Enhancing enzymatic efficiency by attachment to semiconductor nanoparticles for biosensor applications

Abstract

Nanosensors employing quantum dots (QDs) with appended biofunctional moieties offer tremendous promise for disease surveillance/diagnostics and chemical/biological threat activity. Their small size permits cell penetration and their inherent photochemical properties are well-suited for rapid, optical measurement. The effectiveness of enzymes immobilized on QDs, however, are not completely understood, hindering development of chemical/biological sensors and remediation materials. Here, we analyze enzyme effectiveness for the neutralization of a simulant nerve agent when attached to two distinctly-sized QDs. Two sizes of QDs, 525 or 625 nm, were appended with DHLA ligands to improve aqueous stability and prevent aggregation. Various molar ratios of de novo phosphotriesterase trimer (PTE 3 ) were rapidly self-assembled via spontaneous metal coordination of the PTE oligohistidine tag onto the Zn 2+ -rich QD surface. PTE catalyzes the detoxification of organophosphate pesticides (e.g, paraoxon, an analog of sarin) to p-nitrophenol whose absorbance can be measured at 405 nm. The optimal ratio of PTE 3 to 525 nm and 625 nm QD's was determined to be 12 and 24, respectively. The enhanced enzyme performance in both cases is most likely due to increased enzyme-substrate interactions from improvements in 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 enzymatic activity from phosphotriesterase trimer gold nanoparticle bioconjugates for pesticide detection

“…14 Researchers postulate that the immobilization of enzymes in lower density on NPs limits enzyme-to-enzyme neighbor interactions and hence increases the likelihood of proper enzyme conformation and subsequently higher enzyme activity or more specifically increases in the rate of enzyme-substrate to product conversion (k 2 ). 20,24,44 However, our results also demonstrate that PTE 3 activity is higher on larger sized NPs (i.e., 20 nm vs. 10 nm and 5 nm) which we previously postulated may arise in part to the high localized density of PTE 3 on the larger NPs and overall increases in avidity of the enzyme-NP bioconjugate. 20 We do note a subtle but rather interesting discrepancy between the two experimental formats utilized here.…”

“…Bioconjugate activity was assayed and compared to free PTE 3 using a similar method as previously described. 44 It should be noted that the AuNP-PTE 3 ratios chosen were far below surface saturation in most cases to ensure that all enzymes were attached to the AuNPs. Conjugates and enzyme only controls were diluted into a final concentration series of paraoxon ranging from 20 to 5000 µM within 384-well Corning flat bottom, non-binding microtiter plates.…”