Nanoplasmonic Fluorescence Enhancement Applied to Study of Ion Channels

Abstract

A metal nanoparticle can act as an antenna capable of increasing both the excitation rate and quantum yield of a fluorophore in close proximity to the nanoparticle. These properties thus enhance the fluorescence yielding an increase in brightness and decrease of the photobleaching rate - a highly useful tool in fluorescence studies of membrane proteins from the macroscopic to single molecule level. We have applied this technique to image purified membrane proteins in supported bilayer. The biological sample is purified KcsA K-channels labeled with TMR-6-M in the bundle crossing and reconstituted as proteoliposomes. We have also studied the membrane fluorophore DiI C18 in supported bilayer as a control. Among the many approaches towards fabrication of effective nanoparticles, we have synthesized spherical silver nanoparticles of ∼100 nm diameter coated with a thin SiO2 outer layer (Ag@SiO2). We have explored different size particles and various SiO2 thicknesses to find experimental conditions for optimal fluorescence enhancement. The SiO2 layer provides protection from chemical attack, acts as a spacer layer to avoid direct metal-fluorophore quenching, and allows surface functionalization. We have conjugated silica-coated silver particles to glass coverslips via polylysine (PL) in order to achieve a high-density silver nanoparticle monolayer. We record fluorescence from the labeled ion channels in an inverted TIRF microscope configuration imaged with a high-speed EMCCD camera. KcsA proteoliposomes are added to an Ag@SiO2-PL coverslip surface to rupture as supported bilayer patches for single molecule imaging. DiI liposomes were used in the same way. The KcsA-TMR and DiI samples show enhancement of at least 4-fold and 10-fold, respectively, compared to the same sample without nanoparticles. These results demonstrate the utility of this technique in fluorescence studies of ion channels or other membrane proteins. Supported by NIH 1R21MH078822 & 1F31NS054532.