Anal. Chem. 1980, 52, 1935-1937
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Field Effect Transistor Sensitive to Penicillin Steve Caras and Jig Janata" Deparfment of Bioengineering, University of Utah, Salt Lake City, Utah 84 112
A feasibility study of an enzyme-coupled field effect transistor has been done. This device was constructed by depositing a co-cross-llnked penicllllnase-albumin layer over a pH-sensitive field effect transistor. The differential mode of measurement largely eliminates the temperature sensitivity and the effects of ambient pH variation. The new probe has a llfetime of 2 months and time response T~~ = 25 s. The range and sensitivity are comparable to the conventional penicillinsensitive macroelectrodes. The small size of the sensitive gate requires only a minute amount of enzyme (-2.5 X lo-' I U ) which could prove to be an important factor in construction of other enzymatic sensors utilizing more expensive enzymes.
Enzyme electrodes have proved to be useful in biochemical analysis and have been developed and characterized for a variety of substrates ( 1 ) . Papariello e t al. (2) and independently Nilsson et al. (3)developed a penicillin enzyme electrode by immobilizing penicillinase over a p H glass electrode. In these probes penicillinase catalyzes the hydrolysis of penicillin t o penicilloic acid according to the reaction R CO N H
p,---; ",
penicillinase
0
CO2
R C O N H ~ "
Ho-l 0
Penicilloic acid is a strong acid which releases protons and depresses the p H at the surface of the p H electrode. Nilsson e t al. ( 4 ) and Enfors e t al. ( 5 ) have characterized similar penicillin enzyme electrodes and have found that their sensitivity depends on the buffer capacity of the bulk solution. T h e possibility of a n enzymatically coupled ion-sensitive field effect transistor (ENFET) has been postulated ( 6 ) ,and recently Danielsson et al. (7) described a urea-sensitive device on the basis of their gas-sensing FET and called their device a n "enzyme transistor". As with conventional ion-sensitive electrodes (ISEs), ionsensitive field effect transistors (ISFETs) can be made sensitive to different organic substrates by immobilizing a suitable enzyme layer over the surface of an I S F E T gate. T h e primary purpose of this work has been to demonstrate t h e feasibility of a directly operating enzymatically coupled field effect transistor. A penicillin-sensitive transistor was chosen for two reasons: first, a p H ISFET is the simplest ISFET because it does not require an additional ion-selective membrane. Second, analogous penicillin-sensitive electrodes have been well characterized (2-51, and their performance characteristics could serve as a standard for our devices.
EXPERIMENTAL SECTION Materials. Unless stated otherwise, all solutions were prepared from analytical reagents and deionized 15-MR water. Solutions of bovine serum albumin (5, 10, and 15%) (BSA) were prepared in 0.02 M phosphate buffer, pH 6.8. Glutaraldehyde (2.5% v/v) for cross-linking was prepared by diluting 25% stock solution with water. Penicillinase ((3-lactamasefrom Bacillus cereus, EC 3.5.2.6, 350 IU/mg, Sigma, or 66 IU/mg, Calbiochem) was dissolved in 0.02 M phosphate buffer, pH 6.8, to a unit concentration of 16000 0003-2700/80/0352-1935$0 1.OO/O
IU/mL (Sigma) or 3770 IU/mL (Calbiochem). Penicillin G (benzylpenicillin, Na salt, 1675 IU/mg, Sigma) was dissolved in an appropriate buffer to 100 mM. DL-I,ySine monohydrochloride (0.1 M) (Sigma) in 0.02 M phosphate buffer, pH 6.8, was used to terminate the cross-linking reaction. Device Construction. The transistor chip was glued on a 6 F (2.0 mm diameter) dual lumen PVC catheter, wirebonded and encapsulated as described previously (8) During the encapsulation step the gate region of the two ISFETs was separated by an epoxy partition. After testing for leakage and pH response, we applied the albumin membrane. A schematic diagram of an ENFET is shown in Figure 1. 'The ENFET probe consists of the two separate ISFETs described above, one gate having a cross-linked albumin-penicillinase membrane (penicillin-sensitivegate), while the other gate has only a cross-linked albumin membrane and exhibits only pH response (reference gate). The cross-linked albumin membrane for each gate was prepared in the manner described by Broun (91. The probe was washed with water before casting the albumin membrane. Equal volumes of BSA, penicillinase, and glutaraldehyde solutions were mixed (total volume 30 pL) and a drop was applied to one ISFET gate. A similar mixture was prepared except that the penicillinase solution was replaced with an equal volume of 0.02 M phosphate buffer, pH 6.8. A drop of this mixtuw was applied to the other ISFET gate. The probe was allowed to set at room temperature for approximately 10-15 min and was then immersed in DL-lysine for another 10 min, in order to stop the cross-linking reaction. The probe was then placed in 0.02 M phosphate buffer, pH 7.2, for at least 1 h before use. Between measurements, the devices were stored at 4 "C. Zwitterionic buffer system (HEPES-PIPES) which was used in this work was prepared from N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) and piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), Calbiochem. Apparatus. An Ag/AgCl (saturated KC1) reference electrode was placed directly into solution with the probe except for the variable-temperature experiment in which case a saturated KCl salt bridge was used. The pH ISFETs were tested for pH response by titrating a 0.1 M KH,P04 solution with 0.1 M NaOH in a pH range of 6.0-8.0. The titrations were done at 25 OC and were monitored simultaneously with a conventional pH meter (Beckman Expandomatic 11).
All penicillin response measurements were taken in differential current mode (Figure 2), measuring the drain current difference between the penicillin-sensitive gate and the reference gate ( I O ) . These solutions were magnetically stirred in a thermostatically controlled vessel at 37 f 0.1 O C .
RESULTS AND DISCUSSION pH Response. After encapsulation of an ISFET, leakage current and p H response were tested. T h e integrity of the silicon nitride gate and of encapsulation was tested by measuring the leakage current between 0 and -3 V. As shown previously (21) the pH response is reduced when ISFETs exhibit large leakage currents. If significant leakage occurred, the device was discarded. A typical p H response of the ISF E T s between p H 6 and 8 is 47-50 m V / p H which is well within the range of p H ISFETs with silicon nitride gates (8). T h e p H response with a cross-linked albumin membrane applied over the gate was unaffected, but the time response of the device was slower, as expected. T h e time response of the penicillin FET to a step change of 10 m M penicillin is shown in Figure 3. The time constant C 1980 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980 "G
v5 230
i
IO
a 0
II 5 / 3 1
b
3
e A A
4 Flgure 1. Schematic diagram of ENFET (not to scale): (1) drain; (2) source, (3) substrate; (4)insulator; (5)reference electrode; (6)albumin membrane (with or without penicillinase); (7) solution. V, is voltage applied to the gate and V, is voltage applied to the source o f the transistor.
30
25
A
35 TEMPERATURE
A
A
A
40 OC
Figure 4. Temperature dependence of the penicillin ENFET in 0.02 M phosphate buffer: pH 7.2; [penicillin] = 10 mM; ( 0 )penicillin gate: (0)reference gate; (A)difference. 3 I
- LOG
[PENICILLIN1
2
I
I
I
"Y
N-"
Reference Electrode
s Flgure 2. Circuit diagram for measuring differential drain current o f the ENFET probe.
TIME, sec
Flgure 3. Typical differential time response curve of a penicillin ENFET to a step change from 0 to 10 mM penicillin in 0.02 M phosphate buffer: PH 7.2; r = 25 OC;re3 = 11 S; T g 5 = 39 s. is between 10 and 25 s while rg5ranges from 30 to 50 s. As expected, the thickness of the albumin membrane affected t h e time response of the penicillin FET. However, no systematic study of this effect was done since the membrane thickness is a difficult parameter to control under our conditions. T h e degree of cross-linking and the concentration of albumin and enzyme loading showed no consistent effect on t h e time response. T h e differential current measurement of ISFETs provides a n automatic temperature compensation if the temperature sensitivity of both gates is approximately equal (IO). As shown in Figure 4,the dominating effect is the temperature sensitivity of each ISFET. T h e differential response to the penicillin FET is shown in Figure 5. At a buffer capacity of 0.02 M , the penicillin response is linear from 0.1 to 25 mM. At 25 mM the sensitivity begins to decline. The effects of buffer capacities and mixed a decrease in buffers are shown in Table I. As expected (4), t h e buffer capacity of the bulk solution increased the sensitivity of the penicillin FET. Mixed buffers at a buffer capacity of 0.01 M as compared to a 0.01 M phosphate buffer tended
Figure 5. Differential response of the ENFET probe to penicillin plotted on (0)linear and (A)logarithmic scales: 0.02 M phosphate buffer; pH 7.2;T = 37 O C ; averaged over 11 runs. Table I. Slope and Linear Range of Penicillin ENFET in Different Buffer Solutions at pH 7 . 2 0 a pH phosphate buffer
HEPESPIPES citrate 0.005 M 0.01 M 0.02 M 0.01 M 0.01 M slope, m V [peni- 16.00 8.00 3.12 6.60 5.50 cillin ] linear range, mM 0.2-6 0.2-13 0.2-25 0.2-15 0.2-10 a Data included in this table were obtained by using differential measurement (please refer to text and Figure 2). to decrease the sensitivity of the device without extending the linear range of the titration curve. Enzyme loading was also studied and its effect on sensitivity and range was found to be the same as reported before ( 2 , 5 ) . T h e differential mode of operation (10) of the penicillin E N F E T proved to be particularly useful. I t provides automatic compensation for changes of ambient pH and tem-
Anal. Chem. 1980, 52, 1937-1940
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boundary. At low enzyme loading, the sensor becomes stirring sensitive a t a significantly lower penicillin concentration. When another layer of albumin membrane was applied over the penicillin-sensitive gate, stirring sensitivity was supressed but a t 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 IU). This points to a a small amount of enzyme (2.5 X 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, a t 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. T h e sensitivity is again decreased compared to phosphate buffer. T h e 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. T h e 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 a t 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 48824
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
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1980 American Chemical Society
