage conditions required and the minimum storage period for which the tissue sections can be employed in the preparation of several biocatalytic membrane electrodes. It can be seen that tissues generally retain their biocatalytic activity for months if stored in the frozen state. For many electrodes, the excellent lifetime of the stock tissue sections and their inherent low cost make tissue slices the most economicaltype of biocatalyst for the construction of bioselective membrane electrodes.
>
;SO-
5E 'Z60-
m
z E
!40a
n
2o ' U
'
Time, (min)
880
'
io40
Flgure 5. Effect of time on the guanine deaminating activity using a 0.2 M borate, 0.02% sodium azide, pH 9.5 buffer.
220-
->
E "
-
m
g1800
n
I
I
5.0 -Log
I
1
I
4.5 4.0 3.5 G u a n i n e Conc., (M)
Flgure 6. Response of the rabbit liver based guanine electrode to guanine using a 0.2 M borate, 0.02% sodium azide, pH 8.0 buffer at various electrode ages: day 3 (0); day 6 (0); day 10 (A);and day 14 (0).
In the case of the rabbit liver based guanine membrane electrode, the stability of the biocatalytic activity is dependent upon the pH of the working buffer. Figure 5 shows the effect of a pH 9.5 buffer on the guanine deaminating activity with respect to time. Whereas the optimum pH for the electrode activity is 9.5 (see Figure 4), it is clear that at this pH there is a rapid decline in the level of biocatalytic activity. Similar results are obtained when a pH 9.0 buffer is used. When a pH 8.5 buffer is used, this effect is not so dramatic and the electrode possesses a useful lifetime of 6 days before the guanine response begins to decrease at the upper region of linearity. Figure 6 shows the response of this electrode over a 14-day period using a pH 8.0 working buffer. No changes
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982 1o
I
781
o p
0
,I
220
j1
f'
2 20
L' 40
1401
L
A
-
I
I
4.5 4.0 3.5 -Log Substrate Conc., (M)
5.0
Flgure 7. Response of the tissue-based guanine electrode to guanine (0),adenosine (Up, AMP (A), ADP (V),and ATP (O),using a 0.2 M borate, 0.02% sodium azlde, and pH 8.0 buffer.
in the response times are observed over this time period, and as seen in Figure 6, no significant differences in the response are observed when the electrode is stored in the working buffer at room temperature. Although the electrode possesses only 50% of the maximum electrode activity at pH 8.0, this pH is the most practical with respect to overall response characteristics because of tihe enhanced stability. As listed in Table 11, stock rabbit livers can be stored for at least 7 months at -25 "C without any significant loss of biocatalytic activity. Selectivity. Special attention must be given to the selectivity of tissue-based biocatalytic membrane electrodes because of the numerous metabolic paths usually found in tissue materials. 'The main types of interfering activities which are likely to occur include the generation of the measured product from a substrate other than the principal substrate and the utilization of a substrate whose reaction changes the pH at the electrode surface. The first of these is the more common and the more difficult to eliminate. A strategy for eliminatingthis type of interference has been reported (8) and involves determining the specific enzyme or enzymes responsible for the interfering activity followed by repression of that activity with the use of specific inhibitors. A major concern in this strategy is to ensure that the added inhibitor has no adverse effect on the desired biocatalytic activity. The second type of interference is best minimized by employing a working buffer with high buffer capacity. The selectivity of the rabbit liver based guanine electrode was tested by monitoring the response obtained in the presence of compounds structurally related to guanine and to amine containingbiological compounds for which deaminating activities are frequently encountered. When present at a concentration of about 1 mM, the following compounds produced no response from the tissue-based guanine electrode: inosine, adenine, GMP, IMP, creatinine, creatine, asparagine, serine, urea, glutamine, glutamate, ornithine, threonine, lysine, valine, glycine, arginine. Initial studies concerning the selectivity of the rabbit liver based guanine electrode in 0.1 M phosphate-0.1 M borate buffer revealed an interference from guanosine. This interference was thought to be the result of two possible mechanisms, e.g., the reaction shown in eq 2 catalyzed by the enzyme nucleosidase (E.C. 3.2.2.1) or the reaction of eq 3 catalyzed guanosine guanosine
-
+ H20
+ Pod3-
D-ribose + guanine
u-ribose 1-phosphate
(2)
+ guanine (3)
'
TS'O i m e , ( m110 in) ~
"'760
'
7AO
Flgure 8. Effect of manganese(I1)on the adenosine deaminating actlvity of the rabbit liver based guanine electrode with respect to time using a 0.2 M borate, 0.02% sodlum azide, pH 8.0 buffer: (A) prior to the addition of MnCi,; (B) in the presence of 1 mM MnCI,; and (C) after reimmersion of electrode into buffer without MnCI, present.
9
I
I
t 5.0 4.5 4.0 3.5 -Log Substrate Conc., ( M )
Flgure 9. Selectivity enhanced response of the tissue-based guanine electrode to guanlne (0),adenosine (a),AMP (A), ADP (V), and ATP (O),using a 0.2 M borate, 10 mM manganese chloride, 0.2% sodium azlde, and pH 8.0 buffer. by the enzyme guanosine phosphorylase (E.C. 2.4.2.15). In both cases, the guanine produced from the interfering activity would be converted to ammonia by the primary reaction (eq 1). It was found that removal of phosphate from the buffer system also eliminated the interfering activity; a fiiding which suggests that the guanosine phosphorylase pathway was operative. Subsequent work was carried out in 0.2 M borate buffer. The response of the rabbit liver based guanine electrode to guanine, adenosine and various adenosine-containing nucleotides is shown in Figure 7 . It can be seen that this electrode responds significantly to each of these compounds. A possible scheme for this interfering activity is that shown in eq 4 and 5, where 1represents the enzyme alkaline phos-
+ XP adenosine + H 2 0 5inosine + NH, AXP
+ H 2 0 -!.,
adenosine
(4)
(5)
phatase (E.C. 3.1.3.1) and I1 represents adenosine deaminase (E.C.3.5.4.4). For removal of this interfering activity, an inhibitor to adenosine deaminase must be included in the operating conditions. It has been reported that moderate
782
Anal. Chem. 1982, 5 4 , 782-787
concentrations of manganese(I1) ion completely inhibit adenosine deaminase activity at pH 8.0 (17). Figure 8 shows that manganese(I1) ions a t a concentration of 1 mM inhibit about 80% of the adenosine deaminase activity. Because of the complex structure of the tissue slice, it might be expected that this inhibition would require a long incubation period in order to allow sufficient time for the inhibitor to penetrate into and to disperse itself throughout the tissue layer; however, the results presented in Figure 8 suggest that this is not the case since the inhibition occurs in a relatively short period of time. Also shown in Figure 8 is the reversibility of the inhibition with rapid reactivation of the interfering pathway after removal of the inhibitor. As can be seen from Figure 9, a 10 mM concentration of manganese(I1) completely eliminates the interfering response to adenosine and its related compounds. Thus a careful selection of optimum operating pH, buffer constituents, and selective inhibitors results in a tissue electrode for guanine with high selectivity, good lifetime, and other attractive operating characteristics. The combination of such biochemical “tuning” steps with appropriate selection of membrane materials, tissue thicknesses, and immobilization procedures represents the essential elements of the best strategy for electrode optimization available at the present time.
LITERATURE CITED Rechnltz, 0. A. Science 1981, 214, 287-291. Brady, J. E.; Carr, P. W. Anal. Chem. 1980, 52, 980-982. Carr, P. W. Anal. Chem. 1977, 49, 799-802. Hameka, H. F.; Rechnitz, G. A. Anal. Chem. 1981, 53, 1586-1590. Rechnltz, G. A.; Arnold, M. A,; Meyerhoff, M. E. Nature (London) 1979. 278, 466-467. Arnold, M. A.; Rechnitz, G. A. Anal. Chem. 1981, 53, 1837-1842. Nlkolells, D. P.; Papasthopoulos, D. S.; Hadjiioannou, T. P. Anal. Chim. Acta 1981, 126, 43-50. Arnold, M. A.; Rechnitz, G. A. Anal. Chem. 1981, 53, 515-518. Amold, M. A.; Rechnltz, G. A. Anal. Chem. 1980, 52, 1170-1174. IUPAC Analytical Chemistry Division, Pure Appl. Chem. 1978, 48, 127- 132. Albert, A.; Brown, D. J. J. Chem. SOC. 1954, 2060-2071. Ito, S.; Takoaka, T.;Morl, H.; Teruo, A. Clln. Chlm. Acta 1981, 715, 135- 144. Kobos, R. K. I n ”Ion-Selective Electrodes in Analytical Chemistry”; Frelser, H., Ed.; Plenum: New York, 1980; Chapter 1. Gullbauk, G. G.; Smith, R. K.; Montalvo, J. G., Jr. Anal. Chem. 1988, 4 1 , 600-605. Hewitt, E. J.; Nicholas, D. J. D. I n “Metabolic Inhibitors”; Hochster, R. M.. Quastel. J. H., Eds.; Academic Press: New York, 1963; Vol. 11, Chapter 29. Kuriyama, S.; Rechnltz, G. A. Anal. Chlm. Acta 1981, 731, 91-96. Alkawa, T.; Aikawa, Y.; Brady, T. G. Int. J. Blochem. 1980, 12, 493-495.
RECEIVEDfor review November 18,1981. Accepted January 11,1982. We are grateful to the National Science Foundation (Grant No. CHE-8025625) for supporting this research.
Differential Normal Pulse Voltammetry for the Anodic Oxidation of Iron(I I) Timothy R. Brumleve,’ R. A. Osteryoung, and Janet Osteryoung* Department of Chemlstty, State University of New York at Buffalo, Buffalo, New York 14214
Dlfferentlal normal pulse voltammetry has been used to determine klnetlc parameters for the oxidation of Iron( 11) In l M H2S04at a glassy carbon electrode. The data ftt the theory well. Analysk of dependence of peak potential on pulse width produces values of anodlc transfer coefficient of 0.35 and formal rate constant of 1.5 X cm s-’. The enhanced current response over that obtalned In differentlal pulse voltammetry Is shown and reverse dlfferentlal normal pulse techniques are demonstrated.
The technique of differential normal pulse (DNP) voltammetry with alternating sign of the second (differential) pulse provides a number of advantages over conventional techniques for voltammetric analysis and for determination of electrochemical kinetic parameters. The current-potential response is peak-shaped and symmetrical about the peak. Peak position, height, and width are related simply to kinetic parameters for totally irreversible reactions (1). Also, most of the time the electrode is held at a potential at which product is not formed. This may be contrasted with classical differential pulse voltammetry in which the potential is scanned through the entire range of interest, and therefore product is produced continuouslyas in DC voltammetry. This paper is concerned with the application of DNP voltammetry to the totally irPresent address: Anderson Physics Laboratories, 406 N.Busey Av., Urbana, IL 61801. 0003-2700/82/0354-0782$01.25/0
reversible oxidation of Fe(I1) in 1 M H2S04at a stationary glassy carbon electrode. The analytical utility of this technique is demonstrated, and comparisons with normal pulse (NP) and classical differential pulse (DP) voltammetry point out advantages in background discrimination and control of reaction conditions for solid electrodes. This technique is also applied to the quantitative evaluation of electron-transfer kinetic parameters for Fe(I1). The results are in good agreement with the recent theoretical predictions of Brumleve and Osteryoung (1)for the DNP waveform for totally irreversible systems. The use of a glassy carbon electrode is a particularly stringent test of the technique both for analytical determinations and for extracting kinetic information, since residual (background)currents are usually quite large at these electrodes (2). As a final note we consider a variation of the technique which we term reverse differential normal pulse (RDNP) voltammetry and which is analogous to the reverse pulse (RP) variation of NP voltammetry (3, 4). Application of this technique to the anodic oxidation of Fe(I1) reveals that RDNP in the alternate pulse mode embodies many of the features of cyclic voltammetry (such as peak shaped voltammograms) while retaining the advantages of pulse voltammetry (charging current discrimination and superior control of timing parameters and reaction conditions). EXPERIMENTAL SECTION The computer-controlled pulse voltammetric instrument has been described previously (5). 0 1982 American Chemical Society
