Antibody Binding at the Liposome–Water Interface: A FRET Investigation toward a Liposome-Based Assay

Abstract

Different signal amplification strategies to improve the detection sensitivity of immunoassays have been applied which utilize enzymatic reactions, nanomaterials, or liposomes. The latter are very attractive materials for signal amplification because liposomes can be loaded with a large amount of signaling molecules, leading to a high sensitivity. In addition, liposomes can be used as a cell-like “bioscaffold” to directly test recognition schemes aiming at cell-related processes. This study demonstrates an easy and fast approach to link the novel hydrophobic optical probe based on [1,3]dioxolo[4,5- f ]-[1,3]benzodioxole (DBD dye mm239) with tunable optical properties to hydrophilic recognition elements (e.g., antibodies) using liposomes for signal amplification and as carrier of the hydrophobic dye. The fluorescence properties of mm239 (e.g., long fluorescence lifetime, large Stokes shift, high photostability, and high quantum yield), its high hydrophobicity for efficient anchoring in liposomes, and a maleimide bioreactive group were applied in a unique combination to build a concept for the coupling of antibodies or other protein markers to liposomes (coupling to membranes can be envisaged). The concept further allowed us to avoid multiple dye labeling of the antibody. Here, anti-TAMRA-antibody (DC7-Ab) was attached to the liposomes. In proof-of-concept, steady-state as well as time-resolved fluorescence measurements (e.g., fluorescence depolarization) in combination with single molecule detection (fluorescence correlation spectroscopy, FCS) were used to analyze the binding interaction between DC7-Ab and liposomes as well as the binding of the antigen rhodamine 6G (R6G) to the antibody. Here, the Förster resonance energy transfer (FRET) between mm239 and R6G was monitored. In addition to ensemble FRET data, single-molecule FRET (PIE-FRET) experiments using pulsed interleaved excitation were used to characterize in detail the binding on a single-molecule level to avoid averaging out effects.

Photoluminescent Nanoparticles for Chemical and Biological Analysis and Imaging

“…(B) Schematic showing the incorporation of a dye (DBD mm239) with a hydrophobic tail into a liposome. Reproduced from ref . Copyright 2018 American Chemical Society.…”

“…In general, PLMs are associated with lipid NPs through preferential partitioning into the core of SLNs or the lipid bilayer membrane of liposomes via hydrophobic interactions, covalent modification of lipid headgroups, electrostatic interactions with lipid headgroups or SLN-stabilizing surfactants, and encapsulation within the aqueous interiors of liposomes. , An example of the incorporation of a fluorescent dye with a hydrophobic tail into a liposome is shown in Figure B …”

“…423,424 An example of the incorporation of a fluorescent dye with a hydrophobic tail into a liposome is shown in Figure 17B. 425…”

[1,3]-Dioxolo[4,5-f]benzodioxole (DBD) Fluorescent Dyes; Synthesis, Properties, and Applications

“…Upon binding of R6G to the antibody, FRET takes place where DBD acts as the donor. 48 The DBD-labeled fatty acid 22 (cf. Scheme 4) was used for another intermolecular FRET system.…”

Production of giant unilamellar vesicles and encapsulation of lyotropic nematic liquid crystals

“…Giant Unilamellar Vesicles (GUVs) have many potential applications. [1][2][3][4][5][6][7][8] They are widely used as cell models (protocells) 9,10 and in studies in which the ultimate aim is to create artificial life. [10][11][12][13][14][15] As a result, many methods have been developed for their production and for encapsulating materials within them.…”