Passivating quantum dots against histag-displaying enzymes using blocking peptides: salient considerations for self-assembling quantum dot biosensors

Abstract

While tagging proteins and enzymes with metal affinity moieties is a common strategy to facilitate purification, such modifications can be detrimental towards the development of biological assays using nanomaterial reporter agents, especially in cases where such modifications are not indicated by vendors. Particularly, enzymes expressed with polyhistidine tags (histags) may unintentionally and strongly bind to nanoparticles displaying transition metal ions, such as ZnS-coated quantum dots (QDs). Here, we developed a strategy to passivate ZnS-coated QDs using short, histag-containing blocking peptides (BPs), which act to saturate the surface following the addition of functional peptide or nucleic acid substrates. We demonstrated proof of concept for this strategy with the CRISPR-associated LwCas13a enzyme in the presence of QD-based molecular beacons (QD-MBs) for CRISPR-based diagnostics. The BP loading capacity and efficiency of QD surface passivation against dye-labeled peptides and a secondary protein were then characterized for BPs of varied lengths and charges using fluorescence and biophysical approaches. We further found that blocking peptides improved the colloidal stability of QDs in certain environments while having no observable effect on the sterics. These findings suggest that blocking peptides could be a beneficial addition to QDs in a variety of experimental conditions, and they are a crucial variable to consider when designing QD-based biosensors. Importantly the addition of blocking peptides does not require any re-design or modification of the original system and can be tested ad hoc when QD biosensors are not performing as intended.