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.

Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots.

The efficient selection and isolation of individual cells of interest from a mixed population is desired in many biomedical and clinical applications. Here we show the concept of using photoswitchable semiconducting polymer dots (Pdots) as an optical 'painting' tool, which enables the selection of certain adherent cells based on their fluorescence, and their spatial and morphological features, under a microscope. We first develop a Pdot that can switch between the bright (ON) and dark (OFF) states reversibly with a 150-fold contrast ratio on irradiation with ultraviolet or red light. With a focused 633-nm laser beam that acts as a 'paintbrush' and the photoswitchable Pdots as the 'paint', we select and 'paint' individual Pdot-labelled adherent cells by turning on their fluorescence, then proceed to sort and recover the optically marked cells (with 90% recovery and near 100% purity), followed by genetic analysis.

Ultrasensitive Protein Detection on Dot Blots and Western Blots with Semiconducting Polymer Dots.

We demonstrate ultrasensitive fluorescence imaging of proteins on dot blots and western blots using a bright, compact, and orange-emitting semiconducting polymer dot (CNPPV). We achieved a detection limit at the single-picogram level in dot blots; with conventional western blot, we detected 50 pg of transferrin and trypsin inhibitor after SDS-PAGE and transfer onto a PVDF membrane. In this protocol, we describe the preparation of Pdots and its streptavidin bioconjugate and the procedures of dot blot and western blots.

Toxicity and oxidative stress induced by semiconducting polymer dots in RAW264.7 mouse macrophages.

The rapid development and acceptance of PDots for biological applications depends on an in depth understanding of their cytotoxicity. In this paper, we performed a comprehensive study of PDot cytotoxicity at both the gross cell effect level (such as cell viability, proliferation and necrosis) and more subtle effects (such as redox stress) on RAW264.7 cells, a murine macrophage cell line with high relevance to in vivo nanoparticle disposition. The redox stress measurements assessed were inner mitochondrial membrane lipid peroxidation (nonyl-acridine orange, NAO), total thiol level (monobromobimane, MBB), and pyridine nucleotide redox status (NAD(P)H autofluorescence). Because of the extensive work already performed with QDots on nanotoxicity and also because of their comparable size, QDots were chosen as a comparison/reference nanoparticle for this study. The results showed that PDots exhibit cytotoxic effects to a much lesser degree than their inorganic analogue (QDots) and are much brighter, allowing for much lower concentrations to be used in various biological applications. In addition, at lower dose levels (2.5 nM to 10 nM) PDot treatment resulted in higher total thiol level than those found with QDots. At higher dose levels (20 nM to 40 nM) QDots caused significantly higher thiol levels in RAW264.7 cells, than was seen with PDots, suggesting that QDots elicit compensation to oxidative stress by upregulating GSH synthesis. At the higher concentrations of QDots, NAD(P)H levels showed an initial depletion, then repletion to a level that was greater than vehicle controls. PDots showed a similar trend but this was not statistically significant. Because PDots elicit less oxidative stress and cytotoxicity at low concentrations than QDots, and because they exhibit superior fluorescence at these low concentrations, PDots are predicted to have enhanced utility in biomedical applications.

Light-induced Crosslinkable Semiconducting Polymer Dots.

This paper describes a synthetic approach for photocrosslinkable polyfluorene (pc-PFO) semiconducting polymer dots, and demonstrates their superior ability to crosslink and form 3-D intermolecular polymer networks. The crosslinked pc-PFO Pdots are equipped with excellent encapsulating ability of functional small molecules. Optimum conditions of light irradiation on pc-PFO Pdots were investigated and clarified by using polymer thin films as a model. By employing the optimal light irradiation conditions, we successfully crosslinked pc-PFO Pdots and studied their particle sizes, photophysical, and colloidal properties. Single-particle imaging and dynamic-light-scattering measurements were conducted to understand the behaviors of photocrosslinked Pdots. Our results indicate pc-PFO Pdots can be easily photocrosslinked and the crosslinked species have excellent colloidal stability, physical and chemical stability, fluorescence brightness, and specific binding properties for cellular labeling. Considering that optical stimulus can work remotely, cleanly, and non-invasively, this study should pave the way for a promising approach to further develop stimuli-responsive ultrabright and versatile Pdot probes for biomedical imaging.

Single-chain semiconducting polymer dots.

This work describes the preparation and validation of single-chain semiconducting polymer dots (sPdots), which were generated using a method based on surface immobilization, washing, and cleavage. The sPdots have an ultrasmall size of ∼3.0 nm as determined by atomic force microscopy, a size that is consistent with the anticipated diameter calculated from the molecular weight of the single-chain semiconducting polymer. sPdots should find use in biology and medicine as a new class of fluorescent probes. The FRET assay this work presents is a simple and rapid test to ensure methods developed for preparing sPdot indeed produced single-chain Pdots as designed.

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.

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.

Ultrasensitive detection of proteins on Western blots with semiconducting polymer dots.

Ultrasensitive fluorescence imaging of proteins on western blots using a bright, compact, and orange-emitting semiconducting polymer dot (CN-PPV) is demonstrated. A detection limit at the single-picogram level in dot blots is achieved; with conventional western blotting, 50 pg of transferrin and trypsin inhibitor after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer onto a polyvinylidene fluoride membrane are detected. This method does not require any additional equipment or time compared with the conventional procedure with traditional fluorescent probes.

Lyophilization of semiconducting polymer dot bioconjugates.

Semiconducting polymer dot (Pdot) bioconjugates are a new class of ultrabright fluorescent probes. Here, we report a procedure for lyophilizing Pdot bioconjugates so that they successfully retain their optical properties, colloidal stability, and cell-targeting capability during storage. We found that, when Pdot bioconjugates were lyophilized in the presence of 10% sucrose, the rehydrated Pdot bioconjugates did not show any signs of aggregation and exhibited the same hydrodynamic diameters as before lyophilization. The brightness of the lyophilized Pdots was at least as good as before lyophilization, but in some cases, the quantum yield of lyophilized Pdots curiously showed an improvement. Finally, using flow cytometry, we demonstrated that lyophilized Pdot bioconjugates retained their biological targeting properties and were able to effectively label cells; in fact, cells labeled with lyophilized Pdot bioconjugates composed of PFBT, which were stored for 6 months at -80 °C, were ~22% brighter than those labeled with identical but unlyophilized Pdot bioconjugates. These results indicate lyophilization may be a preferred approach for storing and shipping Pdot bioconjugates, which is an important practical consideration for ensuring Pdots are widely adopted in biomedical research.

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.

High-intensity near-IR fluorescence in semiconducting polymer dots achieved by cascade FRET strategy.

Near-IR (NIR) emitting semiconducting polymer dots (Pdots) with ultrabright fluorescence have been prepared for specific cellular targeting. A series of π-conjugated polymers were synthesized to form water dispersible multicomponent Pdots by an ultrasonication-assisted co-precipitation method. By optimizing cascade energy transfer in Pdots, high-intensity NIR fluorescence (φ = 0.32) with tunable excitations, large absorption-emission separation (up to 330 nm), and narrow emission bands (FWHM = 44 nm) have been achieved. Single-particle fluorescence imaging show that the as-prepared NIR Pdots were more than three times brighter than the commercially available Qdot705 with comparable sizes under identical conditions of excitation and detection. Because of the covalent introduction of carboxylic acid groups into polymer side chains, the bioconjugation between NIR-emitting Pdots and streptavidins can be readily completed via these functional groups on the surface of Pdots. Furthermore, through flow cytometry and confocal fluorescence microscopy the NIR-emitting Pdot-streptavidin conjugates proved that they could effectively label EpCAM receptors on the surface of MCF-7 cells, via specific binding between streptavidin and biotin.

A versatile method for generating semiconducting polymer dot nanocomposites.

This paper describes a method, based on co-precipitation, for generating small semiconducting polymer dot (Pdot) nanocomposites, which contain either gold or iron oxide nanoparticles within the Pdot matrix. We demonstrate the utility of Pdot-Au nanoparticles (Au-NP-Pdots) in dual-modality imaging in which co-localization of fluorescence from Pdot and scattering from Au was used to identify Au-NP-Pdot probes for downstream single-particle tracking and cellular imaging. We also demonstrate the potential of employing Pdot-FeO(x) nanoparticles (FeO(x)-NP-Pdots) for both sample preparation, where cells tagged with FeO(x)-NP-Pdots were isolated using an external magnet, and cellular imaging and detection, owing to the intense fluorescence from Pdots. The method we present here should be generalizable to the formation of other Pdot nanocomposites for creating the next generation of multi-functional Pdot probes.

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.

Importance of having low-density functional groups for generating high-performance semiconducting polymer dots.

Semiconducting polymers with low-density side-chain carboxylic acid groups were synthesized to form stable, functionalized, and highly fluorescent polymer dots (Pdots). The influence of the molar fraction of hydrophilic side-chains on Pdot properties and performance was systematically investigated. Our results show that the density of side-chain carboxylic acid groups significantly affects Pdot stability, internal structure, fluorescence brightness, and nonspecific binding in cellular labeling. Fluorescence spectroscopy, single-particle imaging, and a dye-doping method were employed to investigate the fluorescence brightness and the internal structure of the Pdots. The results of these experiments indicate that semiconducting polymers with low density of side-chain functional groups can form stable, compact, and highly bright Pdots as compared to those with high density of hydrophilic side-chains. The functionalized polymer dots were conjugated to streptavidin (SA) by carbodiimide-catalyzed coupling and the Pdot-SA probes effectively and specifically labeled the cancer cell-surface marker Her2 in human breast cancer cells. The carboxylate-functionalized polymer could also be covalently modified with small functional molecules to generate Pdot probes for click chemistry-based bio-orthogonal labeling. This study presents a promising approach for further developing functional Pdot probes for biological applications.

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.

Generation of functionalized and robust semiconducting polymer dots with polyelectrolytes.

We describe a facile method to functionalize semiconducting polymer dots (Pdots) with polyelectrolytes. The polyelectrolyte coating dramatically improves the colloidal stability of the Pdots in solutions which are either of high ionic strength or contain bivalent metal ions: this feature allows Pdots to be used under physiologically relevant environments without losing their functionality. We conjugated the polyelectrolyte-coated Pdots with streptavidin to demonstrate their application in specific cell labeling.

A compact and highly fluorescent orange-emitting polymer dot for specific subcellular imaging.

We demonstrate a new compact CN-PPV dot, which emits in the orange wavelength range with high brightness. The small particle size, high brightness, and the ability to highly specifically target subcellular structures make the CN-PPV dots promising probes for biological imaging and bioanalytical applications.

Probing the interior of synaptic vesicles with internalized nanoparticles.

Synaptic vesicles are subcellular organelles that are found in the synaptic bouton and are responsible for the propagation of signals between neurons. Synaptic vesicles undergo endo- and exocytosis with the neuronal membrane to load and release neurotransmitters. Here we discuss how we utilize this property to load nanoparticles as a means of probing the interior of synaptic vesicles. To probe the intravesicular region of synaptic vesicles, we have developed a highly sensitive pH-sensing polymer dot. We feel the robust nature of the pH-sensing polymer dot will provide insight into the dynamics of proton loading into synaptic vesicles.