I-Che Wu

Multicolor Fluorescent Semiconducting Polymer Dots with Narrow Emissions and High Brightness

Fluorescent semiconducting polymer dots (Pdots) have attracted great interest because of their superior characteristics as fluorescent probes, such as high fluorescence brightness, fast radiative rates, and excellent photostability. However, currently available Pdots generally exhibit broad emission spectra, which significantly limit their usefulness in many biological applications involving multiplex detections. Here, we describe the design and development of multicolor narrow emissive Pdots based on different boron dipyrromethene (BODIPY) units. BODIPY-containing semiconducting polymers emitting at multiple wavelengths were synthesized and used as precursors for preparing the Pdots, where intraparticle energy transfer led to highly bright, narrow emissions. The emission full width at half-maximum of the resulting Pdots varies from 40 to 55 nm, which is 1.5-2 times narrower than those of conventional semiconducting polymer dots. BODIPY 520 Pdots were about an order of magnitude brighter than commercial Qdot 525 under identical laser excitation conditions. Fluorescence imaging and flow cytometry experiments indicate that the narrow emissions from these bright Pdots are promising for multiplexed biological detections.

Hybrid Semiconducting Polymer Dot–Quantum Dot with Narrow-Band Emission, Near-Infrared Fluorescence, and High Brightness

This communication describes a new class of semiconducting polymer nanoparticle-quantum dot hybrid with high brightness, narrow emission, near-IR fluorescence, and excellent cellular targeting capability. Using this approach, we circumvented the current difficulty with obtaining narrow-band-emitting and near-IR-fluorescing semiconducting polymer nanoparticles while combining the advantages of both semiconducting polymer nanoparticles and quantum dots. We further demonstrated the use of this new class of hybrid nanomaterial for effective and specific cellular and subcellular labeling without any noticeable nonspecific binding. This hybrid nanomaterial is anticipated to find use in a variety of in vitro and in vivo biological applications.

Squaraine-Based Polymer Dots with Narrow, Bright Near-Infrared Fluorescence for Biological Applications

This article describes the design and development of squaraine-based semiconducting polymer dots (Pdots) that show large Stokes shifts and narrow-band emissions in the near-infrared (NIR) region. Fluorescent copolymers containing fluorene and squaraine units were synthesized and used as precursors for preparing the Pdots, where exciton diffusion and likely through-bond energy transfer led to highly bright and narrow-band NIR emissions. The resulting Pdots exhibit the emission full width at half-maximum of ∼36 nm, which is ∼2 times narrower than those of inorganic quantum dots in the same wavelength region (∼66 nm for Qdot705). The squaraine-based Pdots show a high fluorescence quantum yield (QY) of 0.30 and a large Stokes shift of ∼340 nm. Single-particle analysis indicates that the average per-particle brightness of the Pdots is ∼6 times higher than that of Qdot705. We demonstrate bioconjugation of the squaraine Pdots and employ the Pdot bioconjugates in flow cytometry and cellular imaging applications. Our results suggest that the narrow bandwidth, high QY, and large Stokes shift are promising for multiplexed biological detections.

Stable Functionalization of Small Semiconducting Polymer Dots via Covalent Cross‐Linking and Their Application for Specific Cellular Imaging

A facile cross-linking strategy covalently links functional molecules to semiconducting polymer dots (Pdots) while simultaneously providing functional groups for biomolecular conjugation. In addition to greatly enhanced stability, the formed Pdots are small (<10 nm), which can be difficult to achieve with current methods but is highly desirable for most biological applications. These characteristics are significant for improving labeling efficiency and sensitivity in cellular assays that employ Pdots.

Photostable Ratiometric Pdot Probe for in Vitro and in Vivo Imaging of Hypochlorous Acid

Developing probes for the detection of reactive oxygen species (ROS), a hallmark of many pathophysiological process, is imperative to both understanding the precise roles of ROS in many life-threatening diseases and optimizing therapeutic interventions. We herein report an all-in-one fluorescent semiconducting polymer based far-red to near-infrared (NIR) Pdot nanoprobe for the ratiometric detection of hypochlorous acid (HOCl). The fabrication takes the advantage of flexible polymer design by incorporating target-sensitive and target-inert fluorophores into a single conjugated polymer to avoid leakage or differential photobleaching problems existed in other nanoprobes. The obtained nanoprobe has improved performance in HOCl sensing, such as high brightness, ideal far-red to NIR optical window, excellent photostability, self-referenced ratiometric response, fast response, and high selectivity. The dual-emission property allows the sensitive imaging of HOCl fluctuations produced in living macrophage cells and peritonitis of living mice with high contrast. This study not only provides a powerful and promising nanoprobe to be potentially used in the investigations of in situ HOCl status of diseases in living systems but also puts forward the design strategy of a new category of ratiometric fluorescent probes facilitating precise and reliable measurement in biological systems.

Yellow Fluorescent Semiconducting Polymer Dots with High Brightness, Small Size, and Narrow Emission for Biological Applications

Cross-linked polymer dots with intense and narrow yellow emission were designed using boron-dipyrromethene (BODIPY) polymer as the acceptor and poly[9,9-dioctylfluorenyl-2,7-diyl-co-1,4-benzo-{2,1′-3}-thiadiazole] (PFBT) polymer as the donor. The emission fwhm’s of the polymer dots (Pdots) were 37 nm. CL-BODIPY 565 Pdots were about 5 times brighter than commercial quantum dots (Qdots) 565 under identical experimental conditions. Specific cellular targeting indicated that the small, bright, and narrow emissive CL-BODIPY 565 Pdots are promising probes for biological applications.

Semiconducting polymer dots with monofunctional groups

This communication describes an approach for preparing monovalent semiconducting polymer dots (mPdots) with a size of 5 nm where each mPdot was composed of precisely a single active functional group.

Improving the Photostability of Semiconducting Polymer Dots Using Buffers

The photostability of fluorescent probes is critical in biological imaging, especially for long-term observational analyses. Here, we describe a simple and universal method to improve the photostability of semiconducting polymer dots (Pdots) and other fluorescent probes by using buffers. Using Pdots as a model system, we found that HEPES or MES buffer can improve the photostability of Pdots by a factor of 20. Through a systematic study, we show that Pdot photobleaching is dominated by photoinduced radicals which can be quenched by the piperazine or morpholine structures of these buffers, which act as radical scavengers. For conditions where choice of buffer is limited, we designed fluorescent polymers conjugated with radical scavengers to improve Pdot photostability. We then demonstrate a practical application in which HEPES buffer is used to improve the photostability of Pdots during cell imaging.

Ratiometric Barcoding for Mass Cytometry

Barcoding is of importance for high-throughput cellular and molecular analysis. A ratiometric barcoding strategy using lanthanide-coordinated polymer dots (Ln-Pdots) was developed for mass cytometric analysis. By using 3 metal isotopes and 4 ratio intensity levels, 16 barcodes were generated to code, and later decode, cell samples in mass cytometry. The ratiometric Ln-Pdot barcodes not only provided high-mass-signal intensities but also eliminated the bias caused by different concentrations of the labeling reagents/barcodes and run-to-run differences in cell labeling efficiency. The ability to distinguish clearly the 16 sets of labeled MCF-7 cells with mass cytometry demonstrated the excellent resolving power of the ratiometric Ln-Pdot barcodes. Furthermore, the results from barcoding PBMC samples via CD45-specific cellular targeting indicated that the ratiometric Ln-Pdot barcodes could facilitate mass cytometry in high-throughput and multiplexed analysis, especially with precious human samples.

Lanthanide-Coordinated Semiconducting Polymer Dots Used for Flow Cytometry and Mass Cytometry

Simultaneous monitoring of biomarkers as well as single-cell analyses based on flow cytometry and mass cytometry are important for investigations of disease mechanisms, drug discovery, and signaling-network studies. Flow cytometry and mass cytometry are complementary to each other; however, probes that can satisfy all the requirements for these two advanced technologies are limited. In this study, we report a probe of lanthanide-coordinated semiconducting polymer dots (Pdots), which possess fluorescence and mass signals. We demonstrated the usage of this dual-functionality probe for both flow cytometry and mass cytometry in a mimetic cell mixture and human peripheral blood mononuclear cells as model systems. The probes not only offer high fluorescence signal for use in flow cytometry, but also show better performance in mass cytometry than the commercially available counterparts.