Stacks of close to 100 phospholipid bilayers fully accessible to proteins: An ATR–FTIR-based chemometric analysis on hydrated phospholipid films

Abstract

A novel method for measuring aqueous solutions of proteins having high affinity for specific membrane components has been developed from attenuated total reflection–Fourier transform infrared (ATR–FTIR) spectra. The method was based on the use of a germanium internal reflection element (IRE) which surface was covered with a PE–biotin film. In order to investigate the potentialities of this approach, streptavidin in aqueous solutions was recirculated continuously in a flow-trough mode on the hydrated phospholipid film. Partial least squares (PLS) and principal component regression (PCR) were used and compared for establishing a multivariate calibration model for streptavidin concentrations ranging from 50 to 500 mg l −1. The best agreement between the known and predicted concentrations of streptavidin by PLS and PCR was obtained when both methods were optimized by selecting the spectral regions having the highest correlation with concentration data. PLS revealed more accurate results than PCR with a standard error of prediction (SEP) of 18.56 mg l −1 (7%). The streptavidin amount effectively bound to the PE–biotin film was estimated separately via a univariate regression between ATR–FTIR absorbance and concentration data representing different protein:phospholipid ratios. The results demonstrated that the protein was capable of diffusing and attaining all of the available sites in the multilayer PE–biotin matrix. Such a phospholipid film-based measurement method is a promising tool for developing new sensing methods for analyzing biomolecules in aqueous solutions.