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Ultrasonic and Optical Sensors

WAVEGUIDE PARAMETER FOR WAVEGUIDE-BINDINGFIBER OPTIC BIOSENSORSRichard B. Thompson and #Lynne Kondracki

BioFIolecular Engineering Branch, Code 6190, Naval Research Laboratory,Washington, D C 20375-5000; and Geo-Centers Inc., 10903 Indian Head Hwy., Ft.Washington, MD 20744.

AbstractFiber optic biosensors based on evanescent wave-excited fluorescence of labeled biomolecules bound to thesurface of the waveguide (waveguide-binding sensors) offergreat promise for detecting a variety of chemical analytes.We have explored factors which influence the sensitivity ofsuch devices. On e impor tant factor is the waveguideparameter, or V number, of the fiber optic itself. Thus, theeffect of varying the V number on the level of fluorescencedetected was measured, and the implications of the resultdiscussed.

IntroductionKronick and Little [l] were the first to show thatevanescent wave-excited fluorescence could be used totransduce an antibody recognition (binding) event as achange in an optical signal. Hirschfeld [2], Andrade [3],and others extended the concept of waveguide bindingsensors to include optical fibers as the waveguide,permitting (in principle) any analyte recognizable by anantibody to be detec ted remotely and continuously. Thiscapability has many possible applications in medicine,environmental monitoring, and biology [4,5], and as a result

has been the focus of much effort.To date, no waveguide-binding sensor is availablecommercially, perha ps because the demonstrated sensitivityhas not been as large as desired. Due to the small sample(a monolayer) and the well-known loss mechanisms in fiberoptic-based fluorometry [6 ] , this may be unsurprising.Recent results suggest that the optical design of fiber opticfluorometric sensors is important to their sensitivity.Factors that are clearly important include the power of theevanescent wave, and the efficiency with which fluorescenceexcited by the evanescent wave at the core surface iscoupled back into the fiber. Unfortunately, these twofactors have opposite dependences on the waveguideparameter (V number) of the fiber optic, For a uniformmode distribution of excitation, the proportion of powerpresent in the evanescent wave decreases with V number[7]. Conversely, the proportion of fluorescence coupledback into the fiber from the core surface increases with Vnumber [8,9]. The opposite V number dependences ofthese two parameters suggests that there may be an

optimum V number (or range of V numbers) for the fiberoptics in waveguide binding sensors.Method

This hypothesis was tested by labeling a declad fusedsilica fiber with a fluorophore on the core surface near thedistal end, and then measuring the evanescent-excitedfluorescence while systematically varying the V number ofthe fiber. This was done by immersing the labeled fiberend e ither in oils having accurately known refractive indices(Cargille), ethanol, or air. The oil or other fluid serves asa cladding, redefining the V number of the fiber in thelabeled area. This procedure afforded V numbers rangingfrom 100 for a 200 micron core fiber in nearly index-matched oil, to greater than 3500 for a 600 micron fiber inair. Since the number of fluorophores attached to the fiberand the entire optical configuration remain constant,variations in measured fluorescence ar e mainly due tochanges in the effective V number of the fiber distal end.The fiber optic sensor configuration was similar to onedescribed previously [6], and is depicted in Figure 1. Thefibers were labeled using a variant of a published proce dure[lo]; further details will appear elsewhere.Results and Discussion

The normalized fluorescence intensities measured forlabeled fibers of 200 and 600 micron core size in variousmedia are depicted in Figure 2. The data are averages forat least five fibers, with the error bars indicating thestandard deviations. The data do not exactly fit ourhypothesis. While the curves evidently go through maximacorresponding to particular V numbers, the curves for thedifferent dia meter fibers a re not superimposable: to withinour experimental error, the maxima differ by nearly afactor of three. A n explanation for this puzzling result wasproposed by Dietrich Marcuse of Bell Labs and CarlVillarruel of the Naval Research Laboratory. Note that thefiber is mounted in the apparatus by its proximal end,which is clad for a few centimeters. They proposed thatthe maxima roughly corresponded to the V numbers of theintact (clad) fibers (thus the factor of three difference), andthat the decrease in observed fluorescence intensity athigher V numbers is due to the higher order modes of thefluorescence being stripped by the short section of cladfiber at the proximal end. It is very likely that fluorescence

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coupled into the fiber is predominantly in weakly guided,high order modes, and where the clad fiber has a lower Vnumber, the modes begin to leak. When the labeled endis in water, approximately 80 % of the fluorescence is lostby this mechanism.This leads to th ree conclusions. First, the fiber mustbe mounted such that it has a higher V number at theproximal end than the unclad (labeled) end. Second, thestandard methods fo r generating a uniform modedistribution (microbending, wrapping around a mandrel) arefutile, since they strip weakly guided modes wherein thefluorescence is most likely to be found. Finally, since thehigher order modes are leakier, waveguide binding sensorsmay be less useful for truly remote sensing. Experimentsare underway to address these issues.

M

Figure 1. Optical Configuration Light from thelaser L passes through the mirror M, and is launchedthrough the objective 0 into the labeled fiber F.Fluorescence (dashed lines) passes back through the fiberand objective, is reflected off the mirror and focusedthrough the filter I onto the detector D.

Wv I OW

w[L0 0 6U

0 8

E 0 42 0 25p 0 0N-

I O 0 IO00 5000

WAVEGUIDE PARAMErERFigure 2. Dependence of Fluorescence on V NumberNormalized fluorescence is plotted as a function of Vnumber for 200 micron (circles) and 600 micron (triangles)core diameter labeled fibers.

AcknowledgementsThe authors would like to thank D. Marcuse and C.Villarruel for many useful discussions, F.S. Ligler forguidance and encouragement, and the Office of NavalTechnology for support.

References[ l ] M.N. Kronick and W.A. Little, "A New ImmunoassayBased on Fluorescence Excitation by Internal ReflectionSpectroscopy," J. Immunol. Meth. 8, 235; 1975.[2] T.E. Hirschfeld, "Fluorescent ImmunoassayEmploying Optical Fiber in a Capillary Tube," US. Patent-o. 4,447,546; 1984.[3] J.D. Andrade, R.A. Van Wagenen, D.E. Gregonis,K. Newby, and J.N. Lin, "Remote Fiber Optic BiosensorsBased on Evanescent-Excited Fluoroimmunoassay: Conceptand Progress," IEEE Trans. Elec. Dev. ED-32, 1175; 1985.

[4] R.B. Thompson and F.S. Ligler, "Fiber OpticBiosensor Technology," NRL Memorandum ReDort No.6182; 988.[5]Optic Sensors," Spectroscopy 2(4), 38; 1987.[6] R.B. Thompson, M. Levine, and L. Kondracki,"Component Selection for Fluorescence-Based Fiber OpticSensors," submitted for publication.[7] R.B. Thompson, "Fluorescence-Based Fiber OpticSensors," in Fluorescence Spectroscopv Vol 11BiochemicalApplications (J.R. Lakowicz, ed.) New York, Plenum Press,in the press.[8] E.-H. Lee, R.E. Benner, J.B. Fenn, and R.K. Chang,"Angular Distribution of Fluorescence from Liquids andMonodispersed Spheres by Evanescent Wave Excitation,"Appl. Opt. 18, 862; 1979.191 D. Marcuse, "Launching Light into Fiber Cores fromSources in the Cladding," IEEE J. Light. Tech. LT-6, 1273;1988.

S.M. Angel, "Optrodes: Chemically Selective Fiber

[lo] S.K. Bhatia, L.C. Shriver-Lake, K.J. Prior, J.Georger, J.M. Calvert, R. Bredehorst, and F.S. Ligler, "Useof Thiol-Terminal Silanes and HeterobifunctionalCrosslinkers for Immobilization of Antibodies on SilicaSurfaces," Anal. Biochem. 178, 408; 1989.

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