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.

Semiconducting Polymer Dots Directly Stabilized with Serum Albumin: Preparation, Characterization, and Cellular Immunolabeling.

Semiconducting polymer dots (Pdots) are brightly fluorescent nanoparticles of growing interest for bioanalysis and imaging. A recurring challenge with these materials is obtaining robust physical and colloidal stability and low nonspecific binding. Here, we prepared and characterized Pdots with bovine serum albumin (BSA) as the stabilizing agent (BSA-Pdots) instead of a more conventionally used amphiphilic polymer, both without and with cross-linking of the protein using glutaraldehyde (BSA(GA)-Pdots) or disuccinimidyl glutarate. Characterization included fluorescence properties; colloidal stability as a function of pH, ionic strength, and solvent perturbation; shape retention and hardness; and nonspecific binding with common assay substrates, fixed cells, and live cells. These properties were contrasted with the same properties for amphiphilic polymer-stabilized Pdots and silica-coated Pdots. On balance, the BSA-stabilized Pdots were similar or more favorable in their properties, with BSA(GA)-Pdots being especially advantageous. Bioconjugation of the BSA-stabilized Pdots was possible using amine-reactive active-ester chemistry, including biotinylation and bioorthogonal functionalization for immunoconjugation via tetrazine-strained-alkene click chemistry. These approaches were used for selective fluorescent labeling of cells based on ligand-receptor and antibody-antigen binding, respectively. Overall, direct BSA stabilization is a very promising strategy for preparing Pdots with improved physical and colloidal stability, reduced nonspecific interactions, and utility for in vitro diagnostics and other bioanalyses and imaging.

Supra-Quantum Dot Assemblies to Maximize Color-Based Multiplexed Fluorescence Detection with a Smartphone Camera.

Photoluminescence (PL) imaging and bioanalysis with smartphone-based devices are of growing interest for point-of-care/point-of-need diagnostics. Strategies for maximizing sensitivity have been explored in this context, but color multiplexing has been very limited, with its maximum level unexplored. Here, we evaluated color multiplexing with smartphone-based PL imaging by using supra-nanoparticle assemblies of quantum dots (supra-QDs). These materials were prepared as composite colors that were tailored to the red-green-blue (RGB) color space of smartphone cameras by coassembling different ratios of R-, G-, and B-emitting QDs on a silica nanoparticle scaffold. The supra-QDs were characterized and used to label cell-sized objects that were measured under flow with a smartphone-based device. Each color followed an approximately linear trajectory in the RGB space, and training of support vector machine models enabled color classification with overall accuracies ≥87% for 10-color multiplexing and better accuracies for fewer colors. Most misclassification occurred at low signal levels, such that establishing a nonclassifiable zone near the origin of RGB color space improved the overall 10-color classification accuracy to ≥94%. Similar improvements in accuracy with greater retention of data were possible with a probabilistic rather than a radial threshold. Simulations that were parameterized by experimental data suggested that ≥14-color multiplexing with accuracies ≥90% should be possible with an optimized supra-QD color set. This study is an important foundation for advancing RGB color-based multiplexing for imaging and analyses with smartphone cameras and related charge-coupled device and CMOS color image sensor technologies.

Tetrameric Antibody Complexes and Affinity Tag Peptides for the Selective Immobilization and Imaging of Single Quantum Dots.

Colloidal semiconductor quantum dots (QDs) are of widespread interest as fluorescent labels for bioanalysis and imaging applications. Single-particle measurements have proven to be a very powerful tool for better understanding the fundamental properties and behaviors of QDs and their bioconjugates; however, a recurring challenge is the immobilization of QDs in a solution-like environment that minimizes interactions with a bulk surface. Immobilization strategies for QD-peptide conjugates are particularly underdeveloped within this context. Here, we present a novel strategy for the selective immobilization of single QD-peptide conjugates using a combination of tetrameric antibody complexes (TACs) and affinity tag peptides. A glass substrate is modified with an adsorbed layer of concanavalin A (ConA) that binds a subsequent layer of dextran that minimizes nonspecific binding. A TAC with anti-dextran and anti-affinity tag antibodies binds to the dextran-coated glass surface and to the affinity tag sequence of QD-peptide conjugates. The result is spontaneous and sequence-selective immobilization of single QDs without any chemical activation or cross-linking. Controlled immobilization of multiple colors of QDs is possible using multiple affinity tag sequences. Experiments confirmed that this approach positions the QD away from the bulk surface. The method supports real-time imaging of binding and dissociation, measurements of Förster resonance energy transfer (FRET), tracking of dye photobleaching, and detection of proteolytic activity. We anticipate that this immobilization strategy will be useful for studies of QD-associated photophysics, biomolecular interactions and processes, and digital assays.

Dextran-Functionalized Super-nanoparticle Assemblies of Quantum Dots for Enhanced Cellular Immunolabeling and Imaging.

Colloidal semiconductor quantum dots (QDs) are a popular material for applications in bioanalysis and imaging. Although individual QDs are bright, some applications benefit from the use of even brighter materials. One approach to achieve higher brightness is to form super-nanoparticle (super-NP) assemblies of many QDs. Here, we present the preparation, characterization, and utility of dextran-functionalized super-NP assemblies of QDs. Amphiphilic dextran was synthesized and used to encapsulate many hydrophobic QDs via a simple emulsion-based method. The resulting super-NP assemblies or "super-QDs" had hydrodynamic diameters of ca. 90-160 nm, were characterized at the ensemble and single-particle levels, had orders-of-magnitude superior brightness compared to individual QDs, and were non-blinking. Additionally, binary mixtures of red, green, and blue (RGB) colors of QDs were used to prepare super-QDs, including colors difficult to obtain from individual QDs (e.g., magenta). Tetrameric antibody complexes (TACs) enabled simple antibody conjugation for selective cellular immunolabeling and imaging with both an epifluorescence microscope and a smartphone-based platform. The technical limitations of the latter platform were overcome by the increased per-particle brightness of the super-QDs, and the super-QDs outperformed individual QDs in both cases. Overall, the super-QDs are a very promising material for bioanalysis and imaging applications where brightness is paramount.

Prototype Smartphone-Based Device for Flow Cytometry with Immunolabeling via Supra-nanoparticle Assemblies of Quantum Dots.

Methods for the detection, enumeration, and typing of cells are important in many areas of research and healthcare. In this context, flow cytometers are a widely used research and clinical tool but are also an example of a large and expensive instrument that is limited to specialized laboratories. Smartphones have been shown to have excellent potential to serve as portable and lower-cost platforms for analyses that would normally be done in a laboratory. Here, we developed a prototype smartphone-based flow cytometer (FC). This compact 3D-printed device incorporated a laser diode and a microfluidic flow cell and used the built-in camera of a smartphone to track immunofluorescently labeled cells in suspension and measure their color. This capability was enabled by high-brightness supra-nanoparticle assemblies of colloidal semiconductor quantum dots (SiO2@QDs) as well as a support vector machine (SVM) classification algorithm. The smartphone-based FC device detected and enumerated target cells against a background of other cells, simultaneously and selectively counted two different cell types in a mixture, and used multiple colors of SiO2@QD-antibody conjugates to screen for and identify a particular cell type. The potential limits of multicolor detection are discussed alongside ideas for further development. Our results suggest that innovations in materials and engineering should enable eventual smartphone-based FC assays for clinical applications.

Polymer Dots with Enhanced Photostability, Quantum Yield, and Two-Photon Cross-Section using Structurally Constrained Deep-Blue Fluorophores.

Semiconducting polymer dots (Pdots) have emerged as versatile probes for bioanalysis and imaging at the single-particle level. Despite their utility in multiplexed analysis, deep blue Pdots remain rare due to their need for high-energy excitation and sensitivity to photobleaching. Here, we describe the design of deep blue fluorophores using structural constraints to improve resistance to photobleaching, two-photon absorption cross sections, and fluorescence quantum yields using the hexamethylazatriangulene motif. Scanning tunneling microscopy was used to characterize the electronic structure of these chromophores on the atomic scale as well as their intrinsic stability. The most promising fluorophore was functionalized with a polymerizable acrylate handle and used to give deep-blue fluorescent acrylic polymers with Mn > 18 kDa and D < 1.2. Nanoprecipitation with amphiphilic polystyrene-graft-(carboxylate-terminated poly(ethylene glycol)) gave water-soluble Pdots with blue fluorescence, quantum yields of 0.81, and molar absorption coefficients of (4 ± 2) × 108 M-1 cm-1. This high brightness facilitated single-particle visualization with dramatically improved signal-to-noise ratio and photobleaching resistance versus an unencapsulated dye. The Pdots were then conjugated with antibodies for immunolabeling of SK-BR3 human breast cancer cells, which were imaged using deep blue fluorescence in both one- and two-photon excitation modes.

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.

Fully Self-Assembled Silica Nanoparticle-Semiconductor Quantum Dot Supra-Nanoparticles and Immunoconjugates for Enhanced Cellular Imaging by Microscopy and Smartphone Camera.

There is a growing need for brighter luminescent materials to improve the detection and imaging of biomarkers. Relevant contexts include low-abundance biomarkers and technology-limited applications, where an example of the latter is the emerging use of smartphones and other nonoptimal but low-cost and portable devices for point-of-care diagnostics. One approach to achieving brighter luminescent materials is incorporating multiple copies of a luminescent material into a larger supra-nanoparticle (supra-NP) assembly. Here, we present a facile method for the preparation and immunoconjugation of supra-NP assemblies (SiO2@QDs) that comprised many quantum dots (QDs) around a central silica nanoparticle (SiO2 NP). The assembly was entirely driven by spontaneous affinity interactions between the constituent materials, which included imidazoline-functionalized silica nanoparticles, ligand-coated QDs, imidazole-functionalized dextran, and tetrameric antibody complexes (TACs). The physical and optical properties of the SiO2@QDs were characterized at both the ensemble and single-particle levels. Notably, the optical properties of the QDs were preserved upon assembly into supra-NPs, and single SiO2@QDs were approximately an order of magnitude brighter than single QDs and nonblinking. In proof-of-concept applications, including selective immunolabeling of breast cancer cells, the SiO2@QDs provided higher sensitivity and superior signal-to-background ratios whether using research-grade fluorescence microscopy or smartphone-based imaging. Overall, the SiO2@QDs are promising materials for enhanced bioanalysis and imaging.

Temperature Mapping in Hydrogel Matrices Using Unmodified Digital Camera.

We report a simple, generally applicable, and noninvasive fluorescent method for mapping thermal fluctuations in hydrogel matrices using an unmodified commercially available digital single-lens reflex camera (DSLR). The nanothermometer is based on the complexation of short conjugated polyelectrolytes, poly(phenylene ethynylene) carboxylate, with an amphiphilic polymer, polyvinylpyrrolidone, which is in turn trapped within the porous network of a gel matrix. Changes in the temperature lead to a fluorescent ratiometric response with a maximum relative sensitivity of 2.0% and 1.9% at 45.0 °C for 0.5% agarose and agar, respectively. The response was reversible with no observed hysteresis when samples were cycled between 20 and 40 °C. As a proof of concept, the change in fluorescent signal/color was captured using a digital camera. The images were then dissected into their red-green-blue (RGB) components using a Matlab routine. A linear correlation was observed between the hydrogel temperature and the green and blue intensity channels. The reported sensor has the potential to provide a wealth of information when thermal fluctuations mapped in soft gels matrices are correlated with chemical or physical processes.

Turning the heat on conjugated polyelectrolytes: an off-on ratiometric nanothermometer.

We report a self-referenced ratiometric nanothermometer based on short conjugated polyelectrolytes. An amphiphilic macromolecule destabilizes the polymer π-π stacking and makes it possible to shift the equilibrium between the less emissive aggregated state (520 nm) and the brighter individual chain (450 nm) within 20.0 °C and 70.0 °C.

Nanohybrid conjugated polyelectrolytes: highly photostable and ultrabright nanoparticles.

We present a general and straightforward one-step approach to enhance the photophysical properties of conjugated polyelectrolytes. Upon complexation with an amphiphilic polymer (polyvinylpyrrolidone), an anionic conjugated polyelectrolyte (poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene]) was prepared into small nanoparticles with exceptional photostability and brightness. The polymer fluorescence intensity was enhanced by 23 -fold and could be easily tuned by changing the order of addition. Single molecule experiments revealed a complete suppression of blinking. In addition, after only losing 18% of the original intensity, a remarkable amount of photons were emitted per particle (∼10(9), on average). This number is many folds greater than popular organic fluorescent dyes. We believe that an intimate contact between the two polymers is shielding the conjugated polyelectrolyte from the destructive photooxidation. The prepared nanohybrid particles will prove instrumental in single particle based fluorescent assays and can serve as a probe for the current state-of-the-art bioimaging fluorescence techniques.