Investigation of micro- and nanoscale hydrogels as protein receptors for use in diagnostic biosensors

Due to the high cost and environmental instability of antibodies, there is precedent for developing synthetic molecular recognition agents for use in diagnostic sensors. Molecular imprinting was first investigated as a method for improving selectivity of crosslinked polymer hydrogels. However, the lysozyme-imprinted polymers exhibited high cross-reactivity and did not afford the polymer with improved selectivity. Instead, the observed cross-reactivity prompted the investigation of charge-containing, non-imprinted polymers as differential protein receptors. To this end, a set nanogels were synthesized by copolymerizing N-isopropylacrylamide with methacrylic acid, followed by a post-synthesis modification. Specifically, a carbodiimide coupling scheme was used to introduce sulfate, guanidinium, secondary amine, or primary amine groups. As expected, modification of the ionizable groups in the network changed the physicochemical and protein binding properties of the nanogels. For high affinity protein-polymer interactions, turbidity of the nanogel solution increased, while for low affinity interactions minimal change in turbidity was observed. Thus, relative turbidity was used as input for multivariate analysis. Turbidimetric assays were performed in two buffers of different pH (i.e., 7.4 and 5.5), but comparable ionic strength, in order to improve differentiation. Using both buffers, it was possible to achieve 100% classification accuracy of eleven model protein biomarkers with as few as two of the nanogel receptors. Subsequently, these polymeric receptors were synthesized on the surface of silica-gold nanoshells to develop a diagnostic biosensor based on localized surface plasmon resonance (LSPR). In particular, the sensitivity of LSPR to changes in local refractive index was exploited to enable detection of three biomarkers (lysozyme, lactoferrin, and IgG) of Sjögren’s syndrome, an autoimmune disease that primarily affects the exocrine system. Overall, the polymer-gold nanomaterial composites developed in this dissertation provide a promising platform for developing affordable and environmentally robust diagnostic biosensors.