Press, New York. N.Y.. 1965. M. J. D. Brand and G. A. Rechnitz, Anal. Chem., 41, 1185 (1969); 42, 478 (1970). W. Jaenicke, E. M. Khairy. and W. Schaefer, 2.Elekhochem., 70, 421 (1966). H. Rickert, “Einfuhrung in die Elektrochemie fester Stoffe”. Springer Verlag, Berlin, 1973. M. Koebel, Dissertation No. 4853, Zurich, 1972. E. Budewski. Electrochim. Acta, 11, 1697 (1967). H. Gerischer, Z.Elektrochem.,54, 366 (1950). K. Camman, Dissertation, University of Munich, 1975. C. Tubandt and H. Reinhold, Z.Elektrochem.,37, 589 (1931). M. S. Frant and J. W. Ross, Science, 154, 1533 (1966). R. P. Buck and I. Krull, J. Electroanal. Chem., 18, 387 (1968). J. W. Ross, Natl. Bur. Stand. (U.S.), Spec. Pub/.,No. 314 (1969).
(19) H. Gerischer, Z.Phys. Chem. (Frankfurtam Mein). 26, 223 (1960): 27, 48 (1961). (20) T. F. Tadros and J. Lyklema, J. Electroam/. Chem., 22, 91 (1969). (21) C. Wagner and W. Trand. Z.Elektrochem., 44, 391 (1938). (22) G. Kimball and A. Glassner. J. Chem. Phys., 8,815 (1940). (23) J. 0’.M. Bockris, “Modern Aspects of Electrochemistry”, Vol. 1, J. 0’. M. Bockris and B. E. Conway, Ed., Butterworths,London, 1954.
RECEIVEDfor review November 10,1975. Accepted February 2, 1976. We thank the National Science Foundation and the Deutsche Forschungsgemeinschaft for financial support of this research.
Highly Selective Enzyme Electrode for 5’-Adenosine Monophosphate D. S. Papastathopoulos and G. A.
Rechnitz’
Department of Chemistry, State University of New York, Buffalo, N. Y. 142 14
A 5’-AMP sensing electrode Is devised using a highly seiective deaminase enzyme in conjunction with an ammonia gas sensing membrane electrode. The resulting nucleotide sensor is very highly Selective for 5’-AMP with good operating sensitivity and dynamic response. Optimum conditions for the concentration and immobilization of the enzyme are explored in terms of electrode operating requirements.
Wide attention has recently been given to the development of membrane electrode sensors selective for various biological substrates using immobilized enzymes in conjunction with potentiometric ion or gas sensing membrane electrodes. Various analytical and biochemical aspects of this field have been the subject of recent reviews and articles (1-7) especially in regard to electrodes for major body fluid constituents and pharmaceuticals. In one of these reviews ( 8 ) , attention was called to the possibility of devising such electrodes as sensors for nucleotides with particular emphasis on an electrode for 5’-adenosine monophosphate (AMP). We now report on the construction and evaluation of the AMP sensor in detail. It will be seen that the AMP sensing electrode has good sensitivity and excellent selectivity characteristics provided optimum enzyme activity and solution conditions are achieved. The new electrode employs a layer of suspended 5’-adenylic acid deaminase enzyme (AMP deaminase), classification number EC 3.5.4.6, in conjunction with an ammonia gas sensing membrane electrode. The substrate is selectively deaminated to inosine 5’-monophosphate (5’-IMP) via the reaction 5’-AMP
+ HzO
Enzyme
5’-IMP
+ NH3
(1)
producing NH3 in stoichiometric quantities and gives rise to a steady-state potential reading reflecting the AMP concentration in the sample to be measured. Use of the gas sensing electrode as a component of the sensor ensures freedom from ionic interferences. EXPERIMENTAL Apparatus. An Orion Model 95-10 ammonia gas sensing electrode was employed in construction of the enzyme electrode. No 862
ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
external reference electrode is required with this design. Potential measurements were carried out with a Corning Model 1 2 meter and were recorded using a Heath-Schlumberger SR-255B recorder. Measurements were made in a thermostated cell held a t 27 f 0.2 “C. A Beckman BD-G spectrophotometer, with thermostated cell compartment, was employed for the optical enzyme activity determinations. Reagents. The AMP deaminase enzyme (grade IV, from rabbit muscle) was obtained from Sigma Chemical Co., St. Louis, Mo., as were the substrates 5’-AMP, 3’,5’-cyclic AMP, 5’-ADP, 5’-ATP, adenosine, and adenine used for selectivity studies. The enzyme is received as a suspension in 66% glycerol, containing 0.33 M KC1 a t pH 7.4, with an enzyme activity of 30 unitdml. Working substrate solutions were prepared in 0.05 M Tris-HC1 buffer, pH 7.5, and stored under refrigeration. 5’-AMP solutions were also prepared in 0.1 M citrate buffer, pH 6.50, 0.1 M succinate buffer, pH 6.40, and in 0.05 M Tris-HC1 buffer a t pH 7.00 and pH 8.4. All substrates were tested for possible ammonia contamination with the Orion 95-10 electrode. Both the 5’-AMP (sodium salt) and 3’,5’-cyclic AMP were found to contain appreciable ammonia background levels and were purified by recrystallization and ion exchange (Baker, ANGC-101 resin), respectively, to negligible ammonia levels prior to use. Enzyme Concentration Procedure. Since the enzyme, as received, had an activity of only 30 units/ml, it was thought desirable to raise enzyme activity by concentration. For this purpose, a molecular filtration procedure was employed (9).One hundred units of the enzyme suspension were passed through a PSED Pellicon molecular filter (25 000 molecular weight cut off) a t 4 OC under 50 psi nitrogen gas pressure over a 16-18 h time period. Enzyme activity was measured spectrophotometrically a t 265 nm using the procedure recommended by the supplier (IO). The concentrated enzyme preparation obtained after 16 h has an activity of -90 units/ml. This preparation was stored at 4 OC and periodically tested for enzyme activity. No significant loss of activity was observed over a 2-month period. Electrode Preparation. The 5’-AMP enzyme electrode was assembled using the general techniques previously described for the urea electrode (11). In the present case, 10 pl of the concentrated enzyme preparation (corresponding to -0.9 unit) was placed between a circular cellophane dialysis membrane and the gas permeable membrane of the ammonia electrode. The resulting electrode was preconditioned by soaking for at least 3 h in 0.05 Tris-HC1 buffer, pH 7.50, and was also stored in this buffer when not in use.
RESULTS AND DISCUSSION Figure 1 shows a schematic representation of the phases comprising the enzyme electrode for 5’-AMP and identifies some of the key steps involved in the overall response of the electrode system to the substrate in the sample solu-
110-
140
-
E
100-
W AMP,
Enzyme,
0
IMP,
"3,
Figure 1. Schematic of 5'-AMP electrode phases and processes -1opAMP i 0 n c . M
~
~
~
~
_
_
~
_
_
_
~
~
~
~
Figure 2. Calibration curve for 5'-AMP electrode
~
Table I. Reproducibility of the 5'-AMP Electrodea
0.05 M Tris-HCI buffer, pH 7.50. 27 OC.
Potential, mV 5'-AMP concn, M
Day 1
Day 2
Day 3
Day 4
8 x 10-5 2 x 10-4 6X 2.2 x 10-3 7.6 x 10-3 1.5 X
176.2 161.0 138.8 109.8 86.8 74.1
177.0 164.2 140.1 107.4 88.9 73.2
179.0 162.3 136.7 110.5 86.1 74.8
178.3 163.0 139.2 110.6 87.2 75.3
-46.6
-46.3
Response slope, -45.8 -46.7 mvldecade (Tris-HC1buffer, pH 7.50, 27 "C)
501
1
4
3
2
-100 A M P C O n C M
tion. The dialysis membrane not only holds the enzyme layer in place but also serves to keep out any high molecular weight materials that might be present in the sample. A typical calibration curve is given in Figure 2, for the electrode response to 5'-AMP in 0.05 M Tris-HC1 buffer, pH 7.50, at 27 "C. The electrode shows a linear response over the 8 X to 1.5 X M substrate concentration range with a slope of -46 mV per decade and a correlation coefficient of 0.9994. Day to day reproducibility of electrode response is given in Table I and is typical of enzyme electrodes using this type of construction. There is no deterioration of electrode response over the 4-day period illustrated, but a gradual decline in response slope and sensitivity was noted with further aging of the electrode. The time required for a steady-state potential to be reached was found to depend upon the substrate concentration level. In the to M concentration range, response times of the order of 6 min were found, while a t conM, response times were centrations approaching shortened to -2 min. These values are comparable to those found for the earlier urea electrode. Since the selection of optimum solution conditions represents, by necessity, some compromise between optimum conditions for the enzyme reaction and those of the "3NH4+ equilibrium (12) involved in the gas electrode, the effects of pH and buffer composition were studied in some detail. The reported (13-15) maximum activity of the deaminase enzyme varies over a wide pH range in different buffers, e.g., pH 5.9-6.6 in succinate, pH 6.4 in citrate, and pH 6.8-7.5 in Tris buffer. Figure 3 shows the results obtained for the response of the 5'-AMP electrode when tested in succinate, citrate, and Tris buffers. Because of the necessary compromise between optimum enzyme activity and optimum response of the NH3 gas electrode, it is clear that the most satisfact.ory range and linearity is found in the Tris buffer. Further investigations in this buffer system
Figure 3. Calibration curves for 5'-AMP electrode in various buffers. Curve A , 0.1 M succinate buffer, pH 6.40; Curve B, 0.1 M citrate buffer, pH 6.50; Curve C,0.05 M Tris-HCI buffer, pH 7.50
t 160-
80
40 -lop AMP con
(19) H. Gerischer, Z.Phys. Chem. (Frankfurtam Mein). 26, 223 (1960): 27, 48 (1961). (20) T. F. Tadros and J. Lyklema, J. Electroam/. Chem., 22, 91 (1969). (21) C. Wagner and W. Trand. Z.Elektrochem., 44, 391 (1938). (22) G. Kimball and A. Glassner. J. Chem. Phys., 8,815 (1940). (23) J. 0’.M. Bockris, “Modern Aspects of Electrochemistry”, Vol. 1, J. 0’. M. Bockris and B. E. Conway, Ed., Butterworths,London, 1954.
RECEIVEDfor review November 10,1975. Accepted February 2, 1976. We thank the National Science Foundation and the Deutsche Forschungsgemeinschaft for financial support of this research.
Highly Selective Enzyme Electrode for 5’-Adenosine Monophosphate D. S. Papastathopoulos and G. A.
Rechnitz’
Department of Chemistry, State University of New York, Buffalo, N. Y. 142 14
A 5’-AMP sensing electrode Is devised using a highly seiective deaminase enzyme in conjunction with an ammonia gas sensing membrane electrode. The resulting nucleotide sensor is very highly Selective for 5’-AMP with good operating sensitivity and dynamic response. Optimum conditions for the concentration and immobilization of the enzyme are explored in terms of electrode operating requirements.
Wide attention has recently been given to the development of membrane electrode sensors selective for various biological substrates using immobilized enzymes in conjunction with potentiometric ion or gas sensing membrane electrodes. Various analytical and biochemical aspects of this field have been the subject of recent reviews and articles (1-7) especially in regard to electrodes for major body fluid constituents and pharmaceuticals. In one of these reviews ( 8 ) , attention was called to the possibility of devising such electrodes as sensors for nucleotides with particular emphasis on an electrode for 5’-adenosine monophosphate (AMP). We now report on the construction and evaluation of the AMP sensor in detail. It will be seen that the AMP sensing electrode has good sensitivity and excellent selectivity characteristics provided optimum enzyme activity and solution conditions are achieved. The new electrode employs a layer of suspended 5’-adenylic acid deaminase enzyme (AMP deaminase), classification number EC 3.5.4.6, in conjunction with an ammonia gas sensing membrane electrode. The substrate is selectively deaminated to inosine 5’-monophosphate (5’-IMP) via the reaction 5’-AMP
+ HzO
Enzyme
5’-IMP
+ NH3
(1)
producing NH3 in stoichiometric quantities and gives rise to a steady-state potential reading reflecting the AMP concentration in the sample to be measured. Use of the gas sensing electrode as a component of the sensor ensures freedom from ionic interferences. EXPERIMENTAL Apparatus. An Orion Model 95-10 ammonia gas sensing electrode was employed in construction of the enzyme electrode. No 862
ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
external reference electrode is required with this design. Potential measurements were carried out with a Corning Model 1 2 meter and were recorded using a Heath-Schlumberger SR-255B recorder. Measurements were made in a thermostated cell held a t 27 f 0.2 “C. A Beckman BD-G spectrophotometer, with thermostated cell compartment, was employed for the optical enzyme activity determinations. Reagents. The AMP deaminase enzyme (grade IV, from rabbit muscle) was obtained from Sigma Chemical Co., St. Louis, Mo., as were the substrates 5’-AMP, 3’,5’-cyclic AMP, 5’-ADP, 5’-ATP, adenosine, and adenine used for selectivity studies. The enzyme is received as a suspension in 66% glycerol, containing 0.33 M KC1 a t pH 7.4, with an enzyme activity of 30 unitdml. Working substrate solutions were prepared in 0.05 M Tris-HC1 buffer, pH 7.5, and stored under refrigeration. 5’-AMP solutions were also prepared in 0.1 M citrate buffer, pH 6.50, 0.1 M succinate buffer, pH 6.40, and in 0.05 M Tris-HC1 buffer a t pH 7.00 and pH 8.4. All substrates were tested for possible ammonia contamination with the Orion 95-10 electrode. Both the 5’-AMP (sodium salt) and 3’,5’-cyclic AMP were found to contain appreciable ammonia background levels and were purified by recrystallization and ion exchange (Baker, ANGC-101 resin), respectively, to negligible ammonia levels prior to use. Enzyme Concentration Procedure. Since the enzyme, as received, had an activity of only 30 units/ml, it was thought desirable to raise enzyme activity by concentration. For this purpose, a molecular filtration procedure was employed (9).One hundred units of the enzyme suspension were passed through a PSED Pellicon molecular filter (25 000 molecular weight cut off) a t 4 OC under 50 psi nitrogen gas pressure over a 16-18 h time period. Enzyme activity was measured spectrophotometrically a t 265 nm using the procedure recommended by the supplier (IO). The concentrated enzyme preparation obtained after 16 h has an activity of -90 units/ml. This preparation was stored at 4 OC and periodically tested for enzyme activity. No significant loss of activity was observed over a 2-month period. Electrode Preparation. The 5’-AMP enzyme electrode was assembled using the general techniques previously described for the urea electrode (11). In the present case, 10 pl of the concentrated enzyme preparation (corresponding to -0.9 unit) was placed between a circular cellophane dialysis membrane and the gas permeable membrane of the ammonia electrode. The resulting electrode was preconditioned by soaking for at least 3 h in 0.05 Tris-HC1 buffer, pH 7.50, and was also stored in this buffer when not in use.
RESULTS AND DISCUSSION Figure 1 shows a schematic representation of the phases comprising the enzyme electrode for 5’-AMP and identifies some of the key steps involved in the overall response of the electrode system to the substrate in the sample solu-
110-
140
-
E
100-
W AMP,
Enzyme,
0
IMP,
"3,
Figure 1. Schematic of 5'-AMP electrode phases and processes -1opAMP i 0 n c . M
~
~
~
~
_
_
~
_
_
_
~
~
~
~
Figure 2. Calibration curve for 5'-AMP electrode
~
Table I. Reproducibility of the 5'-AMP Electrodea
0.05 M Tris-HCI buffer, pH 7.50. 27 OC.
Potential, mV 5'-AMP concn, M
Day 1
Day 2
Day 3
Day 4
8 x 10-5 2 x 10-4 6X 2.2 x 10-3 7.6 x 10-3 1.5 X
176.2 161.0 138.8 109.8 86.8 74.1
177.0 164.2 140.1 107.4 88.9 73.2
179.0 162.3 136.7 110.5 86.1 74.8
178.3 163.0 139.2 110.6 87.2 75.3
-46.6
-46.3
Response slope, -45.8 -46.7 mvldecade (Tris-HC1buffer, pH 7.50, 27 "C)
501
1
4
3
2
-100 A M P C O n C M
tion. The dialysis membrane not only holds the enzyme layer in place but also serves to keep out any high molecular weight materials that might be present in the sample. A typical calibration curve is given in Figure 2, for the electrode response to 5'-AMP in 0.05 M Tris-HC1 buffer, pH 7.50, at 27 "C. The electrode shows a linear response over the 8 X to 1.5 X M substrate concentration range with a slope of -46 mV per decade and a correlation coefficient of 0.9994. Day to day reproducibility of electrode response is given in Table I and is typical of enzyme electrodes using this type of construction. There is no deterioration of electrode response over the 4-day period illustrated, but a gradual decline in response slope and sensitivity was noted with further aging of the electrode. The time required for a steady-state potential to be reached was found to depend upon the substrate concentration level. In the to M concentration range, response times of the order of 6 min were found, while a t conM, response times were centrations approaching shortened to -2 min. These values are comparable to those found for the earlier urea electrode. Since the selection of optimum solution conditions represents, by necessity, some compromise between optimum conditions for the enzyme reaction and those of the "3NH4+ equilibrium (12) involved in the gas electrode, the effects of pH and buffer composition were studied in some detail. The reported (13-15) maximum activity of the deaminase enzyme varies over a wide pH range in different buffers, e.g., pH 5.9-6.6 in succinate, pH 6.4 in citrate, and pH 6.8-7.5 in Tris buffer. Figure 3 shows the results obtained for the response of the 5'-AMP electrode when tested in succinate, citrate, and Tris buffers. Because of the necessary compromise between optimum enzyme activity and optimum response of the NH3 gas electrode, it is clear that the most satisfact.ory range and linearity is found in the Tris buffer. Further investigations in this buffer system
Figure 3. Calibration curves for 5'-AMP electrode in various buffers. Curve A , 0.1 M succinate buffer, pH 6.40; Curve B, 0.1 M citrate buffer, pH 6.50; Curve C,0.05 M Tris-HCI buffer, pH 7.50
t 160-
80
40 -lop AMP con
