Abstract: We have developed a new fluorescence-based biosensor for sensitive detection of species involved in a multivalent interaction. The biosensor system utilizes specific interactions between proteins and cell surface receptors, which trigger a receptor aggregation process. Distance- dependent fluorescence self-quenching and resonant fluorescence energy transfer mechanisms were coupled with a multivalent interaction to probe the receptor aggregation process, providing a sensitive and specific signal transduction method for such a binding event. The fluorescence change induced by the aggregation process can be monitored by different instrument platforms, e.g. fluorimetry and flow cytometry. In this article, a sensitive detection of pentavalent cholera toxin which recognizes ganglioside GMI has been demonstrated through the resonant energy transfer scheme, which can achieve a double color change simultaneously. A detection sensitivity as high as 10 pM has been achieved within a few minutes (c.a. 5 minutes). The simultaneous double color change (an increase of acceptor fluorescence and a decrease of donor fluorescence intensity) of two similar fluorescent probes provides particularly high detection reliability owing to the fact that they act as each other's internal reference. Any external perturbation such as environmental temperature change causes no significant change in signal generation. Besides the application for biological sensing, the method also provides a useful tool for investigation of kinetics and thermodynamics of a multivalent interaction.!19
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