Anal. Chem. 1901, 53, 1837-1842 (9) McLean, W. R.; Stanton, D. L.; Penketh, G. E. Analyst (London)1973, 98, 432-442. (10) Brenner, K. S. J. Chrormfogr. 1978, 767, 365-380. (11) Black, M. S.; Sievers, FI. E. Anal. Chem. 1978, 48, 1872-1874. (12) Quimby, B. El.; Uden, F’. C.; Barnes, R. M. Anal. Chem. 1978, 50, 2112-2118. (13) Beenakker, C . 1. M. Spoctrochlm. Acfa, Part5 1978, 318, 483-486. (14) Beenakker, C. I. M. Spoctrochlm. Acfa, Part5 1977, 328, 173-187. (15) Beenakker, C. I. M.; BaJmans, P. W. J. M. SLmCtr~hh.A&, Pad 5 1978, 338, 53-54. (16) Quimby, B. 0.; Delaney, M. F.; Uden, P. 6.;Barnes, R. M. Anal. Cham -. ... IQTQ .- . - , 51 - . , 875-880. .. - . (17) Quimby, B. 0.; Delaney, M. F.; Uden, P. C.; Barnes, R. M. Anal. Chem. 1980, 52, 259-263. (18) Mulligan, K. J.; Caruso, J. A.; Fricke, F. L. Analyst (London) 1980, 105. 1060-1067. (19) Wasik, S. P.; Schwartz, P. J. Chromafogr. Scl. 1980, 76, 660-683. (20) Estes, S. A.; llden, P. C.; Barnes, R. M. Anal. Chem. 1981, 53, 1336-1340. (21) Sneliman, W.; Rains, T. C.; Yee, K. W.; Cook, H. D.; Menis, 0.Anal. Chem. 1970, 42,394-398. (22) Quimby, B. D. tiewiett-Packard Co., Avondaie, PA, personal communication.
. .
iw
(23) Estes, S. A.; Uden, P. C.; Rausch, M. D.; Barnes, R. M. HRC CC, J. High Resoluf. Chromafogr. Chromafogr. Commun. 1980, 3 , 471-472. (24) Pearse, R. W. B.; Gaydon, A. G. “The Identificatlon of Molecirlar Spectra”, 2nd ed.;Chapman & Hall: London, 1950. (25) Tanabe, K.; Haragiichl, H.; Fuwa, K. Spectrochlm. Acfa, Part5 1081, 365, 119-127. (26) Sarto, L. G., Jr.; Estes, S. A.; Uden, P. C.; Siggia, S.; Barnes, R. M. Anal. Lett. 1981, 14 (A3), 205-218. (27) Beenakker, C. J. M.; Boumans. P. W. J. M.; Rommers, P. J. Phllips Tech. Rev. 1980, 39, 65-77.
RECEIVED for review March 20,1981. Accepted July 6,1981. This study was supported by an A.C.S. Analytical Division Summer Fellowship sponsored by the Olin Corp. Caritable Trust and an A.C.S. Analytical Division Full-Year Fellowship sponsored by the Procter & Gamble Co. (S. Estes). Partial support was also received from US.Department of Energy Contract De-AC02-077EV4320.
Tissue-Based Membrane Electrode with High Biocatalytic Activity for Measurement of Adenosine 5’-Monophosphate M. A. Arnold and G. A. Rechnitz’ Department of Chemistw, #Universityof Delaware, Newark, Delaware 197 1 1
A bioselectlve membrane electrode whlch employs sllces of rabbh muscle tissue held1 at an ammonia gas sensor is shown to have nearly SO times greater blocatalytlc activity for adenoslne 5‘-monophosphate (AMP) than conventional electrodes using commercially available enzyme preparations. As a consequence of thls high specific activity, enzyme concentration steps are elirninated and the rabblt muscle electrode achleves excellent response characteristics wlth a working llfetlme of at least 28 days. Important experimental variables, Including the effect of tissue thickness, are! evaluated In order to optlmire electrode response.
Various types of biocaitalysts have been coupled to ion-selective electrodes for the development of biocatalytic membrane electrodes (1). It has been shown that the opecific biocatalytic activity of these electrodes is critical in determining response slope, dynamic behavior, and useful lifetimes (2, 3). Problems exist when sufficient activities cartnot be achieved because of a low specific activity of the biocatalysts and because of the limited surface area of the ion-selective electrode on which the biocatalyst is immobilized. I n some cases, where an isolated enzyme-based membrane electrode possesses too little biocatalytic activity for the construction of a practical electrode, whole tissue cells might be used as the catalytic layer to increase activity. We now demonstrate this effect through the development of an adenosine 5’monophosphate (AMP) membrane electrode using a thin slice of rabbit muscle as the biocatalytic layer. Both the enzyme-based and tissue-based AMP membrane electrodes utilize the biocatalytic activity shown in eq 1. The
AMP
+ FIZO ----.biocatalyst
inosine 5’-monophosphate + NH3 (1) commercially available enzyme, adenylic acid deaminase (E.C. 3.5.4.6) has been used in the enzyme-based system (4). The
specific activity of thle available enzyme preparation is too low to be used directly and a 16-h concentration step is requiried prior to use of the enzyme in the construction of the electrode. By use of a thin slice of rabbit muscle rich in the biocatalytic activity of interest, this concentration step can be eliminak!d. The specific activity of the biocatalyst within the tissue slice is sufficiently high so that the needed amount of activity is present at the electrode surface. The presence of sufficient activity results in a membrane electrode for AMP whilch possesses outstanding response characteristics, an excellent useful lifetime, and a good dynamic response.
EXPERIMENTAL SECTION Apparatus. All potentiometric measurementswere made with a Corning Model 111 pH/mV meter in conjunction with a Health-Schlumberger Model SR-240 potentiometric recorder. Measurements were made in thermostated cells controlled with a Haake Model FS water bath at 25 “C unless otherwise noted. The Orion Model 95-10 ammonia gas sensing electrode was used for the construction of all bioselective membrane electrodes. Reagents. All solutions were prepared with distilled, deionized water. Analytical grade reagents were used unless otherwise noted. Adenosine 5’-monophosphate (AMP),adenosine 5’-diphosphate (ADP),adenosine 5’-triphosphate (ATP), adenosine 3 ’ 4 cyclic monophosphate (c-AMP),adenosine, adenine, creatine, sodium azide, human control serum, and all amino acids were purchased from Sigma Chemical1 Go., St. Louis, MO. Individual rabbit muscles were obtained frozen and packed individually in plastic containers from Dutchland Laboratory Animals, Denver, PA. The muscles were stored at -25 “C until use. Procedures. Tisciue-Based Membrane Electrode Cornstruction. The rabbit muscle based AMP membrane electrode was constructed by using the procedure previously reported for other tissue-based membrane electrodes (5-7). This procedure involves the immobilizationof a thin slice of tissue at the surface of an ammonia gas sensing electrode by means of a mesh d monofilament of nylon (mesh size 37 pm). The rabbit muscle slice was separated from the gas permeable membrane with a Technicon type “C” dialysis membrane. Unless otherwise noted, the
0003-2700/81/0353-1837$01.25/00 1981 Amerlcan Chemical Society
1838 ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
tissue slices were approximately 0.5 mm thick. The electrode system was allowed to condition for 2-4 h in the working buffer containing 0.1 M Tris-chloride, 0.1 M potassium chloride and 0.02% sodium azide at pH 7.5. The electrode was stored in the working buffer at room temperature between measurements. Determining the Relative AMP Deaminating Activity. The relative AMP deaminatingactivity of the rabbit muscle based AMP electrode was measured while the tissue was coupled to the ammonia gas sensing electrode using the previously described initial rate method (8). The tissue-based AMP electrode was immersed in 3 mL of the buffer system being studied. To this solution, 100 pL of a 0.1 M solution of AMP was added and the resulting production of ammonia was measured directly with the ammonia gas sensing electrode. The initial rate of production of ammonia was obtained from a potential vs. time curve as previously described (9). Measurements of this type yield a measure of the overall efficiency of both the biocatalyst and the ammonia gas sensing electrode, allowing for the simultaneous optimization of solution conditions with respect to each. Estimation of Effective Biocatalytic Activity Units. The effective number of biocatalytic activity units was estimated for the rabbit muscle slices by monitoring the rate of production of ammonia from AMP using the ammonia gas sensing electrode. Rabbit muscle tissue slices were added to 25 mL of a 0.01 M AMP solution made in the working buffer at 25 O C . The moles of ammonia produced were calculated from an ammonia calibration curve at various time intervals. The slope of a plot of micromoles of ammonia produced vs. time was taken as the number of effective biocatalyticactivity units &mol of NH3/min)for the tissue slices measured. Reduction of Background Ammonia from Human Control Serum. The Sigma human control serum was found to contain high levels of ammonia nitrogen. In order to reduce the ammonia nitrogen concentration in the serum, the ammonium ions were exchanged for sodium ions with an ion exchange resin. This was accomplished by adding approximately 10 g of Dowex 5OW-X8 cation exchanger in the sodium form t o 20 mL of the control serum, mixing thoroughly, and decanting the serum. The treated serum when diluted with the buffer was found to contain no ammonia detectable by the ammonia gas sensing electrode.
RESULTS AND DISCUSSION Effective Units of Biocatalytic Activity. A plot of micromoles of ammonia produced (y) w. time ( t )for a 0.5 mm thick rabbit muscle slice reveals a linear relationship described by y = 5.lt - 6.9 (r2= 0.9986). This corresponds to approximately 5 effective bi-talytic activity units in the tissue slice active at the electrode surface. In contrast, a similar quantity (25 pL) of the commercially available adenylic acid deaminase enzyme preparation immobilized at the surface of the ammonia electrode yields a biocatalytic activity of only 0.1 units. These results indicate that a 0.5 mm thick rabbit muscle tissue slice yields nearly 50 times greater biocatalytic activity than the isolated enzyme. Even after extensive concentration of the enzyme suspension, only 0.9 units of the biocatalytic activity could be immobilized (4). It will be seen that the higher biocatalytic activity of the tissue-based electrode results in an AMP with exceptional overall response characteristics. Efect of pH a n d Potassium Ion Concentration. The optimum pH for the rabbit muscle based AMP membrane electrode is a function of the pH dependencies of the biocatalyst and the ammonia gas sensing electrode. In this case, the pH optimum of the system is determined by measuring the relative AMP deaminating activity with respect to changes in pH (see procedures). Figure 1shows the percent maximum activity of the tissue-based AMP membrane electrode with respect to pH using a buffer system composed of 0.1 M Tris-HC1 and 0.02% sodium azide. A broad pH dependency is observed with at least 90% of the maximum deaminating activity being obtained within a pH range of 7.1-7.7. An optimum pH of 7.5 is found which agrees well with an earlier study which involved the isolated enzyme-based AMP membrane electrode ( 4 ) .
4-
1 6.7
7.1
7.5
7.9
PH
Flgure 1. Effect of pH on the AMP deaminating activlty of the rabbit muscle based AMP electrode; error bars represent standard deviation.
?1
.t Flgure 2. Effect of potassium ion concentration on the activity of the rabbit muscle based AMP electrode; error bars represent standard deviation. It is well documented that adenylic acid deaminase activity is activated by the presence of potassium ions (IO). For this reason, the effect of various potassium ion concentrations on the tissue-based AMP membrane electrode is reported. Figure 2 shows the percent maximum activity of the electrode system with respect to potassium ion concentration. From this figure, it can be seen that within the experimental error of the measurement, a potassium ion concentration of 0.1-1.0 M is maximal, a finding which agrees with previous reports (IO). The buffer conditions are selected on the basis of the pH and potassium ion evaluations shown above. In all further studies, unless otherwise noted, a buffer containing 0.1 M Tris-HC1,O.l M potassium chloride, and 0.02% sodium azide a t pH 7.5 is used in order to take advantage of the excellent AMP deaminating activity under these conditions. Sodium azide is required as a tissue preservative (7). Effect of Tissue Thickness. Table I summarizes the results of a study dealing with the effect of tissue thickness on the response of the rabbit muscle based AMP membrane electrode. Tissue thicknesses ranging from 0.14 to 1.08 mm are studied. It can be seen that no significant differences in the slope, linear range, and limit of detection are obtained when tissue thickness ranging from 0.14 to 1.08 mm are used. However, an increase in tissue thickness results in an increase in the response times which increase to unusable levels a t a tissue thickness of 1.08 mm. It is, therefore, recommended that tissue slices less than 0.81 mm thick be used in the construction of this electrode system. Tissue slices of thickness less than 0.5 mm are difficult to use with respect to slicing reproducibility and overall handling.
ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
1839
Table I. Effect of Tissue Thickness on the Rabbit Muscle Based AMP Electrode a tissue thickness, mm
slope, mV/decade
0.14
56.7 ?: 1.2
0.41
57.3 ?: 0.6
0.68
57.4 3: 0.7
0.81
57.0 3: 1.5
1.08
56.4 2: 0.7
linear range, M (9.1 2 2.6) i( lo-!'-
(7.2t 1.9) x 10-3 (6.3 i: 0.6)X lo-!'(9.45 1.0) x 10-3 (8.6 2 3.9)X lo-'(9.6 5 0.6)x 10-3 (6.22 1.5)X lo-"(8.1i: 1.0) x 10-3 (6.82 2.1)X lo-'(9.45 0.6) x 10-3
response time, b min
response time, min
no, of electrodes
2.5 i: 1.0
1.0 i: 0.3
3
(2.05 0 . 3 ) ~10-5
3.5 5 1.3
2.0 5 0.5
3
(2.65 0.7)x
4.0 5 1.0
2.8 i 1.0
3
(2.3i 0.5)x
5.0 5 1.2
3.3
1.5
4
(2.5 i: 1.1)x 10-5
8.2 5 0.3
6.2 5 0.6
3
limit of detection, M (2.3 0.9)x 10-5
i
Values represent averages i: standard deviation. Response time when changing the AMP concentration from 1.41 x to 3.4 X M. Response time when changing the AMP concentration from 3.0 x to 6.6 x M.
c t
120
\
I *------------
5
Flgure 3. Typical response curves for the rabbit muscle based AMP
membrane electrode: 0, AMP; A,ADP. Since there is no advantage with respect to the electrode response characteristics )inusing the thinner slices, all further studies use slices which are about 0.5 mm thick. Tissue slices of this thickness posses8 excellent response characteristics, are easy to handle, and ctm be produced by using a sharp razor blade rather than an expensive microtome. A major factor influencing the response of the AMP tissue-basedmembrane electrode is the degree to which the tissue slice covered the ammonia gas sensing membrane. Best results were obtained when the tissue slice completely covered the gas permeable membrane. Effect of Temperature. The effect of temperature on the response of the AMP tissue-based membrane electrode was found to be negligible for the temperature range tested. No significant differences in the response characteristics, e.g., slope, linear range, limit d detection, and response time, were observed by the electrocle system at temperatures of 20,25, and 30 "C. A temperature of 25 "C was used throughout the remainder of the studies. Typical Response Curve. A typical response curve for the tissue-basedAMP membrane electrode is shown in Figure 3. It can be seen that a slope of approximately57 mV/decade change in AMP concentration is obtained for a liner range of about 1 x lo-' to 10" M AMP with a correlation coefficient of 0.9997. The lower limit of detection for this electrode system is approximately 5 X M AMP.
15 Time [min)
d
- 3.2XlU5M ------M
25
Figure 4. Dynamic response of the AMP tissue-based membrane electrode at various AMP concentrations.
Dynamic Behavior. The dynamic response of the rabbit muscle based AMP membrane electrode at various AMP concentrations is shown in Figure 4. Response times are shown to range from about 8.5 to 2.5 min corresponding to AMP concentrations of 5.0 X and 7.4 X M, respectively. Shorter response times at higher substrate concentrations are commonly observed for biocatalytic membrane electrodes (11). Also shown in Figure 4 is the stability of the steady-state potential for the tissue-based AMP sensor. It can be seen that a constant potential is maintained for at least 15 min at d l AMP concentrations tested ranging from the lower limit of detection to the upper region of linearity. The use of the Oriion internal filling solution (95-1002) in the constructionof thle AMP tissue-based membrane electrode resulted in increasing response times. Over 4-day period, response times were observed to change from 8.5 to 30 min and from 2 to 12 min for AMP concentrations of 5.0 X 10" and 7.4 X M, respectively. Upon disassembly of tlhe electrode after this time period, a thin film of yellow crystals was observed on the side of the gas permeable membrane adjacent to the internal filling solution. By replacement the Orion filling solution with an electrolyte solution composed of 0.17 M sodium chloride and 0.03 M ammonium chloridle, the response times remained constant throughout the lifetime of the tissue-based AMP membrane electrode.
1840 c ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
J'
A B
//I
;
.--
/ , '
cI 1c Flgure 5. Potential vs. time traces for the ammonia gas sensing electrode (-) and the rabbit muscle-based AMP electrode (---): (A) addition of ammonia, final total ammonia nitrogen concentration of 4.5 X lo5 M; (B) addition of AMP, final AMP concentration of 2.5 X lo4 M; (C)reimmersion of electrode in fresh buffer. The recovery time, which is the time required to reestablish the electrode base line potential after the measurement of an AMP solution, has been evaluated for the tissue-based AMP membrane electrodes. Since the recovery time for the tissue-based membrane electrode cannot be better than that of the internal ammonia gas sensing electrode, a comparison of the respective recovery times reveals the effect of the various membrane phases of the tissue-based system on the recovery time. Figure 5 shows a potential vs. time trace for the ammonia gas sensing electrode and for the rabbit muscle based AMP membrane electrode. This shows that the recovery times for the electrodes are similar, with that for the ammonia gas sensing electrode being slightly shorter. Recovery times for the tissue-based AMP membrane electrodes range from 19 to 25 min following measurement in solutions with AMP concentrations ranging from 2.5 X to 9.1 X M. In comparison, the recovery times for the ammonia gas sensine electrode under the same conditions range from 16 to 18 min 'following measurement in ammonia solutions with total ammonia nitrogen concentrations ranging from 4.5 X to 1.6 X M. Total ammonia nitrogen concentrations chosen for comparison are those which produce equivalent potential changes as the corresponding AMP solutions; thus, each electrode senses the same effective ammonia concentration a t the electrode surface prior to the recovery process. These results show that the presence of the immobilized tissue slice and the support membranes result in a slight increase in the recovery time. Selectivity. The selectivity of the tissue-based AMP membrane electrode was tested by monitoring the response obtained in the presence of compounds structurally similar to AMP and of amine containing biological compounds. The possible interferents tested include ADP, ATP, c-AMP, adenosine, adenine, urea, glutamine, creatinine, asparagine, glutamate, arginine, ornithine, and serine. The response of the electrode system for AMP in diluted human serum control samples was also evaluated in order to determine the effect of this complex biological matrix on the electrode performance. Figure 5 shows the response of the tissue-based AMP membrane electrode for AMP in buffer, in buffer also containing 1mM concentrationsof aspargine, arginine, glutamate, ornithine, and serine, and in a pooled human serum control sample diluted 3:l with the buffer. In the latter case, the serum control sample is treated with cation exchange resin to reduce interfering levels of ammonia nitrogen (see proce-
Figure 6. Response of the tissue-based AMP membrane electrode to alone (0),to AMP in the presence of lo3 M each of asperglne, arginine, glutamate, ornithine, and serine (U),and to AMP In diluted human control serum (A). AMP
dures) and the level of buffer components is doubled in order to ensure sufficient buffer capacity for a final pH of 7.5 (i.e., 0.2 M Tris-HC1,0.2 M KC1,0.04% NaN& From Figure 6 it can be seen that the response obtained for AMP with the tissue electrode under each of these conditions is identical throughout the linear range of the electrode response. A least-squares analysis of the combined data from all of the responses reveals a correlation coefficient of 0.9967 and B slope of 55.5 mV/decade change in AMP concentration over the concentration range of 1.48 X to 7.6 X M AMP. These results demonstrate that the tissue-based AMP membrane electrode possesses excellent selectivity for AMP over these compounds and that the complex biological matrix of human control serum has no significant effect upon the response for AMP. The selectivity of the tissue-based AMP electrode was further tested by monitoring the response obtained from the following compounds: ADP, ATP, c-AMP, adenosine, adenine, urea, creatinine, and glutamine. Each of these compounds was tested by making standard additions of each to a final concentration of 6.6 X M to the buffer solution while monitoring the response of the tissue-based AMP membrane electrode. Of the compounds tested, only ADP produced a measurable response, as shown in Figure 3. The electrode responded to AMP 3-5 times more quickly than to ADP. From Figure 3 it can be seen that equivalent potentials are obtained at an ADP:AMP concentration ratio of about 50; therefore, a significant interference will exist only for the measurement of AMP in the presence of ADP concentrations approaching this ADP:AMP concentration ratio. Attempts to completely repress the ADP response of the tissue-based AMP electrode by the addition of various inhibitors proved unsuccessful, The presence of myokinase activity (E.C. 2.7.4.3) in rabbit muscle tissue (12)suggests that the following pathway may be responsible for the ADP deamination: myokinase
2ADP
(E.C.2.7.4.3)'
ATP
+ AMP
adenylic acid deamina8e
AMP -izGij+ IMP I- "3
ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
-
1841
1 1 1
Table 11. Effect of Age on the Response of a Rabbit Muscle Based AMP Sensor electrode age, days
slope, mV/decade
coeff
1
59.8
0.9998
2
Ei7.3
0.9996
3
59.0
0.9997
4
59.0
0.9994
5
Ei6.7
0.9994
av * std dev
b8.4
f
linear range, M
COW
limit of detectmion, M
1.3 x 10-41.1 x 10-2 '7.9 x 10-51.5 X lo-' '1.2 x 10-41.3 X lo-' r3.9 x 10-51.1 X 9.4 x 10-51.3 X
(1.0* 0.2) x 10-4(1.3 f 0.2) x
1.3
a Response time when changing the AMP concentration from 6.0 X changing the AMP concentration from 3.0 x to 6.6 X M.
response time, b min
4.4 x 10-5
6
2
4.3 x 10-5
5
3
4.0 x 10-5
7
3
3.9 x 10-5
7
3
4.9 x 10-5
6
3
6i 1
3
(4.3 * 0.4) x
lo-'
response time, a min
to 1.4 X
M.
f
0.5
Response time when l _ l -
~
Table 111. Rabbit Muscle Based AMP Electrode Reproducibility electrode PO.
slope, mV/decade
corr coeff
linear range, M '1.1 x 10-41.1 x 8.9 x 10-51.0 x 11.3 X loT41.1 x 10-2 1.8x 10-41.3 >: 1.7 x 10-41.1 x 101.5 X 6.7 x 10-3 1.3 x 10-47.9 x 10-3 ( l . 4 f 0.3) x (1.0 * 0.2) x 10-2
1
60.2
0.9997
2
57.6
0.9999
3
59.8
0.9998
4
65.4
0.9990
5
56.0
0.9991
6
59.4
0.9987
7
65.4
0.9998
av * std dev
57.7 * 2.1
a Response time when changing the AMP concentration from 1.4 X changing the AMP concentration from 3.0 x to 6.6 x l o M 3M. -.
On the other hand, an ADP deaminase (E.C.3.5.4.7) has also been reported to be present within rabbit muscle cells ( 1 3 ) and could cause an interference via the pathway
ADP
-ADP deaminase
(E.C.3.5.4.7)
JDP -k NHS
(4)
Myokinase activity in known to be dependent upon the presence of magnesium ions or other divalent cations (22). By addition of a chelating agent to the system for the purpose of complexing any divalent cations which might be present, the interfering ADP deamination may be repressed if the interfering activity is due to myokinase activity. The addition of ethylenediaminetetraacetic acid (EDTA), however, shows no effect on the ADP response up to a final EDTA concentration of 10 mM. The presence of fluoride ions is reported to inhibit both myokinase (12)and ADP deaminase (13) activities; however, no effect is observed on the interfering ADP deamination in the presence of 10 mM potassium fluoride. Moreover, the presence of either 10 mM iodoacetamideor 50 mM phosphate does not alter the interfering activity. Lifetime. The lifetime of the AMP tissue-based membrane electrode is affected by the mesh size of the nylon membrane used in its construction. A rapid loss of biocatalytic activity is observed when using a nylon membrane with a mesh size of 149 pm; whereas, with a mesh size of 37 pm, a stable response is obtained. The physical appearance of the electrode surface after soaking for 4 days with the larger mesh size
limit of detection, M
response time,a min
response time, b min
4.4 x 10-5
6
2
2.5 x 10-5
4
3
4.4 x 10-5
6
2
7 . o ~ 10-5
7
5
5.4 x 10-5
3
2
5.4 x 10-5
5.5
3
4.7 x 10-5
3
2
(4.8 f 1.4) x
to 3.4
X LO-4
-
lo-' M.
5
1.5
3f 1
Response time when _ I -
suggests that a loss of tissue is responsible for the decrealse in activity. Table I1 shows the effect of age on the response of the AMP tissue-based membrane electrode. This table summarizes the various response charact,eristics of the electrode system for the first 5 days of use. It can be seen that excellent reproducibility in the electrode response is obtained over this time period. The AMP tissue-based membrane electrode possessesi a useful lifetime of at bast 28 days. Figure 7 shows the response of an AMP tissue-based membrane electrode at ages of 2,5, and 28 days. No chamges in the response time were observed during this time period, and as can be seen in Figure 5 no significant difference in the electrode response is obtained when the electrode siystem is stored in the working buffer at room temperature. These results were verified by using t h e e individual rabbit muscle based AMP membrane electrodles. The lifetime of the stock rabbit muscles from which the tissue membranes were constructed was found to be more than 7 months. AMP tissue-based membrane electrodes prepared from 7-month old rabbit muscle, stored at -25 O C , possessed excellent response characteristics. Electrode Reproducibility. The reproducibility of the response by several individual AMP tissue-based membrane electrodes is reported. Table I11 summarizes the result3 of a study which compares the slopes, linear ranges, limits of detection, and response times of seven separate tissue-based AMP membrane electrodes which were prepared on seven
1042
Anal. Chem. 1981, 5 3 , 1842-1847
lifetime of 4 days (4). Both the response slope and the lifetime are known to be independent upon the amount of biocatalytic activity present at the electrode surface ( 2 , 3 ) . The use of rabbit muscle tissue slices in the construction of an AMP membrane electrode increases the amount of biocatalytic activity approximately 50 times over the enzyme-based system. This increase in the activity results in a sensor with excellent electrode characteristics including a response slope of 57 mV/decade and a lifetime of at least 28 days. This shows the effectiveness of using tissue slices over isolated enzymes in situations where the latter have insufficient biocatalytic activity.
rsoC
LITERATURE CITED (1) (2) (3) (4) (5)
L - _ - l - . - L 4- -
(8) (7) (8) (9)
4-----4
-Log AMP, (MI
(IO)
Flgure 7. Response of the tissue-based AMP electrode to AMP at various electrode ages: (0)day 2; (A)day 5; (0) and day 28.
(1 1)
different days. It can be seen from these results that the response of this electrode system is quite reproducible and reliable from electrode to electrode. Typical electrode characteristics for the previously reported enzyme-based AMP membrane electrode include a slope of 46 mV/decade change in AMP concentration and a useful
(13)
(12)
Frlcke, G. H. Anal. Chem. 1980, 52, 259R-275% Masclni, M.; Liberti, A. Anal. Chlm. Acta 1974, 68, 177-184. Gullbault, G. G.; Tarp, M. Anal. Chlm. Acta 1974, 73, 355-365. Papastathopoulos, D. S.; Rechnitz, G. A. Anal. Chem. 1978, 48, 862-864. Rechnitz; G. A,; Arnold, M. A.; Meyerhoff, M. E. Nature (London) 1979, 278, 466-467. Arnold, M. A,; Rechnltz, G. A. Ana/. Chim. Acta 1980, 113, 351-354. Arnlod, M. A.; Rechnltz, G. A. Anal. Chem. 1980, 52, 1170-1174. Arnold, M. A.; Rechnitz, G. A, Anal. Chem. 1981, 52, 515-518. D'Orazio, P.; Meyerhoff, M. E.: Rechnitz, G. A. Anal. Chem. 1978, 50, 1531- 1534. Wheeler, T. J.; Lowensteln, J. M. Biochemistry 1980, 19, 4564-4567. Vadegama, P. In "Ion-Selective Electrode Methodology"; Covlngton, A, K., Ed., CRC Press: Boca Raton, FL, 1979; Vol. 2, Chapter 2. Noda, L. In "The Enzymes": Boyer, P. D.,Ed.: Academic Press: New York, 1973 Vol. 8, p 279. Webster, H. L. Nature (London) 1935, 172, 453-454.
RECEIVED for review February 2, 1981. Resubmitted May 4, 1981. Accepted July 20,1981. We are grateful to the National Science Foundation (Grant No. CHE-7728158) for supporting this research.
Pulse Voltammetry with Microvoltammetric Electrodes A. G. Ewing, M. A. Dayton, and R. M. Wightman" Depattmenl of Chemistry, Indiana University, Bloomington, Indiana 47405
Microvoltammetric electrodes can be used to provide reproduclble results In an extremely complex chemlcal medluml the mammalian brain. Because of the small size of the microelectrodes, current on the back step of a chronoamperometric experlment is essentlally nonfaradalc and can be used for residual current correction. The use of normal pulse voltammetry mlnlmlres electrode response deterloratlon caused by fllmlng of the electrode surface by electrogeneratedproducts.
The response Of these electrodes In is to that of other electrodes; it Is not altered by the repetition rate of the pu's@s9and dopaminel a Of prime neurochemlcal Interest, can be partially resolved from 8scorblc add and dihYdroxYPhenYlacetlc acid by in VIVO VOltammetry.
Continuous monitoring of electroactive species in complex media with solid voltammetric electrodes is a very difficult problem because the electrochemical response tends to deteriorate with time. The goal of in vivo voltammetry and of 0003-2700/81/0353-1842$01.25/0
most electroanalysis schemes is to measure the concentration of electroactive species by the observed current and to identify the species via half-wave potential. This goal requires that the electrode retain constant Properties throughout the experiment* When the electrode surface cannot be renewed during the experiment, alternate procedural strategies to this In this paper, we demonstrate problem must be that voltammograms can be Obtained with microvoltammetric electrodes using a pulse potential wave form. The microvo~~etric are fabricakd from carbon fibers and have previously been shown to exhibit several unique properties that facilitate electroanalysis (1,2). The utility of these electrodes is demonstrated here by voltammograms in an extremely complex chemical environment, the mammalian brain. In the early 1970s, €3. N. Adanis recognized that several neurotransmitters (small molecules which relay information between neurons) are easily oxidized and, thus, should be determined with carbon electrodes. This observation led to the development of a number of analytical techniques for the determination of these compounds (3) including the use of 0 1961 American Chemical Society
