Bait and Cleave: Exosite-Binding Peptides on Quantum Dots Selectively Accelerate Protease Activity for Sensing with Enhanced Sensitivity.

The advantageous optical properties of quantum dots (QDs) motivate their use in a wide variety of applications related to imaging and bioanalysis, including the detection of proteases and their activity. Recent studies have shown that surface chemistry on QDs is able to modulate protease activity, but only nonspecifically. Here, we present a strategy to selectively accelerate the activity of a particular target protease by as much as two orders of magnitude. Exosite-binding "bait" peptides were derived from proteins that span a range of biological roles─substrate, receptor, and inhibitor─and were used to increase the affinity of the QD-peptide conjugates for either thrombin or factor Xa, resulting in increased rates of proteolysis for coconjugated substrates. Unlike effects from QD surface chemistry, the acceleration was specific to the target protease with negligible acceleration of other proteases. Benefits of this "bait and cleave" sensing approach included detection limits that improved by more than an order of magnitude, reenabled detection of target protease against an overwhelming background of nontarget proteolysis, and mitigation of the action of inhibitors. The cumulative results point to a generalizable strategy, where the mechanism of acceleration, considerations for the design of bait peptides and conjugates, and routes to expanding the scope of this approach are discussed. Overall, this research represents a major step forward in the rational design of nanoparticle-based enzyme sensors that enhance sensitivity and selectivity.

Assessing the Steric Impact of Surface Ligands on the Proteolytic Turnover of Quantum Dot-Peptide Conjugates.

Proteases are important biomarkers and targets for the diagnosis and treatment of disease. The advantageous properties of semiconductor quantum dots (QDs) have made these nanoparticles useful as probes for protease activity; however, the effects of QD surface chemistry on protease activity are not yet fully understood. Here, we present a systematic study of the impact of sterics on the proteolysis of QD-peptide conjugates. The study utilized eight proteases (chymotrypsin, trypsin, endoproteinase Lys C, papain, endoproteinase Arg C, thrombin, factor Xa, and plasmin) and 41 distinct surface chemistries. The latter included three molecular weights of each of three macromolecular ligands derived from dextran and polyethylene glycol, as well as anionic and zwitterionic small-molecule ligands, and an array of mixed coatings of macromolecular and small-molecule ligands. These surface chemistries spanned a diversity of thicknesses, densities, and packing organization, as characterized by gel electrophoresis, capillary electrophoresis, dynamic light scattering, and infrared spectroscopy. The macromolecular ligands decreased the adsorption of proteases on the QDs and decelerated proteolysis of the QD-peptide conjugates via steric hindrance. The properties of the QD surface chemistry, rather than the protease properties, were the main factor in determining the magnitude of deceleration. The broad scope of this study provides insights into the many ways in which QD surface chemistry affects protease activity, and will inform the development of optimized nanoparticle-peptide conjugates for sensing of protease activity and resistance to unwanted proteolysis.

Developing FRET Networks for Sensing.

Förster resonance energy transfer (FRET) is a widely used fluorescence-based sensing mechanism. To date, most implementations of FRET sensors have relied on a discrete donor-acceptor pair for detection of each analytical target. FRET networks are an emerging concept in which target recognition perturbs a set of interconnected FRET pathways between multiple emitters. Here, we review the energy transfer topologies and scaffold materials for FRET networks, propose a general nomenclature, and qualitatively summarize the dynamics of the competitive, sequential, homoFRET, and heteroFRET pathways that constitute FRET networks. Implementations of FRET networks for sensing are also described, including concentric FRET probes, other single-vector multiplexing, and logic gates and switches. Unresolved questions and future research directions for current systems are discussed, as are potential but currently unexplored applications of FRET networks in sensing.

Preparation and Characterization of Quantum Dot-Peptide Conjugates Based on Polyhistidine Tags.

Quantum dots (QDs) offer bright and robust photoluminescence among several other advantages in comparison to fluorescent dyes. In order to leverage the advantageous properties of QDs for applications in bioanalysis and imaging, simple and reliable methods for bioconjugation are required. One such method for conjugating peptides to QDs is the use of polyhistidine tags, which spontaneously bind to the surface of QDs. We describe protocols for assembling polyhistidine-tagged peptides to QDs and for characterizing the resultant QD-peptide conjugates. The latter include both electrophoretic and FRET-based protocols for confirming successful peptide assembly, estimating the maximum peptide loading capacity, and measuring the assembly kinetics. Sensors for protease activity and intracellular delivery are briefly noted as prospective applications of QD-peptide conjugates.

Photoluminescent Nanoparticles for Chemical and Biological Analysis and Imaging.

Research related to the development and application of luminescent nanoparticles (LNPs) for chemical and biological analysis and imaging is flourishing. Novel materials and new applications continue to be reported after two decades of research. This review provides a comprehensive and heuristic overview of this field. It is targeted to both newcomers and experts who are interested in a critical assessment of LNP materials, their properties, strengths and weaknesses, and prospective applications. Numerous LNP materials are cataloged by fundamental descriptions of their chemical identities and physical morphology, quantitative photoluminescence (PL) properties, PL mechanisms, and surface chemistry. These materials include various semiconductor quantum dots, carbon nanotubes, graphene derivatives, carbon dots, nanodiamonds, luminescent metal nanoclusters, lanthanide-doped upconversion nanoparticles and downshifting nanoparticles, triplet-triplet annihilation nanoparticles, persistent-luminescence nanoparticles, conjugated polymer nanoparticles and semiconducting polymer dots, multi-nanoparticle assemblies, and doped and labeled nanoparticles, including but not limited to those based on polymers and silica. As an exercise in the critical assessment of LNP properties, these materials are ranked by several application-related functional criteria. Additional sections highlight recent examples of advances in chemical and biological analysis, point-of-care diagnostics, and cellular, tissue, and in vivo imaging and theranostics. These examples are drawn from the recent literature and organized by both LNP material and the particular properties that are leveraged to an advantage. Finally, a perspective on what comes next for the field is offered.

Dextran-Functionalized Semiconductor Quantum Dot Bioconjugates for Bioanalysis and Imaging.

The prerequisites for maximizing the advantageous optical properties of colloidal semiconductor quantum dots (QDs) in biological applications are effective surface functionalization and bioconjugation strategies. Functionalization with dextran has been highly successful with some nanoparticle materials, but has had very limited application with QDs. Here, we report the preparation, characterization, and proof-of-concept applications of dextran-functionalized QDs. Multiple approaches to dextran ligands were evaluated, including performance with respect to colloidal stability across a range of pH, nonspecific binding with proteins and cells, and microinjection into cells and viability assays. Multiple bioconjugation strategies were demonstrated and applied, including covalent coupling to develop a simple pH sensor, binding of polyhistidine-tagged peptides to the QD for energy transfer-based proteolytic activity assays, and binding with tetrameric antibody complexes (TACs) to enable a sandwich immunoassay and cell immunolabeling and imaging. Our results show that dextran ligands are highly promising for the functionalization of QDs, and that the design of the ligands is tailorable to help optimally meet the requirements of applications.

Accelerated Structural Prediction of Flexible Protein-Ligand Complexes: The SLICE Method.

Using existing and academically available software, we present a new method for the structural prediction of binding events containing flexible protein targets. SLICE (Selective Ligand-Induced Conformational Ensemble) combines opportunistic stochastic jumps of ligand position with standard molecular dynamics to model the induced-fit binding of ligands starting with unbound host coordinates. To induce the structural adaptations of the complex at the binding site, conformational jumps in ligand position are selected in SLICE from structures generated by a docking software. Multiple binding trajectories from the docking set are followed using molecular dynamics for a set time to relax the host structure and generate new host poses. A new configurational jump is made on the set of newly generated host poses. The process is then repeated. The method was implemented with AutoDock Vina as the docking method, Vina scores as the selection criterion, and Amber code for molecular dynamics and applied to several test systems. A system consisting of Chromobox protein homologue 8 (CBX8) and its small peptide ligand, H3K9Me3, for which the final (bound) configuration is known, is used for verifying SLICE in the present setup. The setup was also applied to several nonpeptide molecules on known difficult flexible targets exhibiting a large disparity between apo and holo host states. The SLICE simulations provide a promising approach to generate induced-fit configurations compared to existing long (microsecond) classical and accelerated dynamics approaches in all the test systems considered here. However, further optimization of SLICE parameters is required for replicating crystal structure coordinates for some systems. We discuss in the following pages the various SLICE parameters and how they can be optimized for the system at hand.

Concentric FRET: a review of the emerging concept, theory, and applications.

Concentric Förster resonance energy transfer (cFRET) is an emerging concept for single-vector multiplexed bioanalysis and imaging. It features a network of competitive and sequential energy transfer pathways, which, to date, has been assembled with a central semiconductor quantum dot (QD) and biomolecular linkers to multiple copies of multiple types of concentrically-arranged fluorescent dyes. In this review, we provide a first-hand account of the concept and development of cFRET, starting from its place in the broader context of FRET probes and assemblies. Topics of discussion include materials for cFRET, with a focus on the enabling properties of QDs and the ideal properties of nominal acceptor dyes; characterization and analysis of cFRET configurations via photoluminescence intensity, emission ratio, lifetime, and photobleaching measurements; semi-empirical modeling to determine the rates and efficiencies of competitive and sequential FRET pathways from overall quenching efficiencies; and archetypical examples of cFRET configurations and their application in bioanalysis and imaging. Most of the latter examples demonstrate multiplexed detection of protease activity or nucleic acid targets. Examples of atypical and cFRET-like configurations are also discussed, including those that utilize time-gated FRET relays and charge-transfer quenching. We conclude with a perspective on challenges and directions for future research with cFRET. Although still emerging as a method, many exciting opportunities in bioanalysis, imaging, and beyond are envisioned for cFRET.

Cationic 2,2'-bipyridine complexes of germanium(ii) and tin(ii).

We present the first systematic study of 2,2'-bipyridine complexes of E(ii) cationic acceptors (E = Ge, Sn). The complexes were comprehensively characterized by spectroscopic and crystallographic methods to yield complexes of ECl1+ and E2+. Computational DFT methods were also employed to survey the bonding in the cations, along with an examination of their molecular orbitals (MOs).

Interplay between adsorbed peptide structure, trapped water, and surface hydrophobicity.

Atomistic molecular dynamics simulations were used to study the influence of interfacial water on the orientation and conformation of a facewise amphipathic α-helical peptide adsorbed to hydrophilic and hydrophobic substrates. Water behavior beneath the peptide adsorbed to a hydrophilic surface was observed to vary with the height of the peptide above the surface. In general, the orientation of water close to the peptide (with the oxygen atom pointing up toward the peptide) was complementary to that observed near the hydrophilic surface in the absence of peptide. That is, no change in orientation of water trapped between the peptide and a hydrophilic surface is required as the peptide approaches the surface. The adsorption of the peptide to the hydrophilic surface was observed to be mediated by a layer of ordered water. Water was found to be largely excluded on adsorption to the hydrophobic surface. However, the small amount of water present was observed to be highly ordered. At the closest point of contact to the hydrophobic surface, the peptide was observed to make direct contact. These findings shed light on the fundamental driving forces of peptide adsorption to hydrophobic and hydrophilic surfaces in aqueous environments.