Smart polymer nanoscale particles for rapid molecular diagnostics

Nanoparticles functionalized with proteins and DNA have shown tremendous potential in increasing the sensitivity of currently available diagnostic tests and in the development of new assay formats. In this dissertation, we have developed a set of block conjugates composed of a temperature-sensitive smart polymer, poly(N-isopropylacrylamide) (PNIPAAm), as one block and a hydrophilic moiety such as protein or DNA as the other block. These PNIPAAm-based block conjugates were shown to reversibly form stable nano- and mesoscale particles in a temperature-controlled fashion. We have demonstrated the use of these reversible nanoparticles in improving the sensitivity of diagnostic assays and in enabling control of protein activity in solution.; A hydrophilic moiety, poly(acrylic acid) (PAA), was attached to PNIPAAm by synthesizing a block copolymer of PNIPAAm and PAA. It was shown that the PAA block prevented continuous aggregation of the hydrophobic PNIPAAm and directed formation of uniform micellar particles. Furthermore, the hydrophilic PAA block was shown to prevent aggregation of PAA-PNIPAAm-streptavidin (SA) conjugates above the lower critical solution temperature (LCST) at pH 7. This allows us to potentially control enzyme activity reversibly by expansion and collapse of the smart polymer (attached to the enzyme) in solution, without the aggregation associated with phase separation.; Biomolecules such as proteins and DNA were also used to control the aggregation properties of PNIPAAm. PNIPAAm-SA and PNIPAAm-DNA block conjugates were found to rapidly form mesoscale particles above the LCST of the PNIPAAm. The particles were stable for days at high temperature, yet could be dissolved within minutes on removal of the temperature stimulus. The particle size could be controlled by manipulating formulation parameters such as heating rate, concentration and polymer molecular weight.; To demonstrate the use of PNIPAAm-protein particles in affinity capture, a PNIPAAm-IgG conjugate was allowed to bind to Chlamydia elementary bodies (EBs) in solution and then induced to form particles by application of a stimulus. The particles containing the EBs were then easily captured for detection by centrifugation. This preconcentration method allows the detection of the EBs at low concentrations, enabling the development of sensitive diagnostic tests for Chlamydia.