SUMMARY
Due to the intensifying threat of the Zika virus to human health, the development of point-of-care methods to
screen for the Zika virus represent an area of urgent need. Current approaches for the clinical testing of the Zika
virus rely on conventional biochemical, genetic, and immunological laboratory assays that require complex
instrumentation and expensive reagents (e.g., antibodies and fluorescent labels). While these methods are
effective, the requirements of these methods, which also often involve multiple steps, making detection
cumbersome, are prohibitive for point-of-care testing. The overall aim of this proposal is to develop a label-free
screening platform for the detection of Zika by integrating stimuli-responsive and optically diffracting materials.
Specifically, in this approach, we will investigate an optical biosensing platform comprised of a hydrogel material
that swells in the presence a protease that is specific to Zika and is impregnated with a crystalline colloidal array
(CCA). To develop such materials, polystyrene particles that self-assemble into a CCA will be co-polymerized
into a polyacrylamide film that contains peptide crosslinks that may be cleaved by the protease (NS2B-NS3).
Cleavage of the crosslinks by the protease is expected to physically alter the crosslinking density of the hydrogel
network, thereby eliciting an increase in the volume of the hydrogel. As a result of swelling, the lattice spacing of
the CCA will be altered, thereby triggering a change in optical diffraction of the hydrogel that can be detected at
visible wavelengths without exogenous labels. The central hypothesis of this work is that the change in lattice
spacing of the CCA-containing hydrogel, and thus the shift in wavelength of peak diffraction, will correlate with
the proteolytic activity of NS2B-NS3. To directly test this hypothesis, the specific aims of this proposal are to: 1)
characterize the optical response of CCA-containing hydrogels with peptide crosslinks in the presence of NS2B-
NS3 (Aim 1) and 2) investigate the selectivity of the CCA-based sensing platform to NS2B-NS3 from the Zika
virus (Aim 2). The sensitivity, including detection range and response time, of the films will be determined by
treating the films, which will be prepared in multi-well plates that allow for high-throughput parallel detection, with
different amounts of NS2B-NS3 and for different times. Additionally, a theoretical model of the swelling and
optical responses will be developed, which will permit rational modification of the hydrogel properties to improve
the overall sensitivity to NS2B-NS3. In aim 2, the selectivity of the screening platform towards the Zika
homologue of NS2B-NS3 will be characterized by quantitatively comparing the optical response elicited by
NS2B-NS3 from Zika with that from genetically related viruses. Furthermore, we will explore avenues to increase
the selectivity of the sensing approach for Zika NS2B-NS3 by altering the peptide crosslinking sequence. In
addition to point-of-care testing, the advantages of this label-free platform present new opportunities for
screening for novel inhibitors of NS2B-NS3, which have immense therapeutic potential.