Chapter 16 - Fluorescence-based intracellular sensing

Abstract

This chapter deals with the myriad structures and functions of all the cellularcomponents and describes how they function in concert. It examines the state-of-the-art biosensors available for such use and focuses on those that utilize fluorescence and fluorescence resonance energy transfer in particular. Under this, it defines fluorescent sensors as sensing moieties that are labeled with one or two fluorophores wherein the binding of target analyte results in a change in fluorescence or FRET. Alternatively, these sensors can take the form of fluorophores that fluoresce or quench in response to specific stimuli. Intracellular targets of interest for monitoring include nucleic acids, small molecule nutrients and ligands, post-translational modifications, intracellular localization, and a variety of biochemical reactions involved in signal transduction cascades. Fluorescent protein sensors typically contain a centrally located protein or peptide-based substrate moiety appended with two fluorescent proteins that function as an efficient FRET D-A pair. Upon the binding of target analyte or the modification of the substrate sequence, a change in fluorescence or FRET is measured. Intracellular or in situ biosensors are anticipated to have tremendous impact upon research into the understanding of basic cellular structure and function.