Confocal Raman Microscopy for Label-Free Detection of Protein–Ligand Binding at Nanopore-Supported Phospholipid Bilayers

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 ∼96% 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.

Ultrasensitive and Label-Free Detection of Cell Surface Glycan Using Nanochannel-Ionchannel Hybrid Coupled with Electrochemical Detector

“…Meanwhile, confocal Raman microscopy also shows the typical vibrations of different groups in Con A attached to PAA surface, including phenylalanine ring breathing mode (1003 cm −1 ) and various backbone amide bands (1233 cm −1 , 1556 cm −1 , 1670 cm −1 ) (SI Figure S3). 46 Finally, EIS was used as a powerful tool to analyze the combining process of Con A with glycan (Figure 1E). Various processes were analyzed using Au electrode as substrate, and the EIS experiments were carried out in 0.1 M KCl solution containing 5.0 mM [Fe(CN) 6 ] 3− / 4− .…”

Towards a protein-selective Raman enhancement by a glycopolymer-based composite surface

“…The peak at 960 cm À1 can be attributed to the phenylalanine ring breathing mode, the peak at 1137 cm À1 can be attributed to the bending vibration of C-O-C, the peak at 1267 cm À1 can be attributed to the C-N stretching vibration and N-H bending vibration of the amide III bands, the peak at 1492 cm À1 can be attributed to the bending vibration of CH 2 , the peak at 1554 cm À1 can be attributed to the C-N stretching vibration and N-H bending vibration of the amide II bands and the peak at 2930 cm À1 can be attributed to the C-H stretching vibration of the aliphatic amino acid group. 60,61 The enhanced Raman signals guarantee the selective detection of Con A lectin.…”

Optical Spectroscopy of Surfaces, Interfaces, and Thin Films: A Status Report

“…Raman spectroscopy, especially Raman imaging, has been utilized in numerous studies to collect critical molecular-level information about biological systems. The Harris laboratory recently created supported phospholipid bilayers deposited onto the internal surfaces of the nanoporous network of high-surface area chromatographic silica particles and used confocal Raman-microscopy to detect specific binding of concanavalin-A (Con-A) to mannose-functionalized phospholipids . Using the Raman scattering from the lipid bilayer as an internal standard, the Raman signal from bound protein was quantified in terms of Con-A absolute surface coverage.…”