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

Summary

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

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 interactions¹. 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 lower⁸. 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.

Protocol

A. Synthesis and Characterization of PDA Liposomes^4,5,6

Note 1: Protect the PDA solution from light using aluminum foil wrapping on every container throughout all the experimental steps.

Note 2: Two different sets of liposome solution (B and C) were prepared following procedure A (Synthesis and characterization of PDA liposomes).

1. Synthesis of N-hydroxysuccinimide Diacteylene (NHS-PCDA)

1. To prepare liposomes, an essential ingredient PCDA-NHS is required. We have synthesized PCDA-NHS using following procedure:
2. Add 10,12-pentacosadiynoic acid (PCDA) (0.267 ....

Discussion

We have performed selective binding of lysine residue of protein on liposome surface using NHS-amine reaction. This FRET based method is capable of doing Real-time monitoring of biotin-streptavidin binding and protein (BSA) binding to the liposome surface. Similar procedure can be applied to study the binding dynamics of various protein interactions with their selective receptors. There is flexibility in choosing fluorophores that will provide changes in the J values depending upon the spectral characteristics o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10,12-pentacosadiynoic acid (PCDA)	GFS chemicals	3261	Light sensitive
N-hydroxysuccinimide (NHS)	Acros organics	157270250	Moisture sensitive
1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC)	Chem-impex International	00050
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)	Avanti Polar lipids	850345P
Rhodamine-tagged Bovine Serum Albumin (BSA-Rh)	Sigma Aldrich	A4537
(1,2-dioleoyl-sn-glycero-3-phosph–thanolamine-N-(biotinyl)(biotin-DOPE)	Avanti Polar lipids	870282