Confocal Raman Microscopy for Label-Free Detection of Protein-Ligand Binding at Nanopore-Supported Phospholipid Bilayers.
Anal Chem
; 90(19): 11509-11516, 2018 10 02.
Article
em En
| MEDLINE
| ID: mdl-30175578
ABSTRACT
Interactions of lectins, proteins that selectively bind carbohydrates, play an important role in many biological processes including cell adhesion, immune response, and cell signaling. Given the range of lectin functions and their potential for application in disease detection, there is a need for methods to investigate lectin-carbohydrate interactions that are rapid, structurally specific, and sensitive to binding from low-concentration samples. In this work, we describe the preparation and application of supported phospholipid bilayers deposited in wide-pore chromatographic silica particles for confocal Raman-microscopy-based detection of specific binding of concanavalin-A to mannose-functionalized phospholipids. The high surface area of porous-silica supports provides an ample concentration of phospholipid and protein for rapid, label-free detection of lectin binding to be carried out in an individual lipid-bilayer-functionalized particle. The Raman spectrum provides structural information on the bound protein as well as the phospholipid bilayer. Using scattering from the supported-lipid bilayer as an internal standard, Raman scattering from accumulated protein can be interpreted quantitatively to determine its absolute surface coverage on the lipid bilayer. At low glycolipid fraction (<1 mol %) in the prepared bilayer, the surface coverage by protein increases linearly with mannose-lipid densities, where the lectin population corresponds to â¼96% occupancy of the mannose ligands. At increasing glycolipid site densities in excess of 1 mol %, the surface-associated protein population saturates at a coverage that is equivalent to a full monolayer of mannose-bound lectin proteins. The results suggest that Raman microscopy of supported phospholipid bilayers in high-surface-area support particles is a promising approach for in situ, label-free, and quantitative investigation of bilayer-localized protein-ligand interactions.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Microscopia Confocal
/
Nanoporos
/
Lectinas
/
Bicamadas Lipídicas
/
Manose
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Estados Unidos