Chiral second harmonic generation for use in monitoring protein adsorption to lipid bilayers and imaging.

摘要

The investigation of molecular and protein adsorption to planar supported lipid bilayers (PSLBs) and imaging of patterned membranes by counter-propagating chiral second harmonic generation (C-SHG) are discussed. The unique aspect of this work is the use of a counter-propagating geometry to selectively isolate the chiral second harmonic emission from a surface, and the application of this novel geometry to produce a new form of chiral imaging. The theoretical framework for both a co-propagating and counter-propagating geometry applied to SHG is discussed, and the differences in the chiral SH emission between the geometries are highlighted through use of model calculations. The counter-propagating C-SHG technique is applied to a model system of (R)-(+)-1,1' -bi-2-naphthol (RBN) and (S)-(+)-1,1'-bi-2-naphthol (SBN) binding to PSLBs of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The thermodynamic binding affinity and kinetics of adsorption were obtained for RBN and SBN to POPC bilayers. Using surface tension measurements the C-SHG response was calibrated for RBN surface density within the POPC bilayer. An enantiomer excess study using RBN and SBN was also conducted to show the role of enantiomeric ratios on the C-SHG response.; The counter-propagating C-SHG technique was also used to measure melittin, a peptide isolated from bee venom, binding to PSLBs of POPC, as a test of the methodology. Using the chiral SH emission from the alpha-helix of melittin a thermodynamic binding affinity was obtained. The role phosholipase A 2 plays in melittin orientation and binding was also revealed through the thermodynamic measurements. Nonlinear SHG spectra for both the achiral and chiral SH emission from melittin bound to POPC were also obtained and compared with UV/Vis absorbance and circular dichroism (CD) spectra. Chiral nonlinear SHG spectra suggest melittin adopts an alpha-helical structure upon insertion into the lipid bilayer.; Finally the counter-propagating C-SHG technique was used to obtain images of the chiral emission of RBN and SBN bound to micro-contact printed PSLBs of POPC. The C-SHG images were compared with fluorescence images obtained through labeling of the lipid bilayer. Comparison of the C-SHG and fluorescence images showed good correlation in both resolution and image quality.; Through the development of a novel counter-propagating geometry the limitations of a co-propagating geometry to measure chiral species at surfaces with SHG have been overcome. These results have important implications for the study of protein adsorption at surfaces and chiral imaging.