Detecting Protein−Ligand Binding on Supported Bilayers by Local pH Modulation

Abstract

Herein, we describe a highly sensitive technique for detecting protein−ligand binding at the liquid/solid interface. The method is based upon modulation of the interfacial pH when the protein binds. This change is detected by ortho-Texas Red DHPE, which is doped into supported phospholipid bilayers and used as a pH-sensitive dye. The dye molecule fluoresces strongly at acidic pH values but not basic ones and has an apparent pK_A of 7.8 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes containing 0.5 mol % biotin-cap-PE. This method was used to detect antibiotin/biotin binding interactions as well as the binding of cholera toxin B subunits to GM₁. Since these proteins are negatively charged under the conditions of the experiment the interface became slightly more acidic upon binding. In each case, the equilibrium dissociation constant was determined by following the rise in fluorescence as protein was introduced. This change is essentially linear with protein coverage under the conditions employed. For the biotin/antibiotin system it was determined that K_D = 24 ± 5 nM, which is in excellent agreement with classical measurements made by total internal reflection fluorescence microscopy involving fluorophore-conjugated antibody molecules. Moreover, the limit of detection was ∼350 fM at the 99% confidence level. This corresponds to 1 part in 69 000 of the K_D value. Such a finding compares favorably with surface plasmon resonance studies of similar systems and conditions. The assay could be run in imaging mode to obtain multiple simultaneous measurements using a CCD camera.