Anal. Chern. 1988, 6 0 , 4 3 3 - 4 3 5 (21) (22)
Peadon, P. A.; Lee, M. L. J . Chrometogr. 1983, 259, 1-16. Chalmers, J. M.; Mackenzie, M. W.; Sharp, J. L.; Ibbet, R. N. Anal.
Cham. 1987. 5 9 . 415-418. (23) Wright, B. W:; FGe, S. R.; McMinn, D. 0.; Smith, R. D. Anal. Chem. 1987, 5 9 , 640-644.
RECEIVED for review July 22, 1987. Accepted November 7,
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1987. The authors gratefully acknowledge support for this project, which was provided by the Science and Engineering Research Council Grant GR/E/00556 and Imperial Chemical Industries PLC, UK. This paper was presented in part at the Spectroscopy Across The Spectrum Conference, University of East Anglia, Norwich, UK, 12-17th July 1987.
Single Fiber-optic Fluorescence Enzyme-Based Sensor Ming-Ren S. Fuh, Lloyd
W.Burgess, a n d Gary D. Christian*
Center for Process Analytical Chemistry, Department of Chemistry, BG-10, university of Washington, Seattle, Washington 98195
Penlcllllnase Is lmmoblllzed on a single fiber-optic pH sensor by cross-llnklng In a thin glutaraldehyde membrane on a porous glass bead, to whlch a fluorescent pH dye has been bound. Penlclllln In solutlon Is enzymatlcally converted to penlclllolc acld at the probe surface, causlng a decrease In pH wlth a concomltant decrease In the fluorescent lntenslty of the dye. An argon Ion laser Is used to exclte fluorescence and the fluorescent radlatkn is collected In the same flber for transmlsslon to the detector vla a coupllng devlce. The slgnal reaches 95% of total response In 20-45 8. The detectlon llmH Is 0.1 mM penlclllln.
The development of biosensors as analytical tools is a growing field in analytical chemistry (1,2). This is due to the ability of biological molecules or organisms to react specifically with a target analyte. In use, these materials are frequently immobilized directly onto a suitable transducer. One example is the enzyme electrode, first introduced by Updike and Hicks (3). Several different types of enzyme electrodes have been developed and have proven to be useful in biochemical analysis (4). In these devices the enzyme reacts with its substrate on or near the electrode surface, and the product of the reaction is detected by the electrochemical transducer. A good example of this approach is the penicillin enzyme electrode, first described by Papariello et al., based on a glass pH electrode (5). Caras and Janata later investigated a penicillin-sensitive field effect transistor (6). All of these electrochemical devices share two potential problems: the necessity for a stable reference electrode and the possibility of a shock hazard if the device is to be used in vivo. Recently biosensors have begun to appear that are based on fiber optics (7-9), and enzyme-based fiber-optic sensors for p-nitrophenyl phosphate (10) and penicillin (11) have been developed. The advantages of fiber-optic sensors include miniaturization, electromagnetic immunity, geometric flexibility, and the fact that there is no direct electrical connection to the sensing site. Here, we describe a miniature fiber-optic sensor suitable for monitoring penicillin or potentially any acid- or base-producing enzyme reactions. The probe itself is a fiber-optic pH sensor consisting of a porous glass bead bound with an immobilized fluorescent dye (12). Penicillinase is immobilized on the probe by cross-linking in a thin glutaraldehyde membrane. The immobilized penicillinase catalyzes the hydrolysis of penicillin to penicilloic acid. The increase of hydrogen ion concentration from the
production of penicilloic acid modulates the bead fluorescence at the fiber tip. EXPERIMENTAL SECTION Materials. Penicillinase, penicillin G (potassium benzylpenicillin), penicillin V (potassium phenoxymethylpenicillin), glutaraldehyde,bovine albumin, and sodium azide were purchased from Sigma Chemical Corp. Potassium chloride was Baker Analyzed grade. Citric acid, Na2HP04,and NaH2P04were obtained from Mallinckrodt. All buffer solutions were calibrated by using a pH electrode and a Beckman SelectIon 5000 pH meter (Beckman Instruments, Inc., Irvine, CA). The pH values of penicillin standards and the blank buffers were monitored by using both the pH meter and the pH fluorescence sensor. Since penicillin is an acid, a high concentration of penicillin will change the pH of the buffer solution. Those penicillin solutions having a 3% (0.1 pH unit) difference in normalized fluorescence intensity from the blank buffer solution were excluded for enzyme probe studies. Preparation of Enzyme Probe. The preparation of the fluorescencepH fiber-opticprobe is described in ref 12. It consists of a single multimode optical fiber (SG-820, Radiant Communication, MA) which conducts both excitation and fluorescence radiations, and a FITC-immobilized porous glass bead (FITC = fluorescein isothiocyanate). The porous glass bead is attached to the end of the optical fiber by using Norland no. 63 W-curable optical epoxy. The immobilization of penicillinase by covalent cross-linking has been described elsewhere (13). Penicillinase and bovine serum albumin (1:2in weight; total about 30 mg) were dissolved in 500 pL, pH 7 phosphate buffer at 4 "C. Then 200 p L of 5% glutaraldehyde was added and the mixture was stirred for 1 min. The end of the fiber optic with the attached dry FITC-immobilized porous glass bead was put into the mixture for several seconds, removed, and left approximately 30 min for cross-linkingat room temperature. The probe configuration is shown in Figure 1. Apparatus. The experimental measurements were obtained by using the instrumental configurationdescribed previously (12). RESULTS AND DISCUSSION The pH optrode without the enzyme membrane has been described in a previous report (12). The probe response is based on the change of the fluorescencecharacteristics of the immobilized fluorescein isothiocyanate (FITC), with change in pH. The strongest fluorescence appears at high pH; lowering the pH reduces the fluorescence intensity. Two linear response regions are observed, one from pH 2.8 to 5.0, and a second with a steeper slope between pH 5.5 and 7.0. This is due to the unequal fluorescence quantum efficiency of the different proteolytic forms of the dye. After depositing the penicillinase membrane, the pH response remains the same, as shown in Figure 2. In addition, the rate of response is
0003-2700/6S/0360-0433$01.50/00 1988 American Chemlcal Soclety
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988
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unaffected. The present pH sensor incorporates the chromophore on a rigid matrix that is not subject to changes in optical path since the matrix itself will not swell. In addition, preparation of the enzyme layer is very simple, involving merely dipping the pH probe in the protein-glutaraldehyde mixture and then removing it and allowing cross-linking to occur a t room temperature. The activity of penicillinase is pH dependent, with a maximum at pH 7.0 (14). The pH sensor has been shown to be most sensitive in the pH range of 5.5 to 7. An operating pH of 6.8 is chosen to take advantage of the high activity and to assure that the probe response remains in the linear range. Because the pH sensor is sensitive to the ionic strength, 0.1 M KCl is added to control the ionic strength of the buffer and penicillin solutions. In addition, 0.01% (w/v) NaN3 is added as a preservative. M buffer is shown The rate of response of this probe in in Figure 3. The signal is observed to reach 95% of total response within 20-45 s of immersion in a penicillin solution, depending on the penicillin concentration. The slowest response in solutions tested was observed at low buffer concentrations, requiring approximately 180 s to reach a steady state. As expected, a difference was observed in the rate of responses for different penicillin species (15). These differences can be attributed to the varying susceptibility of the particular penicillin species to penicillinase catalyzed hydrolysis. Washout time (after dipping in buffer solution), which is the time for fluorescence to return to the blank value, is 1-2 min and is buffer-capacity dependent. Both the rate of response and washout time for this small-volume enzyme
Flgure 3. Rate of responses of enzyme probe to penicillin 0 and penicillin V in 0.001 M citric acid/Na,HPO, buffer. The numbers on the curves represent the concentrations of solutions in millimolar.
optrode are faster than reported for most enzyme electrodes (16-18). These times would be expected to decrease in a flowing system. When sensors based on immobilized pH indicators me used, it is important that solution conditions be well defined (19, 20). Because the bound indicator can only exchange protons with other cations in solution, the equilibrium constant of the indicator will be dependent on the cation(s) present (20). We observed that alkali metal and alkaline earth cations can affect the pH response of the probe if present at suitably high concentrations (12). In the present configuration, the enzyme activity can also be influenced by the matrix constituents. Hence, it is best to dilute the sample in a suitable background matrix to maintain constant conditions, or else match sample and standard matrices. In the enzyme-based sensor, measurement of the initial rate of pH change could be employed to avoid changes in background pH and so forth (21). The observed responses of this optrode to penicillin G and penicillin V in various buffer systems are summarized in Table I. Because the penicillinase activity is dependent upon the buffer system, the detection limit and the working range of the optrode are also buffer dependent. The sensitivity of the sensor appears to be increased by decreasing the buffer capacity, at the expense of decreasing the working range of penicillin concentration. In 0.01 M citric acid/Na2HP04buffer, a working range of 0-10 mM penicillin is obtained, although only a small fluorescence intensity change per mM penicillin is observed. In 0.001 M phosphate buffer, a large fluorescence change is observed between 0.2 and 2.5 mM and then a decreased response slope up to 10 mM. With a 0.001 M citric acid/
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988
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Na2HP04buffer, a detection limit of 0.1 mM penicillin is observed. A similar response is obtained in 1 X lo4 M phosphate buffer. The effect of temperature on this sensor was tested at 8, 24, and 35 "C and the response is shown in Figure 4. The local temperature of the glass bead is expected to be higher than the solution temperature due to the absorption of excitation light. The sensitivity of this probe does not appear to change significantly: less than 1% over this temperature range. A temperature effect on the pH optrode (12) is observed.
CONCLUSION This fluorescence enzyme fiber-optic sensor has a detection limit of approximately 0.1 mM penicillin and a response range of 0.1-10 mM. This is comparable to penicillin electrodes and other penicillin analysis methods (17). The optrode exhibits a slight decrease in activity over time, dropping to approxi-
mately 95% of original activity over a 5-day period. The response time of this optrode is on the order of 30 s, which is faster than response time for most enzyme electrodes. The response time of the sensor could be enhanced by binding FITC to the penicillinase first and then cross-linking onto the glass bead. Because of the irregular shape and size of the porous glass bead, the amount of fluorescein isothiocyanate and enzyme on the bead varies from probe to probe. Thus a variation of absolute response is expected and could be improved by using glass beads of more uniform shape and size. However, the relative responses of different probes are essentially the same, as shown in Figure 5. Similar responses and detection limits for different probes were obtained, and each could be easily calibrated. We have shown the feasibility of a single fiber-optic fluorescence enzyme-based sensor that could easily be adapted to other enzyme-substrate systems. The small sensing volume (about 2 nL) and nonelectrical nature of this device make it suitable for many special applications. Registry No. Pen-G, 61-33-6;Pen-V, 87-08-1;Pen, 1406-05-9; penicillinase, 9001-74-5;penicilloic acid, 11039-68-2.
LITERATURE CITED Peterson, J. 1.; Vurek, G. 0. Science 1984, 224, 123. Schuk, J. S. Med. Instrum. 1985, 19, 158. Updlke, G. P.; Hicks, S. P. Nature (London) 1987, 214, 986. Gullbauk, G. G. I n Comprehenslve Anawicai Chemistry, Svehla, G., Ed.; Elsevier: Amsterdam, 1977; Vol. 8. (5) Papariello, G. J.; Mukherjl, A. K.; Shearer, C. M. Anal. Chem. 1973, 45. 700. (6) Caras, S.; Janata. J. Anal. Chem. 1980, 5 2 , 1935. (7) Raju, T. N. K.; Vldyasagar, D. Med. Instrum. 1982, 16, 154. (8) Lubbers, D. W.; Opitz, N. 2.Naturforsch. C : Biochem., Biophys., Biol., Viroi. 1975, 30C, 352. (9) Peterson, J. I.; Fltzgerald, R. V.; Buckhold, D. K. Anal. Chem. 1984, 56, 2. (10) Arnold, M. A. Anal. Chem. 1985, 5 7 , 565. (11) Kulp, T. J., personal communication, 1987. (12) Fuh, M. S.;Burgess, L. W.; Hlrschfeld, T.; Christian, G. D.; Wang, F. Analyst (London) 1987. 112, 1159. (13) Durand, P.; Davu, A,; Thomas, D. Biochlm. Biophys. Acts 1978, 527, 277. (14) Waley, S. G. Biochem. J. 1975, 149, 547. (15) Hou, J. P.; Poole, J. W. J. fharm. Sci. 1971, 6 0 , 503. (16) Nilsson, H.; Akerlund, A. C.; Mosbach, K. Blochim. Biophys. Acta 1973, 320, 529. (17) Nlllson, H.; Mosbach, K.; Enfors, S. 0.; Molin, N. Biotechnol. BIoeng. 1978, 2 0 , 527. (18) Tor, R.; Freeman, A. Anal. Chem. 1986, 5 8 , 1042. (19) Janata. J. Anal. Chem. 1987, 5 9 , 1351. (20) Woods, B. A., Ruzicka, J.; Christian, G. D.; Rose, N. J.; Charlson, R. J. Analyst (London), In press. (21) Yerlan. T. D.; Christian, G. D.; Ruzicka, J. Analyst (London) 1988, 1 1 1 , 865. (1) (2) (3) (4)
RECEIVED for review July 20,1987. Accepted November 12, 1987.
