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RECEIVED for review May 24,1991. Accepted October 8,1991. This work was financially supported in part by a Grant-in-Aid of Research from Sigma Xi and a Northern Illinois University Graduate School Dissertation Completion Award. Presented in part at the 42nd Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy, Chicago, 1991, Paper No. 537.
Planar Waveguide Immunosensor with Fluorescent Liposome Amplification Steven J. Choquette,* Laurie Locascio-Brown, and Richard A. Durst’ National Institute of Standards and Technology, Gaithersburg, Maryland 20899
A regenerable planar waveguide lmmunosensor for the cHnkal analyte theophylline has been developed. Regeneration Is accomplished under flow conditions uslng a moderate afflnity antibody, and muitlple analyses can be performed with a single wavegulde sensor. Sensors capable of more than 15 sequential measurements have demonstrated better than 10% precision. The use of theophylllnelabeled llposomes In this Competitive immunoassay provides 1 order of magnitude greater dgnal enhancement over theophylline derivatlzed with fluoresceln.
INTRODUCTION Planar optical waveguides are typically thin dielectric films (0.1-10 pm) supported on a rigid substrate of lower refractive index. In the thicker (1 mm), geometrically similar total internal reflection (TIR) element, the propagating light makes discrete contacts with the dielectric interface. Light guided by a planar or fiber waveguide, however, is more accurately described as a continuous energy distribution along the path of propagation ( I ) . The potential advantage of planar and fiber waveguides over TIR elements is the increased optical path length of the evanescent wave. The advantages of planar versus fiber waveguides are increased durability, potential for miniaturization, and ease of fabrication using a variety of materials and methods. As a result, the optical properties of planar guides may be potentially engineered for a particular chemical measurement, an attribute that is currently not enjoyed by contemporary fiber-based sensors. The first planar waveguide sensors to be developed were based upon attenuation of the guided beam (absorption spectrometry) (2-4). Interest in these sensors waned with the development of inexpensivefiber components until the early 1980s when planar waveguides were “rediscovered” for applications in Raman spectroscopy (5,6). Applications of planar waveguides to absorption spectrometry are continuing (7-11) and planar waveguide fluorescence techniques are currently under investigation (12, 13). Recent novel applications of planar waveguides include refractive index sensors based on
* To whom correspondence should be addressed.
Present address: Cornell University, NYAES, Geneva, NY 14456.
input grating couplers (14) and integrated optic Mach Zehnder interferometers (15). These sensors respond to general optical phenomena (Le. refractive index and color) and, as such, lack chemical specificity. Attachment of antibodies to an optical sensor surface however can provide the chemical specificity required for analytical measuremeats. Sutherland et al. (16)were among the first to demonstrate both slab and cylindrical totalinternal reflection fluorescence (TIRF) immunosensors. Highly sensitive fluorescence immunosensorsemploying evanescent wave and distal tip excitation were then extended to multimode and single-mode fibers by Vo Dinh (In,Tromberg and Sepaniak (I@, and Bright (19),among others. Planar waveguides employing immunospecific reactions have also recently been reported with evanescent fluorescence excitation (20, 21). Quantitative analytical measurements made with immunosensors require either regeneration of the antibody or calibration based on a series of single-use devices. High-affinity antibodies are typically used because the analytical sensitivity of the assay is related to the affinity constant. However, facile dissociation of the antigen-antibodycomplexes formed with such antibodies is not favored, making regeneration of the intact antibody difficult to achieve. The use of chaotropic reagents to dissociate the antigen-antibody complex has often resulted in significant loss of antibody activity when used with immunosensors, with the recent work of Bright (19) being a notable exception. As a result, the majority of immunosensors are single use devices and calibration curves are obtained using multiple disposable sensors. The higher dissociation rates exhibited by moderate affinity antibodies (K,< lo8) make regeneration of complexed antibodies feasible using mass action in a flow system. Recent investigations in this laboratory, using a flow injection immunoanalysis (FIIA)system (221, have demonstrated quantitative regeneration of a surface-immobilized antibody in an affinity column. Flowing antigen-free buffer over the antibody-antigen complex for a period of time greater than 10 half-lives ( N 15min) of the dissociation rate constant can lead to quantitative regeneration of the immobilized antibody. We have adapted this approach to optical waveguide sensors and developed an immunosensor for theophylline in a competitive assay using antigen-tagged liposomes. Liposomes are spherical phospholipid structures that possess an internal cavity. For this assay, the aqueous interior
0003-2700/92/0364-0055$03.00/00 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY 1, 1992
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