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Anal. Chem. 1987, 59, 1226-1230 Fiber-optic Chemical Sensors for Competitive Binding Fluoroimmunoassay B r u c e J. Tromberg' a n d Michael J. Sepaniak* Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600 T u a n Vo-Dinh* a n d Guy D. Griffin Advanced Monitoring Development Group, Health and Safety Research Division, Oak Ridge National Laboratory, P.O. Box X , Oak Ridge, Tennessee 37831 This paper describes the development of a fiber-optic chem- ical sensor based on the principle of competitive-binding fluorescence immunoassay. Rabbit immunoglobin G ( IgG) is covalently lmmoMlized on the distal sensing tip of a quartz optical flber. The sensor is exposed to fluorescein Isothlo- cyanate (FITC) labeled and unlabeled anti-rabbit IgG. The 488-nm line of an argon-ion laser provides excitation of sen- sor-bound anaiyte. This results in fluorescence emission at the optical fiber's sensing tip. Sensor response is inversely proportional to the amount of unlabeled anti-IgG in the sam- pie. Limits of detection (LOD) vary with incubation time, sample size, and measurement conditions. For 10-kL sam- ples, typical LOD are 25 fmol of Unlabeled antibody in a 20- min incubation period. These results indicate that each fi- ber-optic fluoroimmunosensor can be constructed to perform a single sensitive, rapid, low-volume immunoassay, in in situ or benchtop applications. Fiber-optic chemical sensors (FOCSs) have been designed to provide simple, rapid, in situ analyses of trace chemicals (1-8). FOCSs are characterized by their chemically selective immobilized reagent phase at the fiber's sampling terminus. This reagent phase distinguishes these devices from their less selective physical sensor counterparts (9-11). In order to detect trace amounts of chemicals with high sensitivity and specificity, the principles of solid-phase im- munoassay can be applied to FOCS design. This involves the covalent immobilization of receptor molecules (antibody or antigen) to the distal face of a single-strand gw-~m-diameter quartz optical fiber. The fiber and immobilized receptor molecules form a stable, selective fluoroimmunosensor (FIS). Careful selection of the proper immobilization procedure enhances FIS stability by minimizing receptor leakage. Provided there is sufficient immobilized reagent for reasonable sensitivity, this direct attachment of antibody or antigen via organosilanating reagents is preferable to those techniques which utilize membrane or gel-entrapped reagent phases. This is due to the fact that sensor response times are limited by mass transport to the fiber and immunochemical kinetics. Despite their potential for higher loadings, these processes may be slower in membrane or gel systems (1, 2). Several types of immunoassays can be performed. The simplest involves in situ FIS incubation followed by direct measurement of a naturally fluorescent analyte (12). For nonfluorescent materials, in situ incubation is followed by development in fluorophor-labeled second antibody. The resulting antibody sandwich produces a fluorescence signal Also Oak Ridge Associated Universities Graduate Fellow, Ad- vanced Monitoring Development Group, Health and Safety Research Division, Oak Ridge National Laboratory. that is directly proportional to bound antigen. The sensitivity obtained when using these techniques increases with increasing amounts of immobilized receptor (13). A third detection scheme involves competition between fluorophor-labeled and unlabeled antigen. In this case, the unlabeled analyte com- petes with labeled analyte for a limited number of receptor binding sites. Assay sensitivity therefore increases with de- creasing amounts of immobilized reagent (13). The appropriate FIS immunoassay method is determined by the chemical characteristics of the antigen/antibody system of interest. For example, naturally fluorescent antigens and haptens should be analyzed by direct measurement. Non- fluorescent haptens should be detected via competitive binding or sandwich techniques, though the scarcity of hapten epitopes (antibody recognition sites) may diminish the sensitivity of sandwich analyses. An alternate means of gaining sensitivity in direct and sandwich assays entails increasing the absolute amount of immobilized receptor by increasing the fiber surface area. This can be accomplished with small-diameter fibers by utilizing evanescent-wave excitation of biomolecules bound to the circumference of the sensing tip. These sensors are characterized by large exposed surface areas and low eva- nescent wave penetration depths ( 5 1 4 , 15). Their primary limitation is the fact that evanescently excited fluorescence is coupled less efficiently into the optical fiber than distal-face (vide infra) excitation. In this paper, the FIS is exposed to fluorophor-labeled and unlabeled ligand. This results in competition for FIS binding sites. Signal intensity in this competitive binding immu- noassay scheme is inversely proportional to a n a l e (unlabeled ligand) concentration. Though the fiber's diminutive surface area restricts the absolute amount of bound analyte, excellent sensitivity is achieved since an efficient distal-face fluorescence collection geometry is used. In addition, the small amount of immobilized receptor serves as the limiting reagent in the competitive assay. As a result, rapid, nonequilibrium low- volume analyses can be performed. Moreover, factors that alter fluorescence signals, such as quenching, matrix inter- ferences, and self-absorption are relatively insignificant for the FIS. This is partly due to the fact that the sensing ter- minus is thoroughly washed following analyte incubation and all measurements are made in a controlled matrix. EXPERIMENTAL SECTION Materials. Rabbit immunoglobin G (IgG), polyclonal anti- rabbit IgG, polyclonal fluorescein isothyocyanate (FITC) conju- gated anti-rabbit IgG, bovine serum albumin (BSA), and oval- bumin were purchased from Cooper Biomedical, Inc., Malvern, PA. Spectrapor cellulose dialysis membrane (MW cutoff = 12000) was acquired from Thomas Scientific, Philadelphia, PA. (3- Glycidoxypropy1)trimethoxysilane (GOPS) was obtained from Aldrich Chemical Co., Milwaukee, WI. Phosphate-buffered saline (PBS), pH 7.4, and all other reagents were supplied by Sigma Chemical Co., St. Louis, MO. @ 1987 American Chemical Society

Fiber-optic chemical sensors for competitive binding fluoroimmunoassay