Ultrasonic and Optical Sensors

WAVEGUIDE PARAMETER FOR WAVEGUIDE-BINDING FIBER OPTIC BIOSENSORS Richard B. Thompson and #Lynne Kondracki BioFIolecular Engineering Branch, Code 6190, Naval Research Laboratory, Washington, DC 20375-5000; and Geo-Centers Inc., 10903 Indian Head Hwy., Ft. Washington, MD 20744.

Abstract Fiber optic biosensors based on evanescent waveexcited fluorescence of labeled biomolecules bound to the surface of the waveguide (waveguide-binding sensors) offer great promise for detecting a variety of chemical analytes. We have explored factors which influence the sensitivity of such devices. One important factor is the waveguide parameter, or V number, of the fiber optic itself. Thus, the effect of varying the V number on the level of fluorescence detected was measured, and the implications of the result discussed.

optimum V number (or range of V numbers) for the fiber optics in waveguide binding sensors. Method This hypothesis was tested by labeling a declad fused silica fiber with a fluorophore on the core surface near the distal end, and then measuring the evanescent-excited fluorescence while systematically varying the V number of the fiber. This was done by immersing the labeled fiber end either in oils having accurately known refractive indices (Cargille), ethanol, or air. The oil or other fluid serves as a cladding, redefining the V number of the fiber in the labeled area. This procedure afforded V numbers ranging from 100 for a 200 micron core fiber in nearly indexmatched oil, to greater than 3500 for a 600 micron fiber in air. Since the number of fluorophores attached to the fiber and the entire optical configuration remain constant, variations in measured fluorescence are mainly due to changes in the effective V number of the fiber distal end. The fiber optic sensor configuration was similar to one described previously [6], and is depicted in Figure 1. The fibers were labeled using a variant of a published procedure [lo]; further details will appear elsewhere. Results and Discussion The normalized fluorescence intensities measured for labeled fibers of 200 and 600 micron core size in various media are depicted in Figure 2. The data are averages for at least five fibers, with the error bars indicating the standard deviations. The data do not exactly fit our hypothesis. While the curves evidently go through maxima corresponding to particular V numbers, the curves for the different diameter fibers are not superimposable: to within our experimental error, the maxima differ by nearly a factor of three. An explanation for this puzzling result was proposed by Dietrich Marcuse of Bell Labs and Carl Villarruel of the Naval Research Laboratory. Note that the fiber is mounted in the apparatus by its proximal end, which is clad for a few centimeters. They proposed that the maxima roughly corresponded to the V numbers of the intact (clad) fibers (thus the factor of three difference), and that the decrease in observed fluorescence intensity at higher V numbers is due to the higher order modes of the fluorescence being stripped by the short section of clad fiber at the proximal end. It is very likely that fluorescence

Introduction Kronick and Little [l] were the first to show that evanescent wave-excited fluorescence could be used to transduce an antibody recognition (binding) event as a change in an optical signal. Hirschfeld [2], Andrade [3], and others extended the concept of waveguide binding sensors to include optical fibers as the waveguide, permitting (in principle) any analyte recognizable by an antibody to be detected remotely and continuously. This capability 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 available commercially, perhaps because the demonstrated sensitivity has not been as large as desired. Due to the small sample (a monolayer) and the well-known loss mechanisms in fiber optic-based fluorometry [6], this may be unsurprising. Recent results suggest that the optical design of fiber optic fluorometric sensors is important to their sensitivity. Factors that are clearly important include the power of the evanescent wave, and the efficiency with which fluorescence excited by the evanescent wave at the core surface is coupled back into the fiber. Unfortunately, these two factors have opposite dependences on the waveguide parameter (V number) of the fiber optic, For a uniform mode distribution of excitation, the proportion of power present in the evanescent wave decreases with V number [7]. Conversely, the proportion of fluorescence coupled back into the fiber from the core surface increases with V number [8,9]. The opposite V number dependences of these two parameters suggests that there may be an

1102--IEEE ENGINEERING IN MEDICINE & BIOLOGY SOCIETY llTH ANNUAL INTERNATIONAL CONFERENCE

coupled into the fiber is predominantly in weakly guided, high order modes, and where the clad fiber has a lower V number, the modes begin to leak. When the labeled end is in water, approximately 80 % of the fluorescence is lost by this mechanism. This leads to three conclusions. First, the fiber must be mounted such that it has a higher V number at the proximal end than the unclad (labeled) end. Second, the standard methods for generating a uniform mode distribution (microbending, wrapping around a mandrel) are futile, since they strip weakly guided modes wherein the fluorescence is most likely to be found. Finally, since the higher order modes are leakier, waveguide binding sensors may be less useful for truly remote sensing. Experiments are underway to address these issues.

Acknowledgements The authors would like to thank D. Marcuse and C. Villarruel for many useful discussions, F.S. Ligler for guidance and encouragement, and the Office of Naval Technology for support. References [l] M.N. Kronick and W.A. Little, "A New Immunoassay Based on Fluorescence Excitation by Internal Reflection Spectroscopy," J. Immunol. Meth. 8, 235; 1975. [2] T.E. Hirschfeld, "Fluorescent Immunoassay Employing Optical Fiber in a Capillary Tube," US. Patent No. 4,447,546; 1984. [3] J.D. Andrade, R.A. Van Wagenen, D.E. Gregonis, K. Newby, and J.N. Lin, "Remote Fiber Optic Biosensors Based on Evanescent-Excited Fluoroimmunoassay: Concept and Progress," IEEE Trans. Elec. Dev. ED-32, 1175; 1985.

[4] R.B. Thompson and F.S. Ligler, "Fiber Optic Biosensor Technology," NRL Memorandum ReDort No. 6182;1988.

S.M. Angel, "Optrodes: Chemically Selective Fiber [5] Optic Sensors," Spectroscopy 2(4), 38; 1987.
[6] R.B. Thompson, M. Levine, and L. Kondracki, "Component Selection for Fluorescence-Based Fiber Optic Sensors," submitted for publication. [7] R.B. Thompson, "Fluorescence-Based Fiber Optic Sensors," in Fluorescence Spectroscopv Vol 11: Biochemical Applications (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 and Monodispersed Spheres by Evanescent Wave Excitation," Appl. Opt. 18, 862; 1979.
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Figure 1. Optical Configuration Light from the laser L passes through the mirror M, and is launched through the objective 0 into the labeled fiber F. Fluorescence (dashed lines) passes back through the fiber and objective, is reflected off the mirror and focused through the filter I onto the detector D.

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D. Marcuse, "Launching Light into Fiber Cores from 191 Sources in the Cladding," IEEE J. Light. Tech. LT-6, 1273; 1988.
[lo] S.K. Bhatia, L.C. Shriver-Lake, K.J. Prior, J. Georger, J.M. Calvert, R. Bredehorst, and F.S. Ligler, "Use of Thiol-Terminal Silanes and Heterobifunctional Crosslinkers for Immobilization of Antibodies on Silica Surfaces," Anal. Biochem. 178, 408; 1989.
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Figure 2. Dependence of Fluorescence on V Number Normalized fluorescence is plotted as a function of V number for 200 micron (circles) and 600 micron (triangles) core diameter labeled fibers.

IEEE ENGINEERING IN MEDICINE & BIOLOGY SOCIETY llTH ANNUAL INTERNATIONAL CONFERENCE--1103