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Enzyme-Based Fiber Optic Zinc Biosensor Richard B. Thompson' and Eric R. Jones Department of Biological Chemistry, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, Maryland 21201, and Center for BiolMolecular Science and Engineering, Code 6900, Naval Research Laboratory, Washington, DC 20375-5000
especially given the high cost ($10 OOO per day) and A f k o r ~ f k r o p t k ~ c a p a b i e o f d e t change, ~ limited availability of ships where such analytical chemistry zinc( I I ) at nanmoiarconcmtratha bdescrhd. The sensor may be performed.8 A continuously monitoring in situ sensor tranmducos the apecifk rocognltlon of the lon by an enzyme would be desirable for these studies since it could be (carbonk anhydrase) a8 a change in the fluorescence of an maintained at a desired depth and could continuouslyreport lnhibttor whkh MI& to the zinc In the actlve site. The analyte levels as the ship proceeded along ita course. Fiber concontration of metal Ion is proportlonai to the ratlo of optic chemical sensors have precisely these qualities, and fluorescence Int.nrlty at two waveiongths, corresponding to recognizing these advantages Walt, Lieberman, and others tho omldon from bound and free inhibttor. Sensing of zinc have described fiber optic chemical sensors for use at sea.9JO may be p.rl0nn.d through a single optical fiber.
INTRODUCTION Fiber optic chemical sensors are devices of growing importancein fields as diverse as oceanography, chemical process control, in vivo clinical diagnosis, and environmental monitoring. Fiber optic sensors have the capability of continuously monitoring the level of an analyte in a sample that may be up to kilometers away from the instrument or in an inaccessible space. A large fraction of these sensors exploit a change in fluorescence in response to the analyte as a means to transduce ita presence or level. Fluorescence-basedsensing is desirable due to its high sensitivity, good selectivity, and relatively straightforward instrumentation. Recent reviews, books, and symposium proceedings'-5 are available. A very important potential application of fiber optic chemical sensors is in the field of chemical oceanography. One of the current issues in chemical oceanography is the difficulty of determining certain chemical constituents of seawater, such as trace metals, at many points in the ocean. The basis of the problem is the slow rate at which discrete samples can be obtained from deep in the water column and analyzed, usually by preconcentration followed by atomic absorptionor emission spectrophotometry,or electrochemical methods.6J Such techniques permit analysis of only a few dozen samplesper day because they cannot usually be stored, the analyses require skilled labor, and moreover the sophisticated instrumentation necessary is ill-suited to shipboard operation. The rate and ease of measurement are important issues in designing data collecion efforts to understand phenomena occurring on a large scale, such as global climate
* To whom correspondence should be addressed at Maryland.
(1)Thompson, R. B. In Topics in Fluorescence Spectroscopy, Vol. 2: Principles; Lakowicz,J. R., Ed.; Plenum: New York, 1991: pp 345-365. (2)Wolfbeis, 0.S.,Ed. Fiber Optic Chemical Sensors and Biosensors; CRC Press: Boce Raton, FL, 1991 (2volumes). (3)Wise, D., Wingard, L., Eds.; Biosensors with Fiberoptics; Humana Press: Clifton, NJ, 1991. (4) Hansmann, D. R.; Milanovich, F. P.; Vurek, G. G.; Walt, D. R. Proceedings of Fiber Optic Medical and Fluorescent Sensors and Applications; Vol. 1648 Soc. Photopt. Instr. Engrs.: Bellingham, WA, 1992. (5) Wolfbeis, 0. S., Ed. Chemical and Medical Sensors; R o c . SPIE Vol. 1510;Soc. Photoopt. Instrum. Engrs.: Bellingham, WA, 1991. (6)Wong, C. S.,Boyle, E., Bruland, K. W., Burton, J. D., Goldberg, E. D., Eds.; Trace Metals in Seawater; Plenum Press: New York, 1983. (7) VanGeen, A.;Boyle, E. Anal. Chem. 1990,62,1705-1709. 0003-2700/s3/0385-0730$04.00/0
Among metal ion constituents of seawater, zinc(I1) is one of a handful including Fe and Mn having particular importance because they serve as nutrients and indeed are required as enzyme cofactors by many taxa of organisms for survival. These ions typically exhibit a concentration dependencewith depth characteristic of nutrients (low levels in the photic zone where most plankton are, then increasing to a constant level at greater depths). Much Zn(I1) is dissolved in the form of complexes with ill-defined organic ligands, such that free concentrationsof these ions are typically in the subnanomolar range.11-15 Indeed, there are large regions of the ocean where low concentrations of one or more of these ions are believed to limit primary productivity of microorganisms.15 Zinc(I1) levels in sea water have only recently been measured with accuracy, because of the great difficulty of avoiding contamination of the samples during collection and subsequent analysis.llJ2 Stripping voltammetry has been the method of choice,14with some effort being devoted to understanding the speciation of zinc ion in the ocean.11J3J6 For trace metal analyses in sea water, the classical fluorometric indicators such as morin or hydroxyquinolinesulfonate that have been incorporated into fiber optic sensors by pioneers such as Seitz"J8 are ill-suited mainly because they are insufficiently selective. Even the excellent chelatometric indicators for calcium developed by Tsien and his colleagues19 do not discriminate againat similar concentrations of Zn or Mn when these are present, nor Mg at 100-foldhigher concentrations.20 By comparison, an indicator for Zn in sea
(8)Walt, D.R., Ed. National Research Council Panel Report on New Measurement Technologies for the Oceans; National Academy of Sciences: Washington, DC, in press. (9)Goyet, C.; Walt, D. R.; Brewer, P. G. Deep-sea Res. 1992,39,10151026. (10)Lieberman, S.H.; Inman, S.M.; Stromvall, E. J. InProc. Sympos. Chem. Sensors; Electrochemical Soc.: Pennington, NJ, 1987;pp 464473. (11)Bruland, K.W. Limnol. Oceanogr. 1989,34,269-285. (12)Sherrell, R. M.; Boyle, E. A. Deep-sea Res. 1988,35,1319-1334. (13)Bruland, K.W. Appl. Geochem. 1988,3,75. (14)Donat, J. R.; Bruland, K. W. Mar. Chem. 1990,243,301-323. (15)Brand, L.E.; Sunda, W. G.; Guillard, R. R. L. Limnol. Oceanogr. 1983,243,1182-1195. (16)Midorikawa, T.; Tanoue, E.; Sugimura, Y. Anal. Chem. 1990,62, 1737-1746. (17)Zhujun, Z.;Seitz, W. R. Anal. Chim. Acta 1985,171, 251-8. (18)Saari, L. A.; Seitz, W. R. Anal. Chem. 1983,55,667-670. (19)Tsien, R.Y. Annu. Reu. Neurosci. 1989,12,227. (20)Haugland, R. P. Handbook Fluorescent Probes and Research Chemicals, 5th ed.; Molecular Probes: Eugene, OR, 1992;Chapters 20 and 22. 0 iSS3 American Chemical Society
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Flguro 1. Blnding of dansylamlde to carbonic anhydrase. In this cartoon dansylamlde binds to carbonk anhydrase ontY when zlnc is prin the acthre site and emits a blue fluorescence: in the absence of zlnc the dansylamlde does not blnd and exhlbb weak, Ipeenlsh-yeWow fluorescence In buffer.
water must exhibit million-fold(60dB) selectivitywithrespect to magnesium, as well as other cations. Such selectivity is not to be found in typical synthetic chelators such as these, or ionophores.21 However, some metalloenzymes exhibit exceptional binding selectivity for the metal ions which participate as cofactors in catalysis. An example of this is the carbonic anhydrase from mammalian erythrocytes (carbonate hydro-lyase, EC 4.2.1.1),22v23which functions in vivo by dehydrating bicarbonate to give CO2 (and vice versa), only the latter of which can pass through cell membranes. From our standpoint carbonic anhydrase is of interest because it binds ita Zn2+ cofactor in the active site with substantial specificity: only Co and Mn are reported to bind with activity and reduced affmity,24~2~ and these ions are also present only at trace levels in sea water and are unlikely to interfere in the assay. It was essential that a means be found to transduce the binding of Zn2+to the enzyme as a change in fluorescence; in fact, this requirement dictated the choice of enzyme for this analyte. Moreover, it was also desirable that the fluorescence change not be simply a change in intensity, but a wavelength shift in fluorescence which would permit the ratio of emission at two different wavelengtb to be correlated with the analyte level. The virtues of ratiometric measurementa are by now widely appreciated19J0 they include insensitivity to excitation level, fluorophore loading, photobleaching, scattering, and inner filter effecta. For fiber optic sensors, the relative insensitivity to signal variation due to microbending, mode transformation, and passing through slip rings makes ratiometric measurements attractive. For these reasons, workers have measured pH through optical fibers using ratiometric indicators such as hydroxypyrenetrisulfonateeZ6Our approach was based on the well-known properties of aryl sulfonamides as competitive inhibitors of carbonic anhydrase,27and in particular the work of Chen and Kernohan.% They found that NJV-dimethyl-2-aminonaph(21) Ammann, D.Ion-Selective Microelectrodes: Rinciples, Design, Application; Springer-Verlag: Berlin, 1986. (22) Dodpon, S. J., Taehian, R. E., Gros, G., Carter, N. D., Eds. The Carbonic Anhydrases; Plenum Press: New York, 1991. (23) Lindskog, S.; Henderson,L. E.; Kannan, K. K.; Liljae, A.; Nyman, Ed.;Academic Press, P. 0.;Strandberg,B. In The Enzymes; Boyer, P. D., New York, 1971; Vol. 5, pp 587-665. (24) Coleman, J. E. Biochemistry 1965,4, 2644-2655. (25) Lindskog, S.; Nyman, P. 0. Biochim. Biophys. Acta 1964, 85, 462-474. (26) Zhujun, Z.; Seitz, W. R. A d . Chim. Acta 1984, 160, 47-55. (27) Lindskog, S.; Thorslund, A. Eur. J. Biochem. 1968,3,453-460. (28)Chen, R. F.; Kernohan, J. C. J. Biol. Chem. 1967,242,5813-23.
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Flgm2. Fiwxescencotltratknofcerbonkanhybeseanddansylamide wtth zinc. A solution of 9 pM carbonic anhydrase, 9 pM dansylamlde in HEPES buffer was tltrated wtth ZnCi2,and the fluorescenceemlsslon spectrum measured (excltatlon at 326 nm, 2-mm emlsslon slits) after
each successive addltion. thalene-5-sulfonamide (dansylamide) bound as an inhibitor to the Zn in the active site of the enzyme (Figure 11, with a concomitant blue shift and enhancement of ita fluorescence emission in comparison to ita emission in water. The dansylamide was not seen to bind elsewhere on the enzyme at concentrations below millimolar. We reasoned that if the Zn were removed fromthe enzymeactive site, the dansylamide would not bind and would exhibit ita typical weak, red-shifted emission in aqueous solution. In fact this is the case (Figure 2), and we are able to measure the occupancy of the active site by Zn by measuring the ratio of the fluorescenceintensities at two wavelengths correspondingto emission from the bound and free forms of the sulfonamide inhibitor and to adapt this scheme to an optical fiber sensor.
EXPERIMENTAL SECTION Materials and Reagents. NJV-Dimethyl-2-aminonaphthalene-5-sulfonamide (dansylamide) was from Aldrich, HEPES (Ultrapure grade) was from Baker, and other buffer salts were reagent grade or better. Deionized water was further purified by passage through a Millipore system until it exhibited resistance of 18 MQ,and interfering cations were removed from buffers by passage through a bed of chelating resin (Sigma). Following passage through the chelatingresin,solutionswere stored in clean polyethylene containers and pipetted using plastic pipets or metal-freepipet tips;fluorescence was measured in cuvettesmade of synthetic fused silica. Carbonic anhydrase from bovine erythrocytes (Sigma C-7500) was stripped of ita Zn atom by prolonged dialysisat 4 O C in 50 mM sodiumacetate pH 5.4 buffer
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F@wr 3. Fluorescencebesedfiber optlc sensor. Exdtatbn Isindicated by the solM arrows, emlsslon by the dashed arrows.
containing approximately 5 mM 2,6-dipicolinic acid (Aldrich). No attempt was made to differentiate the responses of or purify the isozymes likely to be present in this preparation. Following dialysis into 50 mM Na HEPES pH 7.3,150 mM sodium sulfate, the enzyme was subjected to affinity chromatography on (aminomethyl)benzenesulfonamide-derivatizedSepharose 4B resin (Sigma)*gin the same buffer. This resin binds enzyme which still contains zinc ions and lets apoenzyme pass through. The unretarded peak corresponding to the apoenzyme exhibits practically no fluorescenceenhancement in the blue when added to saturatingsolutionsof dansylamide,suggestingthat it is almost entirely zinc free and relatively free of dansylamide-binding impurities. Apparatus. Fluorescence emission spectra and emission intensities in cuvettes were measured on an ISS K2 fluorometer (Champaign, IL) using a Liconix 4214NB laser emitting approximately 5 mW multimode at 326 nm for excitation. The fiber optic sensor optical configuration was similar to those described previously (Figure 3).30 Briefly, excitation from the laser passes through a 2-mm hole in the mechanical axis of the off-axis paraboloid (Janos A-8037-205) and is launched into the proximal end of the fiber through a chopper (Stanford Research SR-540) by a 25-mm focal length f/l fused silica lens (Newport Corp. SBX-019). The excitation passes down the fiber (General Fiber Optics 16-200s 200-pm core plastic clad fused silica step index fiber) and excites the dansylamide at the distal end. The fluorescencepasses back down the fiber to its proximal end,where the light spreads out according to the numerical aperture of the fiber; the objective nearly recollimates the fluorescence, which reflects off the off-axis paraboloid and is focused through the filters and onto the detector (a New Focus Model 1801 PIN photodiode) whose output at the chopper modulation frequency of 267 Hz is amplified and displayed by a Stanford Research SR 510lock-in amplifier. For some measurements through the fiber the off-axis paraboloid, objective lens, and fiber holder were mounted in the ISS fluorometer in place of the cuvette holder, as described elsewhere;3I in these experiments the chopper and lock-inwere not used and the dc output of the Hamamatsu R928 photodetectorwas directlyquantitated on the instrument's digital voltmeter after amplification. The off-axis paraboloid and synthetic fused silica objective contribute to background to a negligible extent compared to the photoluminescence of the optical fiber, which is not filtered out by the chopper;30thus the lack of the chopper contributed negligiblyto the sensitivitylevel achieved here. Other fibers tested exhibited greater attenuation, worse photoluminescence,or both. The emission filters were a liquid fiiter3O of 2 mm 1M NaNOzin water closest to the off-axis paraboloid, and an interference filter (Melles Griot) with peak transmission at either 460 or 560 nm to isolate the emissions from the enzyme bound and free forms of the dansylamide, respectively. In practice, the interference filters were rapidly interchanged and successive measurements ratioed. (29) Whitney, P. L. Anal. Biochem. 1974,57, 467-476. (30)Thompson, R. B.; Levine, M.; Kondracki, L. Appl. Spectrosc. 1990,44, 117-121. (31)Thompson, R.B.;Lakowicz, J. R. Anal. Chem, in press.
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Flgurr 4. Ratio of fluorescence lntenslties as a function of rlnc concentration. Data are shown for tltrations like that In Flgure 2 In a cuvette (solM line) and through a fiber using the apparatus of Flgure 3 (dashed ilne).
RESULTS Our goal is to determine zinc in solution by measuring the ratio of fluorescenceemission intensities of dansylamide when bound to the carbonic anhydrase (when it is bound to the zinc in the active site)to that of free dansylamide in solution; the fraction of dansylamide bound (and exhibiting strong, blue emis8ion) is determined by the fraction of enzyme active sites containing zinc and therefore the metal's concentration in solution. Thus for highest sensitivity, it is desirable to detect fluorescence from a few molecules of dansylamide bound to zinc ions in the active site of the enzyme, in the presence of a much greater number free in solution. That this can be done is seen in Figure 2, which is a titration of 9 p M apoenzyme in HEPES buffer in the presence of 9 pM dansylamide with low concentrations of zinc. The buffer and enzyme alone exhibited negligible fluorescence, and ZnClz concentrations below 100 rM did not measurably affect the fluorescence of dansylamide in the absence of enzyme. The fluorescence enhancement observed here by titrating with zinc closely corresponds to the 15-foldenhancement observed by Chen titrating the enzyme with dansylamide.28 Furthermore, fluorescence lifetimes of the free and bound dansylamide measured in this laboratory32 (3.4 0.5 and 20.3 0.2 ns, respectively; data not shown) matched very closely with the values measured by Chen and Kernohan (3.9 and 22.1 ns, respectively) 25 years Data obtained in cuvettes in similar fashion to those in Figure 2, but with only 1 pM apoenzyme and the modest, essentially constant background of intensity due to free dansylamide subtracted from the intensity at 450 nm, are plotted as a log-log plot in Figure 4. The concentration of dansylamide was chosento be high enough to assure saturation of the binding site when zinc is present there (Kd = 2.4 X 10-I M);28 the enzyme concentration was chosen to be about 10fold lower than the dansylamide concentration to assure that at saturation with zinc, the broad emission from the bound form would contribute only modestly at the emission wavelengths of the free dansylamide and that the intensities would be comparable. Evidently the plot is linear (correlation coefficient = 0.9975) over about 2 decades in concentration. Moreover, the linear range corresponds roughly to the concentration range at which zinc is commonly found in the ocean.11J2 The enzyme, even in zinc-free form, was stable at 4 OC for more than 6months; dansylamide, on the other hand, is not stable for more than 1 week in dilute solution under these conditions. Taken together, these data suggest that a fiber optic sensor of useful sensitivity can be constructed using carbonic anhydrase/dansylamide in the sensing tip.
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(32)Thompson, R.B.;Gratton, E. Anal. Chem. 1988,60, 670-674.
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SEMI-PERMEABLE MEMBRANE EPOXY OPTICAL FIBER
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GLASS CAPILLARY O-RIN(
Flgurr 5. Senslngtip for dlstalend of the flber optic. Laser light enters
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equilibrium in some minutes, with the ratio of intensities stabilizingafter some time. In fact the signal did not stabilize reproducibly, and the dansylamide leaked out as judged by monotonic drops in the fluorescence intensity at both wavelengths (results not shown) and the appearance of the dansylamide in the surrounding medium as determined by its fluorescence. As in the case of the pH sensor,31 dialysis tubing is an unsatisfactory selective membrane, and might be replaced with a different semipermeablemembrane such as those used for ion-selective electrodes,35which would only permit passage of small cations, not the fluorescent inhibitor nor the enzyme. Alternatively, the dansylamide might somehow be coupled to a polymer support. Experiments are underway to address this issue.
the tip fromthe left and excltesfluorescence of the carbonicanhydrase/ dansylamlde mixture In the chamber at the right: some of the fluorescence reenters the fiber at the dlstal face and travels back to the left to the detector.
We decided to assess the utility of a fiber optic sensor using the above approach. The ratio of intensities corresponding to the free and bound forms were measured with the apparatus of Figure 3 by dipping the cleaved end of the fiber into solutions of apoenzyme with dansylamide in HEPES buffer and with zinc ion at varying concentrations. For these experiments intensities were measured through interference filtersas describedin apparatus; the 460-nm filter was chosen (asopposed to 440 nm) to avoid interference from the 441.6nm plasma emission line of the HeCd laser. The results of one such experiment are also depicted in Figure 4. The most important feature is that the apparent detection limit is at least ten-fold worse than in cuvettes. The principal reason for this is substantial interfering photoluminescence1 of the optical fiber itself when excited with 326-nm light. This photoluminescence contributed to intensities at both 460 and 560 nm and at low zinc concentrations overwhelmed the emission from the bound dansylamide. Other fibers tried exhibited greater photoluminescence,greater attenuation of the excitation, or both. Apart from this the calibration curve appears roughly linear at higher concentrations (correlation coefficient = 0.975). Since the optical conditions are not the same (filters versus an emission monochromator),we do not expect the two calibration curves to be identical. Indeed, by judicious choice of wavelengths the most sensitive range may be selected at will. Several workers have described means to introduce indicator reagents continuouslyat the distal end of optical fibers, but since neither the enzyme nor the dansylamide is consumed aa a reagent (e.g., this is an equilibrium binding-type sensor), it was only necessary to maintain the dansylamide and apoenzyme at the distal end of the fiber. Thus apoenzyme and dansylamide were encapsulatedwithin an all-glass sensing tip with a dialysis membrane barrier similar to that described for pH measurement31 (Figure 5). The possibility of the borosilicate glass leaching out contaminating zinc was viewed as negligible over a period of a few minutes at neutral pH. The dialysis tubing used to close the opening (Spectrapor 1, Spectrum Medical Industries) had a molecular weight cut off of approximately 6000, which is adequate to retain the apoenzyme but not zinc ion nor the dansylamide. While no dialysis membrane known willdiscriminate between two such small species for any length of time, we hoped that the zinc would diffuse in more quickly than the dansylamide would leak out. In view of the known rate constants for zinc binding,33-34we anticipated that the reaction would come to (33) Henkens, R.W.;Sturtevant, J. M. J. Am. Chem. Soe. 1968,90, 2669-2676. (34) Henkens,R.W.;Watt, G. D.;Sturtevant,J. M. Biochemistry 1969, 8,1874-1878.
DISCUSSION We have demonstrated a fiber optic metal ion biosensor which is relatively sensitive and specific for zinc. Despite the advantages of this approach and the encouraging initial results, much remains to be done to make a workable sensor for low levels of zinc in the ocean or other environments.Two isues of particular importance, in addition to the form of the sensing tip, are reversibility and fiber attenuation. Perhaps first and foremost is reversibility. The off rate of Zn from carbonic a n h ~ d r a s e ~ is ~roughly J~ s-l, which is too slow for even for very long-term, low data rate applications like buoy-based sensors which take one data point per day. There are various approaches possible for dissociating zinc once it is bound. For instance, a temperature jump or pH drop induced by a light flash through the fiber or introduction of a reagent might be used to dissociate the zinc. A second issue is the applicability of this sensing scheme to ocean monitoring at depth in view of the high attenuation of fiber for ultraviolet light. The fiber used here has attenuation of greater than -100 dB/km at 300 nm, which is largely due to Rayleigh scattering from inhomogeneities in the fiber; it is unclear how much this might be improved upon, if at all. Two approaches are possible to circumvent this. First, other fluorescent aromatic sulfonamides may be found which also serve as inhibitors and exhibit a large shift in emission while being excited at much longer wavelengths. At 600 nm the attenuation of typical fibers is approximately -10 dB/km, making measurements at greater depths feasible. Moreover, the strong photoluminescence of the fiber whichcircumscribed our detection limit (Figure 4) is very much reduced at longer wavelengths; the fraction of this photoluminescence attributable to Raman scattering will exhibit the same (frequency)4 dependence as Rayleigh scattering and will be reduced at least 16-fold. While long wavelength fluorescent sulfonicacids are known, such as Sulforhodamine 101 and Lissamine Rhodamine B, the wavelengths of their emission are less “solvent sensitive” than the dansyl moiety, and therefore the corresponding sulfonamides seem unlikely to exhibit the same large shift of emission wavelength upon binding that dansylamide does. Efforts are underway to identify and test other candidate fluorophores. The second approach is twophoton excitation of fluorescence, which has been dernonstrated in optical fiber sensing by Tromberg et al.36 and by L ~ t l e . ~While ’ this approach is feasible, generating the required very high peak power laser pulses remains an expensive, nontrivial task. While the ultimate goal of this study has been the development of a sensor useful in the oceans, the device may (35) Ammann, D. Ion-Selective Microelectrodes: Principles, Design, and Application; Springer-Verlag: Berlin, 1986. (36) Tromberg, B.J.;Eastham, J. F.; Sepaniak,M. J. Appl. Spectrosc. 1984,38, 38. (37) Steffen, R.L.; Lytle, F. E. Anal. Chirn. Acta 1987,200,491-502.
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be applicable elsewhere as well. In particular, zinc is viewed as an important micronutrient, and ita metabolism and physiological roles apart from being an enzyme cofactor are the subject of current investigation. Zinc can also be a pollutant derived from mine tailings and other sources, but is bf less interest due to ita low toxicity. Finally, the use here of a fluorescent inhibitor in an enzyme-based fiber optic biosensor is novel and may be applicable to the determination of other enzyme substrates, inhibitors, and cofactors of interest.
ACKNOWLEDGMENT The authors wish to thank Dr. Jeff Deschamps of the Naval Research Laboratory for the dipicoliiate zinc stripping procedure and Dr. Joseph Lakowicz for material support. This work was supported by the Office of Naval Research.
RECEIVEDfor review September 22, 1992. Accepted December 1, 1992.
