Anal. Chem. 1980, 52, 1937-1940
1937
boundary. At low enzyme loading, the sensor becomes stirring sensitive at a significantly lower penicillin concentration. When another layer of albumin membrane was applied over the penicillin-sensitive gate, stirring sensitivity was supressed but at the expense of the response time of the probe. When not in use the probes were immersed in 0.02 M phosphate buffer and stored in the refrigerator. Under these conditions the probes had a lifetime of 2 months with only a slight loss of activity. Failure was usually due to excessive leakage current through the gate or encapsulation. Due to its small sensing area (0.5 mm2),the E N F E T requires only a small amount of enzyme (2.5 X IU). This points to a significant savings when more expensive enzymes are used. Because the membrane adheres well to the epoxy encapsulation, no retaining membrane is needed. This is another advantage of the ENFET probe as compared to a conventional macroelectrode. Although the overall performance characteristics of penicillin E N F E T are similar to those observed with analagous enzyme macroelectrodes, there are some significant improvements. The differential mode of measurement is easily accomplished with resulting temperature and ambient pH stability of the probe. Long lifetime, rapid time response, small size (economy), and no need for a retaining membrane are other positive facets of this new probe.
perature. This is particularly important for operation in low buffer capacity media where the sensitivity is the highest (Table I). The differential current operation also provides temperature compensation ( I O ) if the temperature sensitivity of the two gates is approximately equal. Our results have shown that there is no effect on differential temperature sensitivity in the presence or in the absence of penicillin. Therefore, the temperature sensitivity of the penicillin E N F E T probe depends mainly on the temperature characteristics of the individual ISFETs and not on the temperature dependence of the penicillinase kinetics within the temperature range studied. The lower limit depends on the lowest detectable steady-state change of p H which is due to the enzymatically catalyzed reaction. This limit, which is a complex function of diffusion rates and enzyme activity (12),was found to be approximately 0.2 m M for our sensors. T h e upper limit of the linear range of the response curve increases with high buffer capacity; however, the sensitivity of the electrode decreases. Figure 5 shows that the upper limit is -70 mM. This corresponds to a pH a t the albumin membrane/Si,N4 interface of 4.7. Since the pH dependence of penicillinase activity is bell shaped with maximum at pH 7.0, this would correspond to a reduction of the penicillinase activity (13) by 72%. Since the p H varies as a function of distance through the membrane, the steady-state enzyme activity is also a complex function of distance, substrate concentration, and diffusion rates. Thus, at high penicillin concentrations the increase in sensitivity due to the decrease in buffer capacity is balanced by the loss of activity of the enzyme. When a more acidic buffer like citrate is used instead of a 0.01 M phosphate buffer, the sensitivity and linear range of the E N F E T probe are decreased. In the HEPES-PIPES buffer system, the buffer capacity is constant between pH 6.8 and 7.55. The sensitivity is again decreased compared to phosphate buffer. The numerical solution of the diffusion kinetic model for enzymatic potentiometric probes for general steady-state conditions has been published recently (12). In that model the rate constants are independent of the p H and, therefore, the upper limits are much higher than those found for the penicillin sensors. The penicillin E N F E T is sensitive to stirring a t high penicillin concentrations. This is apparently due to the variation of fluxes of H+ and penicillin at the membrane/solution
LITERATURE CITED Guilbault. G. G. In "Comprehensive Analytical Chemistry"; Svehla, G., Ed.; Elsevier: Amsterdam, 1977; Vol. 8. PaDariello, G. J.: Mukherhi. A. K.: Shearer. C. M. Anal. Chem. 1973. 45, 790. Nilsson, H.; Akerlund, A. C.; Mosbach, K. Biochim. Biophys. Acta 1973. 320, 529. Nilsson, H.; Mosbach. K. Biotechnol. Bioeng. 1978, 20, 527. Enfors, S. 0.; Nilsson, H. Enzyme Microb. Techno/. 1979, 1 , 260. Janata, J.; Moss S. Biomed. Eng., 1976, 11, 241. Danielsson. B.; Lundstrom. I . ; Mosbach, K.; Stiblert, L. Anal. Lett. 1979, 12, 1189. Janata, J.; Huber, R. Ion-Sei. Electrodes, Rev. 1979, 1, 31. Broun, G. 6 . Methods Enzymol. 1976, 4 4 , 263. Comte, P. A.; Janata, J. Anal. Chim. Acta 1978, 101, 247. Moss, S. D.; Smith, J. B.; Comte, P. A,; Johnson, C. C.; Astle, J. J . Bioeng. 1976, 1 . 11. Brady, J. E.; Carr, P. W. Anal. Chem. 1980, 52, 977. Waley, S. G. Biochem. J . 1975, 149, 547.
RECEIVED for review May 28, 1980. Accepted July 21, 1980. The authors gratefully acknowledge support of this work from the National Institutes of Health, Grant No. NIGMS 22952.
Adenosine-Selective Electrode Inna Deng and Chris Enke" Department of Chemistty, Michigan State University, East Lansing, Michigan
An adenosine (adenosine riboside)-selective electrode has been devised which uses the enzyme adenosine deaminase in conjunction with an ammonia gas sensing membrane electrode. The resulting electrode is capable of detecting adenosine at the micromolar level at pH 9.0 and 37 O C . Operating variables have been critically examined to define conditions for optimum linearity and sensitivity.
can be reused hundreds or thousands of times. Many organic and biological compounds for which simple analyses were not available can now be determined by reaction with immobilized enzymes and electrodes sensitive to a reaction product (1,2 ) . The electrodes, which are often ion- or gas-selective electrodes, provide simple and fast analyses. Further, the sample solutions remain appreciably uncontaminated and can be reused for other tasks. An electrode developed as sensor for the nucleotide 5'adenosine monophosphate (5'-AMP) has been reported ( 3 ) as well as electrodes that determine adenosine deaminase activity ( 4 ) . We now have developed a sensitive electrode for
T h e development of immobilized enzymes has given the analytical field a powerful new process. Immobilized enzymes 0003-2700/80/0352-1937$01 .OO/O
48824
CZ
1980 American Chemical Society
1938
ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
the nucleoside adenosine (adenine riboside), which has a detection limit in the range of 0.7-5.0 pM and is 10-20 times more sensitive than similar electrodes reported by other workers (3, 5-8). The present paper is concerned with the design and characteristics of the adenosine sensor. Mention of a n adenosine electrode has also been made in a recent paper by Rechnitz e t al. (9). T h e adenosine electrode is constructed with a layer of a suspended mixture of adenosine deaminase and bovine serum albumin between a dialysis membrane and the gas permeable membrane of an ammonia-sensing membrane electrode. Adenosine is deaminated according to the equation
t
adenosine deaminase
adenosine (adenine riboside) + H 2 0 inosine (hypoxanthine riboside)
, + NH3
When the electrode is in contact with a sample solution containing adenosine, the NH3 concentration produced at the electrode surface by the above reaction is proportional to the concentration of the adenosine. Thus the electrode potential responds to the adenosine concentration. The resulting electrode has excellent sensitivity and selectivity for adenosine. This study shows that the sensor response function depends upon pH and temperature and that the optimal pH for the electrode is much higher than that for the enzyme reaction alone. EXPERIMENTAL SECTION Apparatus. The ammonia gas sensing electrode was an Orion Model 95-10. The electrode consists of a combination pH electrode outfitted with a gas-sensitive membrane and an internal solution M NH4Cl. Electrode potential measurements were of 5 X carried out with a Markson ElectroMark analyzer. The readings were recorded manually. Measurements were made in a thermostated water bath with a precision of k0.2 "C. A Model 2400 Beckman spectrophotometer was employed for the spectrophotometric rate determination of enzyme activity. Reagents. All chemicals used were reagent grade. Freshly deionized ammonia-free water was used to prepare all solutions. The enzyme used in the present study was adenosine deaminase (type 111, from calf intestinal mucosa, activity 235 units/mg of protein, about 5 mg/mL solution, Sigma Chemical Co.). The enzyme was used without further purification. A 15% solution of albumin (from serum bovine, crystallized and lyophilized, containing 1-37' globulin, Sigma) was prepared in 0.5 M Tris-HC1 buffer pH 7.5. The stock solutions of adenosine, adenine, 5'-AMP, 2'-AMP, 3'-AMP, and 2',3'-cyclic AMP were prepared in Tris-HC1 buffer, 0.05 M. Adenosine was found to have a solubility limit of about lo-* M in the buffer at room temperature. T o test the NH3 response of the NH, electrode, we prepared a stock solution of 0.1 M ammonium chloride with deionized water. Procedure. The enzyme electrode used in the present study was prepared by dispersing 7 pL of enzyme solution (corresponding to - 8 units at pH 7.5 and 25 "C) and 3 pL of 15% albumin solution evenly over the gas permeable membrane of the ammonia electrode. A piece of dialysis paper was placed over the enzyme layer to trap the enzyme and albumin from diffusion into the solution. The enzyme electrode was soaked with stirring in 0.05 M Tris-HC1 buffer for at least 1 h before use. Leaking sometimes occurred, but it was easily detected by observing a yellow coloration of the buffer solution. The electrode was stored in 0.05 M Tris-HC1 buffer, pH 7.0, and refrigerated at 4 "C. All the solutions prepared were tested for ammonia contamination with the ammonia sensor before use. No ammonia contamination was found in any of the solutions prepared. RESULTS AND DISCUSSION The adenosine electrode in 0.05 M Tris-HC1 buffer. pH 9.0, at 37 "C has a linear response over the 7 X to lo-* M substrate concentration range with a slope of 55 mV per decade. The potential measured is the difference of the potential response in the buffer background and the sample solution. Below 2 x M, no potential difference is observed
'
I e 6
IO
PH
Effect of pH on enzyme activity: (0)0.05 M Tris-HCI buffer, (A)0.02 M phosphate buffer. Figure 1.
120t
t
\
W
L I
0'
-6
8I
IO
PH
Adenosine electrode response as function of pH. All responses at 25 'C: ( 0 )0.05 M Tris-HCI buffer, (A)0.02 M phosphate M adenosine: curve buffer; curve A, buffer background: curve B, C. the differences of curve A and curve B. Figure 2.
between the buffer and sample solutions. Effect of p H on Electrode Sensitivity. The concentration of ammonia in an aqueous solution is pH dependent, as is the enzyme activity of a given amount of enzyme. In aqueous solutions, the pH has a positive effect on the ratio of ammonia to ammonium with ammonia being the predominate form a t pH 11 or above ( I O ) . Unlike many other enzymes, adenosine deaminase is active over a very wide pH range (11). Our determination of enzyme activity as a function of pH is shown in Figure 1. Although the p H of maximum activity is observed near the neutral point, the activities at pH 9 and pH 6 are 63% and 78% relative to that of pH 7. Since the enzyme activity remains high a t pH greater than 7, and the ammonia/ammonium ratio is greater a t higher pH, the maximum sensor sensitivity is expected to occur at a p H appreciably greater than 7 . As shown in Figure 2, the potential measured in both the buffer background and the sample solution decreases with rising pH. However, the difference in voltage between buffer and sample rises rapidly with p H and achieves a maximum near pH 9.0. The pH of maximum sensor sensitivity is far more basic than the optimum p H for the enzyme reaction itself, which is at pH 7 . At pH 9.0, the sensor is 8 times more sensitive than a t pH 7.0 and 4 times greater than at pH 7.5. Further investigations of the response in various concentrations a t pH 9.0 and p H 7 . 5 (Figure 3) demonstrate that
ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
r
1939
Table I. Selectivity Studies for Adenosine Electrodea AmV ( m v buffer - mV solution) 3'5'substrate adeno- ade- 2',3'- 2'concn, M sine nine AMP AMP AMP AMP 36 80 130 171
10-5 10-4 10-3 a
0 0
0
0
o
0 0 2
-1
1
0
-4
0
o
0 0
0 0 0 2
Tris-HC1 buffer. pH 9.0, 37 "C.
Table 11. Long-Time Behavior of the Adenosine Electrodea response slope, detection day mVIdecade limit, M 1
I
6
1
4
c
130
A
55 46 43 42 46 43 27
7 x 10.' 2 x 5x 5x 5x 5 x lo+ 10-5
2
[adenosine] Figure 3. Calibration curves for adenosine electrode as function of pH and temperature (all curves in 0.05 M Tris-HCI buffer): curve A , PH 9.0, 37 0C; curve B, pH 9.0, 25 "C; curve c, PH 7.5, 37 "c; curve D, pH 7.5, 25 "C. -log
401
3 4 6 7 9 32
1
1
1
30
35
40
Temperoture ("C!
Effect of temperature on response of adenosine electrode (all curves in 0.05 M Tris-HCI buffer, pH 9.0): curve A, buffer background, curve B, lo-' M adenosine; curve C, the differences of curve A and curve B. Figure 4.
higher sensitivity and greater linearity are obtained a t pH 9.0. A rational explanation of the observed maximum response at p H 9.0 is that the 37% decrease in enzyme activity at pH 9.0 is offset by the increased fraction of the NH3 produced that remains in the NH3 form. Effect of Temperature on the Electrode Sensitivity. A temperature increase has a positive effect on the diffusion rate, the partial pressure of ammonia, and the enzyme activity. Therefore, the electrode sensitivity is expected to vary with temperature. The electrode response to M of adenosine was measured at 25, 30, 32, 35, and 37 "C (Figure 4). The response is essentially constant between 25 and 30 "C, but
a
Tris-HC1 buffer, pH 9.0, 37 "C.
above 30 "C, a dramatic increase in response was observed. The effect of temperatures greater than 37 "C (body temperature) was not studied because of the increasing risk of causing denaturation of the enzyme. The electrode response to different concentrations of adenosine was measured a t 25 and 37 "C in solutions of pH 7.5 and pH 9.0; the results are shown in Figure 3. The electrode exhibits higher sensitivity and more linear response at 37 "C for both pH 7.5 and p H 9.0. Response Time. The response time, i.e., the time required for a steady-state potential to be reached, varies with substrate concentration. In this experiment, the potential is considered to have reached a steady state when it changes less than 1 mV in a 2-min period. The response time is about 7-12 min for concentrations below M and shortens to 6-10 min in the concentration range of to M and to 2-4 min in M solution at p H 9.0 and 37 "C. The time required for steady-state potential to be reached was longer at lower temM adenosine at pH 9.0 were perature. Response times for 7, 20, and 27 min at 37, 30, and 25 "C, respectively. Higher temperatures thus have a positive effect on both electrode sensitivity and response speed. Selectivity Study. It was reported that the adenosine deaminase from the intestine is specific for adenosine and will not deaminate other adenine derivatives or inosine (hypoxanthine riboside) (12, 13). However, it is difficult to completely separate the deaminase from the potent phosphatase present in the intestine (13). Sigma Chemical Co. has indicated that the commercial deaminase used in this research has a trace (
