Stable, ligand-doped, poly(bis-SorbPC) lipid bilayer arrays for protein binding and detection

Abstract

A continuous-flow microspotter was used to generate planar arrays of stabilized bilayers composed of the polymerizable lipid bis-SorbPC and dopant lipids bearing ligands for proteins. Fluorescence microscopy was used to determine the uniformity of the bilayers and to detect protein binding. After UV-initiated polymerization, poly(lipid) bilayer microarrays were air-stable. Cholera toxin subunit b (CTb) bound to an array of poly(lipid) bilayers doped with GM(1), and the extent of binding was correlated to the mole percentage of GM(1) in each spot. A poly(lipid) bilayer array composed of spots doped with GM(1) and spots doped with biotin-DOPE specifically bound CTb and streptavidin to the respective spots from a dissolved mixture of the two proteins. Poly(bis-SorbPC)/GM(1) arrays retained specific CTb binding capacity after multiple regenerations with a protein denaturing solution and also after exposure to air. In addition, these arrays are stable in vacuum, which allows the use of MALDI-TOF mass spectrometry to detect specifically bound CTb. This work demonstrates the considerable potential of poly(lipid) bilayer arrays for high-throughput binding assays and lipidomics studies.

FIGURE 1

Epifluorescence images of a PSLB microarray consisting of UV-irradiated Rh-DPPE/bis-SorbPC (a and c) and Rh-DPPE/DOPC (b and d) before (a and b) and after (c and d) exposure to air. The Rh-DPPE loadings in bis-SorbPC and DOPC bilayers are 1.9% and 1.0% (mol/mol), respectively. (e) Plot of normalized average intensities of the two types of PSLB spots before and after air exposure. The error bars represent the standard deviations of five trials.

FIGURE 2

(a) Epifluorescence images of a lipid microarray of 0%, 0.5%, 2%, and 10% GM₁ in poly(bis-SorbPC) after adsorption of Alexa488CTb. (b) Plot of the normalized average intensities of the gradient array shown in part a. The error bars represent the standard deviations of six trials.

FIGURE 3

Plot of normalized average fluorescence intensities of Alexa488CTb and TRITC-SA adsorbed from a mixture of the two proteins to a lipid microarray consisting of 30% biotin-DOPE in poly(bis-SorbPC), 10% GM₁ in poly(bis-SorbPC), and pure poly(bis-SorbPC). The error bars represent the standard deviations of six trials.

FIGURE 4

Plot of the average fluorescence intensity of Alexa488CTb adsorbed to a 10% GM₁/poly(bis-SorbPC) PSLB through three regeneration cycles followed by air exposure. The error bars represent the standard deviations of four to six trials.

FIGURE 5

MALDI-TOF mass spectra of Alexa488CTb adsorbed to bilayers of (a) 10% GM₁/poly(bis-SorbPC) and (b) pure poly(bis-SorbPC).