Study of cholesterol derivative and phospholipid (DPPC) mixed film using LB technique and FRET: Design of cholesterol sensor

Abstract

In this work mixed film behavior of Cholesterol derivative 5α, 6α-Epoxycholestan-3β-ol (Chol) and a Phospholipid named 1,2-Dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) have been investigated using Langmuir-Blodgett (LB) technique. Surface pressure-area (π-A) isotherms, UV–vis, and fluorescence spectroscopy and Brewster Angle Microscopy (BAM) studies were carried out for characterizations of the films at air – water interface and solid substrate. π-A isotherms as well as analysis of excess area and Gibb’s free energy suggested interaction between Chol and DPPC in the mixed monolayer. However, interaction between Chol and DPPC balances at Chol mole fraction (XChol) 0.77 resulting in a stable and almost ideally mixed monolayer. BAM investigations give visual evidence of interaction showing change in domain structure/nature in the DPPC-Chol mixed monolayer compared to their pure counterpart. Fluorescence Resonance Energy Transfer (FRET) was employed to sense the presence of Chol in the lipid bilayer film. Acriflavine (Acf) and Rhodamine B (RhB) were used as donor and acceptor respectively. It was found that FRET efficiency linearly changes with Chol concentration in the lipid bilayer. With proper calibration, it may be possible to use this present system under investigation to sense Chol in cell membrane. Proposed sensing system has also been tested using real serum sample.