Recognitive hydrogel coated gold nanoshells for label-free detection of tear biomarkers

Despite the high prevalence of dry eye, many challenges exist when diagnosing the symptom and related diseases. Thus, novel biosensors are needed to detect changes in dry eye specific biomarker expression for screening and diagnostic applications. For this purpose, crosslinked synthetic polymers and plasmon-based sensing, were used as the recognition and signal transducing elements in the development of a low cost, label-free biosensor for detection of dry eye biomarkers in human tears. A library of poly(N-isopropylacrylamide)-based nanogels containing a custom aldehyde monomer, which forms covalent bonds with solvent accessible lysine residues present on the protein surface, and/or an anionic monomer (methacrylic acid) were fabricated. The concentrations of anionic and aldehyde monomers in the synthesized nanogels impacted their swelling behavior, charge character, and their protein binding behavior. The protein binding results obtained at the nanogel level indicated that electrostatic interactions are the main driving force behind protein-polymer interactions. Specifically, anionic nanogels possess recognition affinities towards high isoelectric point proteins (namely, lysozyme, lactoferrin, and IgG) in non-competitive buffered environments. In contrast, the incorporation and concentration of the aldehyde monomer capable of forming covalent interactions with select high isoelectric point tear proteins in the hydrogel coated gold nanoshells (AuNS), resulted in significant increases in protein detection through changes in the maximum localized surface plasmon resonance wavelength exhibited by the AuNS in non-competitive environments. Proof-of-concept testing of the developed hydrogel coated AuNS was performed in commercially available pooled human tears for label-free detection of two high isoelectric point proteins, lysozyme and lactoferrin, containing solvent accessible lysines. In a competitive diluted tear sample, protein binding to hydrogel coated AuNS was primarily governed by electrostatic interactions, similar to results obtained at the nanogel level. While our results revealed significant differences in protein binding when both ionic and covalent monomers were incorporated into the hydrogel coated AuNS in non-competitive protein environments, this result does not predict the outcome of the biosensor performance in a competitive biological environment. Instead, the results obtained demonstrate the cross-reactivity of the synthesized nanogels and hydrogel coated AuNS, an aspect which could be useful for future applications