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.

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.

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.

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.

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.

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.

Ratiometric temperature sensing with semiconducting polymer dots.

This communication describes ultrabright single-nanoparticle ratiometric temperature sensors based on semiconducting polymer dots (Pdots). We attached the temperature sensitive dye-Rhodamine B (RhB), whose emission intensity decreases with increasing temperature-within the matrix of Pdots. The as-prepared Pdot-RhB nanoparticle showed excellent temperature sensitivity and high brightness because it took advantage of the light harvesting and amplified energy transfer capability of Pdots. More importantly, the Pdot-RhB nanoparticle showed ratiometric temperature sensing under a single wavelength excitation and has a linear temperature sensing range that matches well with the physiologically relevant temperatures. We employed Pdot-RhB for measuring intracellular temperatures in a live-cell imaging mode. The exceptional brightness of Pdot-RhB allows this nanoscale temperature sensor to be used also as a fluorescent probe for cellular imaging.

Development of ultrabright semiconducting polymer dots for ratiometric pH sensing.

Semiconducting polymer-based nanoparticles (Pdots) have recently emerged as a new class of ultrabright probes for biological detection and imaging. This paper describes the development of poly(2,5-di(3',7'-dimethyloctyl)phenylene-1,4-ethynylene) (PPE) Pdots as a platform for designing Förster resonance energy transfer (FRET)-based ratiometric pH nanoprobes. We describe and compare three routes for coupling the pH-sensitive dye, fluorescein, to PPE Pdots, which is a pH-insensitive semiconducting polymer. This approach offers a rapid and robust sensor for pH determination using the ratiometric methodology where excitation at a single wavelength results in two emission peaks, one that is pH sensitive and the other one that is pH insensitive for use as an internal reference. The linear range for pH sensing of the fluorescein-coupled Pdots is between pH 5.0 and 8.0, which is suitable for most cellular studies. The pH-sensitive Pdots show excellent reversibility and stability in pH measurements. In this paper, we use them to measure the intracellular pH in HeLa cells following their uptake by endocytosis, thus demonstrating their utility for use in cellular and imaging experiments.

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.

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.

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.

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.

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.

Near-infrared fluorescent dye-doped semiconducting polymer dots.

Near-infrared (NIR) fluorescence sensing is desirable for in vivo biological measurements, but the method is currently limited by the availability of NIR fluorescent markers as well as by their poor performance, such as self-aggregation and dim fluorescence, in a physiological environment. To address this issue, this paper describes a NIR fluorescent polymer dot (Pdot) that emits at 777 nm. This Pdot was comparable in size to a water-soluble NIR quantum dot that emits at 800 nm (ITK Qdot800) but was about four times brighter and with a narrower emission peak. We formed the NIR Pdot by doping the NIR dye, silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775), into the matrix of poly (9,9-dioctylfluorene-co-benzothiadiazole) (PFBT) as the Pdot formed using a nanoscale precipitation technique. Free molecules of NIR775 aggregate in aqueous solution, but encapsulating them into the hydrophobic Pdot matrix effectively introduced them into aqueous solution for use in biological studies. Most importantly, the brightness of NIR775 was dramatically enhanced because of the excellent light-harvesting ability of PFBT and the very efficient energy transfer from PFBT to NIR775. We anticipate this bright NIR Pdot will be useful in biological measurements and cellular imaging where strong NIR emission is beneficial.