Ultralow Fouling and Functionalizable Surface Chemistry Based on a Zwitterionic Polymer Enabling Sensitive and Specific Protein Detection in Undiluted Blood Plasma

Abstract

A crucial step in the development of implanted medical devices, in vivo diagnostics, and microarrays is the effective prevention of nonspecific protein adsorption from real-world complex media such as blood plasma or serum. In this work, a zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) biomimetic material was employed to create a unique biorecognition coating with an ultralow fouling background, enabling the sensitive and specific detection of proteins in blood plasma. Conditions for surface activation, protein immobilization, and surface deactivation of the carboxylate groups in the polyCBAA coating were determined. An antibody-functionalized polyCBAA surface platform was used to detect a target protein in blood plasma using a sensitive surface plasmon resonance (SPR) sensor. A selective protein was directly detected from 100% human blood plasma with extraordinary specificity and sensitivity. The total nonspecific protein adsorption on the functionalized polyCBAA surface was very low (<3 ng/cm (2) for undiluted blood plasma). Because of the significant reduction of nonspecific protein adsorption, it was possible to monitor the kinetics of antigen-antibody interactions in undiluted blood plasma. The functionalization effectiveness and detection characteristics using a cancer protein marker candidate of polyCBAA were compared with those of the conventional nonfouling oligo(ethylene glycol)-based surface chemistry.

Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites

“…26). The specificity of biosensors and discrimination against the unwanted signal of background proteins are usually achieved via surface chemistry and the binding selectivity 27,28 . In practice, a small fraction of the background proteins do bind to the surface, creating a residual signal.…”

Ultralow‐Fouling, Functionalizable, and Hydrolyzable Zwitterionic Materials and Their Derivatives for Biological Applications

“…[50] Results show that antibodies can be immobilized onto pCB surfaces in high capability and used to detect antigens in 100% blood plasma with very low background noise. [47] This has not been achieved by any other material including PEG. For many applications, such as multi-functional nanoparticles, protein microarrays, and tissue scaffolds, it is highly desirable to develop surface coatings or bulk materials that combine an ultralow-fouling background with abundant bio-recognition elements immobilized.…”

“…[38] Until recent years, studies of pCB have been limited to its chemical and physical properties. It was demonstrated recently that pCB shows undetectable nonspecific protein adsorption (<0.3 ng cm À2 ) from single-protein solutions [45] or complex media [46,47] by SPR. The structure of CB is similar to that of glycine betaine, which is one of the compatible solutes vital to the osmotic regulation of living organisms.…”

“…1d), when uniformly distributed at the molecular level, are equivalent to zwitterionic materials and highly resistant to nonspecific protein adsorption. It is shown that an ultralow fouling surface can be achieved when the surface contains a nanometer-scale homogenous mixture of balanced charged groups not only from zwitterionic (both positively and negatively charged moieties connected in the same group) [46,47,50,51] but also mixed positively and negatively charged moieties from different groups, such as mixed-charge SAMs, [52,53] polymer coatings, [54] or hydrogels. [55] Based on the mixed-charge principle, it is possible to design and prepare a wide spectrum of new nonfouling materials, such as ultralow-fouling natural peptides in the form of either alternating or randomly mixed charge.…”

“…[32,43,58,59] The ''graft-to'' method involves (i) the preparation of polymers containing nonfouling and adhesive groups, such as 3,4-dihydroxyphenylalanine (DOPA) groups [24,60] and polylysine segments, [61] and (ii) the direct attachment of the polymers onto the surface. pSB and pCB have been grafted to or from surfaces via several common adhesive linkers, including thiols for a gold surface, [43,45,47,50,51,62,63] silanes for a glass surface, [64] hydrophobic moieties for a hydrophobic surface, [41,65,66] or DOPA for various surfaces, [67,68] or via interpenetrating networks (IPNs) into polyurethane (SPU) matrices. [69] pSB-based hydrogels have also been prepared.…”