Multifunctional Zwitterionic Surface Chemistry for Applications in Complex Media

Abstract

The realization of personalized medicine relies on the discovery of clinically relevant biomarkers as well as on the development of corresponding assays. Due to the complexity of human blood plasma and serum, the most common sources for biomarker analysis, current attempts to integrate existing biosensing assays with analyte detection has resulted in two major shortcomings: high rates of false-positives, from non-specific binding, and a lack of assay sensitivity, due to low ligand loading. Taken together, these two factors indicate that a high signal-to-noise ratio (S/N) is vital for achieving sensitive biomarker-based diagnostics. Furthermore, a single material that can (1) exhibit non-fouling properties from undiluted human blood, (2) present abundant and easily functionalizable chemical groups for ligand attachment, and (3) possesses high immobilization capacities, would offer the most promising approach to achieving this goal. Such idealities were addressed using zwitterionic poly(carboxybetaine) (pCB) surface chemistry. First, an important parameter was realized for identifying surface coatings suitable for real-world applications involving undiluted complex media. It was found that ultra low fouling properties using a thin film is possible if it is densely packed. While such prevention of non-specific adsorption is important, the detection of biomarkers also hinges on the ability to immobilize biologically active ligands all while maintaining the original ultra low fouling background noise of the surface coating. Hence, the dual-functionality of pCB, which provides both protein resistance and ligand functionalization, was then applied to protein arrays. Here, uniform spot morphology as well as excellent non-fouling properties following antibody immobilization was achieved. This enabled improvements in the sensitivity for multiplexed detection of target analytes directly from undiluted human plasma. As even the best non-fouling background combined with the highest affinity ligand would still have a limited S/N ratio due to the 2-dimensional (2-D) structure of polymer films, two efforts to improve the "signal" component were also investigated. The first method led to the development of a hierarchical architecture consisting of a thin and highly dense first layer and a loose but controlled second layer, for low fouling and high ligand loading, respectively. The second approach for improving biomarker assay performance involved taking advantage of new biosensor devices. Such novel sensor designs exhibit decreasing surface dimensions with unique geometries and enhanced theoretical sensitivities. Due to these distinct characteristics, the development of a dual-functional "graft-to" surface coating was necessary. Here, the conjugation of the adhesive molecule DOPA with pCB enabled the successful attachment to a biosensor surface while also demonstrating ultra low fouling and functionalization properties. This "graft-to" technology can be readily extended to other device platforms. Finally, while normal immobilization conditions for pCB allow for the attachment of acidic and neutrally charged ligands, two strategies for expanding the range of ligands, to include basic proteins (i.e. with high isoelectric points), which can be coupled to an ultra low fouling zwitterionic background were also investigated. It was found that the use of reversible citraconic anhydride protection enabled the coupling of the highly basic protein lysozyme to the pCB surface. The second strategy, involving a novel zwitterionic ultra low fouling material, led to an initial characterization which indicated promising results. In summary, this work represents a multifunctional zwitterionic surface chemistry readily suitable for applications in undiluted complex media.