Construction of hybrid bilayer membrane (HBM) Biochips and characterization of the cooperative binding between cytochrome-c and HBM.

We constructed multi-channel hybrid bilayer membrane (HBM) biochips and characterized them by surface plasmon resonance imaging. Each channel in the biochip was prepared using vesicles with different proportions of negative, neutral, and positive lipids. The HBM surfaces were tested by interaction with two globular proteins that recognize surfaces covered with opposite charges. Spots modified with the same HBM show responses within a relative standard deviation of 10% or smaller. These devices were also used to study in detail the interaction between cytochrome-c (cyt-c) and HBMs. Cooperative binding between cyt-c and negative HBMs was demonstrated. Using an adaptation of the Hill model, we calculated a Hill coefficient of 5 and a 10-fold increase in the binding constant with the increase in cyt-c concentration. We propose that this treatment can be used to evaluate the cooperative binding of surface proteins to membranes.