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We have used an evanescently excited fiber optic immunoassay in order to study the kinetic response of an immunosens anddetermine its absolute sensitivity. We have estimated that when binding sites are saturated approximately 10" antigen are1xind to a fiber probe of 0.57 cm2 active area using optical techniques and without resorting to a radioassay. The number oflabelled antigen for a threshold signal is approximately 10g. We can easily detect fluorescently labelled antigen at subnanomo- lar concentrations and have measured the probe response to antigens over me than two orders of magnitude. The overall response time for this system and labelled antigen (Msw100,000) is on the order of 10 minutes for a 40 pM solution. At higherconcentrations, a response may be obtained in a few minutes. 1. INTRODUCTION A fiber optic sensor using evanescent wave excitation distinguishes specific surface binding (made specific by appropriatetreatment of the surface) from bulk fluorescence in solution. This capability to separate the signal of interest from backgroundinterferences is of interest in applications such as rapid screening of environmental samples for specific contaminants. Theabsolute sensitivity of this type of probe is seen to be relatively low; however, it is adequate for many applications in whichspecificity, ease of use, and speed are iniportantThe probe cnsists of an optical fiber which has been stripped of its cladding and had its core coated with antibodies targetedagainst a specific analyte. The antibodies bind only to that specific analyte, removing it from solution and holding it withina few hundred angstroms of the core of the fiber. Other contaminants will remain free in solution, but will not preferentiallybind to the fiber. Since the evanescent field decays quickly as one moves away from the fiber surface, little fluorescence isexcited in the bulk solution from free fluorophores. Although for a given number of fluorophores the evanescently excitedfluorescence is weak compared to the signal that could be generated from that same number offluorophores located at the distalfiber face, the larger surface area available on the side of the fiber allows for a greater quantity of antibody to be immobiliZeLHence the optical signal from either a distal or an evanescent configuration are couiparable,1 and the evanescent configurationsuffers less interference from fluorophores in the bulk solution. The combination of preferential binding and low interferingfluorescence give this sensor its specificity.2'The technology is flexible and may be used to develop assays for any chemical for which an antibody is available, includingPcBs, solvents such as benzene, toluene, and xylene, and thtroaromatic compounds. The fiber approach was chosen becauseof its potential to develop a sensor capable of real time performance and automated readont capabifity that does not requireextensive operator intervention nor complicated sample preparation. Our goal is to develop a specific sensor which could beused to characterize mixed waste streams by a relatively untrained user at low cost. We expect that the first GE applicationofthis sensor will be to assay for PCBs during site characterization and cleanup in an effort to increase the efficiexxy of remedi-ation activities.This paper describes the experimental configuration that we have developed for a fluorescent fiber immunoassay. We presentdose response curves for antigens in the concentration of 40 pM to 8 nM and demonstrate that our probes show no measurableresponse to control antigen nor to fluorophores in the bulk solution. We describe our technique for estimating the quantity ofantigen (and hence antibody) bound to the probe and compare the experimental results to the theoretical expectation. |
