Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

Abstract

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions 1 . In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA 2,3,4,7 . This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower 8 . Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

Investigating Ligand–Receptor Interactions at Bilayer Surface Using Electronic Absorption Spectroscopy and Fluorescence Resonance Energy Transfer

“…One of the consequences of this increase in the absorbance led to significant errors in the calculations of spectral overlap ( J ). Based on our recent work 61 , we would like to emphasize that the calculations of J , E and CR provide very useful qualitative information and our interpretations of the data do not change significantly due to errors resulted from light scattering.…”

Micro-motors: A motile bacteria based system for liposome cargo transport

“…To investigate the ligand-receptor attachment, we utilized recently reported FRET-based methods, where bacterial interaction with glycolipids probed on the cell membrane surface were analyzed 22 29 . FRET only occurs when a donor molecule is in close proximity (typically less than 10 nm) of an acceptor molecule 30 31 .…”

Citral nanocontainers applied to guava fruits (Psidium Guajava L.) in postharvesting

“…[21] The dispersed solution obtained was immersed in an ultrasound bath for 15 min and homogenized by ultrasonic probe at 75 °C, 25% amplitude, and a pulse cycle of 5:1 seconds on:off for 15 min. [22] The dispersed solution was then passed through a polycarbonate membrane in order to remove any lipid aggregate. [23] Finally, the dispersed solution of liposomal nanocontainers was refrigerated for 8 h, at 4 °C and protected from light.…”

“…The free citral was removed from the liposomal nanocontainers through dialysis with a cellulose ester membrane in an aqueous medium for 48 h with water changes every 4 h. Prior to use the liposomal nanocontainers were polymerized using UV radiation at 254 nm, for 2 min at 4 °C. [22] The dialyzed and polymerized liposomal nanocontainers of PDA:Lecithin, were characterized using UV-Vis spectroscopy scanning in the 400-800 nm range with a scanning speed of 0.5s. The samples were analyzed before and after being exposed to a thermal stimulus of 50ºC with distilled water being used as a blank.…”