Enzymes have many important roles in biology and industry, and proteases are one of the most important classes of enzymes. Semiconductor quantum dots (QDs) are attractive materials for developing protease activity probes because of their advantageous physical and optical properties; however, interactions between a protease and a QD conjugated with its substrate can affect the turnover of that substrate. Here, we study the turnover of multivalent QD-peptide substrate conjugates as a function of multiple parameters: (i) the ligand coating on the QD, including dihydrolipoic acid (DHLA), glutathione (GSH), DHLA-poly(ethylene glycol) (DHLA-PEG), and DHLA-zwitterionic sulfobetaine (DHLA-SB); (ii) the identity of the protease, including trypsin, thrombin, and plasmin; and (iii) the number of substrate and nonsubstrate biomacromolecules conjugated per QD. We show that limiting protease adsorption on QDs is critical for optimizing the turnover of conjugated peptide substrates. Protease adsorption is inhibitory, and very strong adsorption leads to an apparent "scooting" mode of activity with limited turnover. In contrast, with weaker adsorption, enhancements in the turnover rate likely result from a "hopping" mode of activity. The putative hopping mode is thought to feature processive turnover of all substrates in multivalent conjugates with a rate-limiting step of diffusion between individual conjugates, and the magnitude of such enhancements increases with decreases in adsorption. Although it was possible to passivate DHLA- and GSH-coated QDs with high densities of conjugated biomacromolecules, the most effective strategy for reducing adsorption was the substitution of these ligands. Whereas passivation incrementally increased turnover, DHLA-PEG and DHLA-SB ligands converted the mode of turnover with plasmin from scooting to hopping and the DHLA-SB enhanced the turnover rates with thrombin and trypsin by approximately an order of magnitude relative to GSH ligands. The new insights from the broad scope of this study provide an important framework for designing optimized QD conjugates as probes and sensors for enzyme activity.
Enzymes are important biomarkers for molecular diagnostics and targets for the action of drugs. In turn, inorganic nanoparticles (NPs) are of interest as materials for biological assays, biosensors, cellular and in vivo imaging probes, and vectors for drug delivery and theranostics. So how does an enzyme interact with a NP, and what are the outcomes of multivalent conjugation of its substrate to a NP? This invited feature article addresses the current state of the art in answering this question. Using gold nanoparticles (Au NPs) and semiconductor quantum dots (QDs) as illustrative materials, we discuss aspects of enzyme structure–function and the properties of NP interfaces and surface chemistry that determine enzyme–NP interactions. These aspects render the substrate-on-NP configurations far more complex and heterogeneous than the conventional turnover of discrete substrate molecules in bulk solution. Special attention is also given to the limitations of a standard kinetic analysis of the enzymatic turnover of these configurations, the need for a well-defined model of turnover, and whether a “hopping” model can account for behaviors such as the apparent acceleration of enzyme activity. A detailed and predictive understanding of how enzymes turn over multivalent NP-substrate conjugates will require a convergence of many concepts and tools from biochemistry, materials, and interface science. In turn, this understanding will help to enable rational, optimized, and value-added designs of NP bioconjugates for biomedical and clinical applications.
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.
Supramolecular ‘gluing’ of quantum dots is demonstrated with cucurbituril and we present the opportunity to create molecular host–guest sensing schemes.
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