Cross-linked Phospholipid Coatings in Micron-Sized Channels for Spatially Discrete Bioassays and Protein Separations

Abstract

Supported phospholipid bilayers (SPBs) are attractive surface coatings since they are resistant to non-specific protein adsorption, self-assemble, support the function of many integral and membrane bound proteins, and can be functionalized with a variety of available headgroup chemistries. Phospholipid capillary coatings have been reported for applications in capillary electrophoresis (CE), although these coatings suffer from inherent stability problems, which arise from the fragility of the interactions between the bilayer and supportive substrate. Patterned immobilized domains can be functionally diversified, enabling for the screening of multiple analytes in a condensed region. The application of polymerized phospholipids was investigated for applications in CE and preparation of low volume biosensing arrays. A polymerized phospholipid, bis-SorbPC, was used to stabilize the phospholipid bilayer through cross-linking on the surface of fused silica capillaries and compared to other phospholipid capillary coatings for stability and separation quality. Polymerized bis-SorbPC coatings have significantly longer stability against degradation and can be stored for > one year with improved separation efficiency of proteins over bare capillary separations.Three-dimensional fluidic channels were modified with polymerized phospholipid features, which can be chemical "tuned" to introduce specific affinity binding, such as biotin-avidin or antibody-antigen binding, while minimizing nonspecific protein binding to the area of interest. Patterning of bis-SorbPC channels was achieved and the void regions between poly(bis-SorbPC) regions can be filled with other phospholipids to monitor the non-covalent binding (e.g. biotin-avidin, NTA-Ni2+-6xHis-protein) and covalent binding of proteins to DOPE-PEG-pNP and maleimide functionalized lipids, while minimizing non-specific adsorption and protein denaturation. Analytes can be immobilized on the surface via immunobinding events for detection and immunoassay applications. SPBs were used to support membrane proteins within patterned regions of the channel. Functionality can be added to the poly(bis-SorbPC) regions with copolymerization allowing regions of distinct functionality to reside adjacently in the channel. Through multiple polymerization steps, channels with multiple functionalities are demonstrated, with the possibility for higher-throughput multi-functional biosensing channels in the future.