Mimicking Cell Surface Enhancement of Protease Activity on the Surface of a Quantum Dot Nanoparticle

Nature has evolved mechanisms to increase the specificity of enzymes and enhance their activity to support particular biological functions. Mimicking these mechanisms with artificial systems, such as nanoparticles, would greatly benefit bioanalysis. Here, we have taken inspiration from platelet cells to create a fluorescent nanoparticle probe with enhanced sensitivity toward its protease target. Platelets use protease-activated receptor 1 (PAR1) to enhance thrombin activity that initiates their aggregation in the early stages of blood clotting. We therefore coconjugated multiple copies each of a peptide substrate and a fragment of PAR1 to a semiconductor quantum dot (QD) to mimic this behavior. Thrombin activity toward conjugates with and without the PAR1 fragment were tracked via Förster resonance energy transfer (FRET). The co-conjugated PAR1 increased thrombin-catalyzed hydrolysis of the substrate by several-fold up to multiple orders of magnitude, albeit dependent on the surface chemistry of the QD. The enhancement effect arose from a combination of selective binding between the PAR1 fragment and thrombin, and from colocalization of the substrate and the PAR1 fragment at the QD interface. Substrate-receptor co-conjugation is thus a promising strategy for the rational design of nanoparticle bioconjugates with optimized sensitivity and specificity for biosensing and imaging.