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.

Color-Tunable Thermally Activated Delayed Fluorescence in Oxadiazole-Based Acrylic Copolymers: Photophysical Properties and Applications in Ratiometric Oxygen Sensing.

Polymer-based emitters are a promising route to the production of low-cost, scalable solution-processable luminescent materials. Here we describe a series of acrylic oxadiazole-based donor-acceptor monomers with tunable emission from blue to orange, with quantum yields as high as 96%. By introducing structural constraints that limit donor-acceptor orbital overlap, thermally activated delayed fluorescence (TADF) was observed in these materials. Polymerization by Cu(0) reversible deactivation radical polymerization (RDRP) gave high-molecular-weight copolymers (Mn > 20 kDa) with dispersities ranging from 1.10 to 1.45, using a room-temperature procedure with Cu wire as a catalyst. One of these materials, which had phenothiazine as donor moiety, exhibited conformationally dependent dual emission, giving a mixture of prompt fluorescence and delayed fluorescence peaks, whose relative ratios varied based on the amount of O2 present during measurement. We demonstrate that this material can combine prompt and delayed fluorescence to act as a single-component, all-organic, ratiometric oxygen sensor without external calibrant. Application to ratiometric oxygen sensing is demonstrated both using a polymer thin film and via incorporation of this material into water-soluble polymer dots (Pdots), with a ratiometric response to O2 throughout the range of partial pressures relevant to biological environments.

Dextran Functionalization of Semiconducting Polymer Dots and Conjugation with Tetrameric Antibody Complexes for Bioanalysis and Imaging.

Brightly fluorescent semiconducting polymer dots (Pdots) are emerging as very useful probes for bioanalysis and imaging. Unfortunately, Pdot materials often suffer from limitations such as poor colloidal and physical stability, nonspecific adsorption, and relatively few reported surface chemistries and bioconjugate chemistries. To help address these limitations, we have developed dextran-functionalized Pdots (Dex-Pdots). This functionalization improves particle stability over a range of pH and at high ionic strength, hinders surface-induced unfolding, and enables the preparation of immunoconjugates via tetrameric antibody complexes (TAC). The utility of TAC-conjugated Dex-Pdots is demonstrated through a proof-of-concept fluorescence-linked immunosorbent assay (FLISA) for human erythropoietin (EPO), and through immunolabeling of human epidermal growth factor receptor 2 (HER2)-positive SK-BR3 breast cancer cells. The conjugates exhibited less nonspecific binding and greater specific binding than Pdots without dextran functionalization. Overall, dextran functionalization is a highly promising surface chemistry for biological applications of Pdots.

Comparison of Semiconducting Polymer Dots and Semiconductor Quantum Dots for Smartphone-Based Fluorescence Assays.

Fluorescent nanoparticles have transformative potential for smartphone-based point-of-need diagnostics because an optimal material can reduce the technical burden to meet assay performance requirements. Semiconductor quantum dots (QDs) are a now well-established example of such a material. Semiconducting polymer dots (Pdots) and conjugated-polymer nanoparticles (CPNs) are emerging materials that bring the advantages of being bright, easy to synthesize, and metal-free when compared with QDs, but they frequently present the trade-off of spectrally broad emission and less well-defined surface chemistry. Here, we compare these two classes of nanoparticles in the context of a "bare bones" device that uses a smartphone for all-in-one excitation and imaging of fluorescence. The greater per-particle brightness of Pdots provides orders of magnitude better imaging sensitivity versus QDs, and this advantage translates to a model lateral flow assay. Our data suggest that Pdots will support multicolor imaging on a smartphone in an optimized assay, although QDs are likely superior for this purpose. These pros and cons lead to discussion of how physicochemical differences between QDs and Pdots may influence assay performance beyond differences in optical properties. Overall, Pdots have great potential for enabling smartphone-based fluorescence assays with high sensitivity and low detection limits.