Paper BI+AS+NS-ThA6
The Role of Liposomes in Fluorescent Based Microarrays: From Surface Immobilization of Membrane Proteins to Highly Fluorescent Labels

Thursday, November 12, 2009, 3:40 pm, Room K

Technologies utilizing arrays of immobilized biomolecules on planar surfaces are emerging as powerful high throughput tools for bioanalytical measurements. Nowadays, optical sensors based on fluorescence detection are the most widespread. However, many applications especially in the area of protein sensing, rely on the availability of optimized sensing interfaces and signal amplification strategies. Liposomes, due to their hollow particle-like structure and their unique chemical and physical properties, have greatly contributed to the development of sensitive and accurate biological assays.

In this presentation, we highlight with results obtained recently, the contributions of phospholipid vesicles to the development of high performance fluorescence based biosensors.

First, liposomes are an optimal platform for the surface immobilization of membrane proteins since they provide the natural environment required for the functional surface immobilization of these fragile molecules. We demonstrate the creation of a functional, heterogeneous array of G-protein coupled receptors. Vesicles obtained from cellular membrane extracts containing either the H1R-histamine receptor or the M2R-muscarinic receptors were immobilized on a conventional oligonucleotide microarray via complementary tags. Fluorescent ligand binding assays were then performed illustrating that the receptors kept their native conformation. As an alternative platform, we introduce a novel approach for the creation of vesicle multilayers using zirconium phosphate chemistry. As demonstrated in a model biomolecular binding assay, such three dimensional constructs increase the protein loading capacity of a sensor surface.

Liposomes are also excellent candidates as labels for biological assays: phosphocholine-based vesicles are non-fouling and biomolecules or marker molecules (e.g. fluorophores or enzymes) can be easily attached to their surface or encapsulated in their inner cavity. We show that fluorescently labeled phospholipid vesicles provide simple and cheap means for signal amplification and sensitive protein detection on a microarray format. Using vesicles, up to 100 fold increase in sensitivity was observed in a model protein microarray with confocal read-out, compared to a conventional assay performed with fluorophore labeled antibodies.

The various approaches presented here will contribute to the development of sensitive and high performance microarrays for a variety of applications including the investigation of membrane proteins.