Nanosensors employing quantum dots (QDs) and functional moieties such as enzymes offer tremendous promise for disease surveillance/diagnostics and chemical/biological threat activity as their small size permits cell penetration and their inherent photochemical properties (i.e., size tunable fluorescence, ease in attaching functional moieties, resistance to photobleaching, etc) are well-suited for rapid, optical measurement. The effectiveness of biorecognition agents (e.g., enzymes) immobilized on QDs are not completely understood, hindering development of in vitro, in vivo, or in situ chemical/biological sensors and remediation materials. We analyze enzyme effectiveness when attached to QDs using one model system and one illustrating an important biological threat application�the toxicity neutralization of paraoxon, a simulant nerve agent. Two sizes of QDs, 525 or 625 nm, were appended with DHLA ligands to improve aqueous stability and prevent aggregation. Rapid self-assembly of enzymes to the QD surface via spontaneous metal coordination of the oligiohistidine tag onto the Zn2+-rich QD surface, required only the mixing of molar ratios of two reactants. We first use alkaline phosphatase, a hydrolase enzyme that converts 4-methylumbelliferyl phosphate to fluorescent methylumbelliferone, to illustrate improved enzyme kinetics on QDs (e.g., ~20 to 30% increase in Vmax). Next, we immobilize phosphotriesterase (PTE) onto distinctly sized QDs with various enzyme-to-QD ratios. PTE catalyzes the detoxification of organophosphate pesticides (e.g, paraoxon, an analog of sarin) to p-nitrophenol. The optimal ratio of PTE to 525 nm and 625 nm QD's was determined to be 6 and 8, respectively. At these ratios, we saw a ~50 to 60% increase in Vmax for PTE and other kinetic parameters. The enhanced enzyme performance in both cases is most likely due to increased enzyme-substrate interactions from enzyme orientation, enzyme density, and substrate diffusion on or near the QD. Development of these nansosensors as optical-based biosensors (e.g., within compact microfluidic devices) may greatly improve the sensitivity of conventional biological/chemical detection schemes.

Enhanced kinetics of enzymatic nanosensors

248th National Meeting (2014)

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