Direct, ultrasensitive, and selective optical detection of protein toxins using multivalent interactions

Three highly sensitive, selective, and reagent-free optical signal transduction methods for detection of polyvalent proteins have been developed by directly coupling distance-dependent fluorescence self-quenching and/or resonant-energy transfer to the protein-receptor binding events. The ganglioside GM1, as the recognition unit for cholera toxin (CT), was covalently labeled with fluorophores and then incorporated into a biomimetic membrane surface. The presence of CT with five binding sites for GM1 causes dramatic change for the fluorescence of the labeled GM1. (1) In the scheme using fluorescence self-quenching as a signal-transduction mechanism, the fluorescence intensity drops significantly as a result of aggregation of the fluorophore-labeled GM1 on a biomimetic surface. (2) By labeling GM1 with a fluorescence energy transfer pair, aggregation of the labeled GM1 results in a decrease in donor fluorescence and an increase in acceptor fluorescence, providing a unique signature for selective protein-receptor binding. (3) In the third scheme, using the biomimetic surface as part of signal transduction and combining both fluorescence self-quenching and energy-transfer mechanisms to enhance the signal transduction, a signal amplification was achieved. The detection systems can reliably detect less than 0.05 nM CT with fast response (less than 5 min). This approach can easily be adapted to any biosensor scheme that relies on multiple receptors or co-receptors. The methods can also be applied to investigate the kinetics and thermodynamics of the multivalent interactions.