Development of a novel FRET immunosensor for detection of Listeria

Abstract

A fluorescence resonance energy transfer (FRET)-based Listeria biosensor has been developed. Sensors utilizing FRET switch their fluorescence wavelength between donor and acceptor fluorophores as the distance between the two fluorophores change. This change in distance is a result of the conformational change in the 3D structure of the antibody as it binds to the target antigen. Listeria antibodies were labeled with FRET donor fluorophores (Alexa Fluor 546) while protein G was labeled with the acceptor fluorophores (Alexa Fluor 594). The labeled antibody-protein G complex was formed via incubation of the labeled antibody with protein G which specifically attaches to the Fc fragment of antibodies. The labeled antibody-protein G complex was tested in solution and specific and non-specific antigens were exposed to the in solution complex. Changes in fluorescence were monitored by a spectrofluorometer. For "in-solution" tests, the optimal acceptor/donor fluorophore (A/D) ratio was 1.0 for Listeria, and Listeria antigen detection limits were 2.0 Hg/ml. Immobilization studies were also performed where the protein/antibody complex was immobilized to an optical fiber and interface with a benchtop fluorometer. The results showed the limit of detection was 103 cells/ml of Listeria monocytogenes at a surface packing density of 0.033 mg/ml.