Stable functionalization of small semiconducting polymer dots via covalent cross-linking and their application for specific cellular imaging

Abstract

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.

Figure 1

Stability of functionalized Pdots. The absorption (left) and fluorescence (right) spectra of Pdots doped with Rhodamine. (a, b) PFBT/PSMA-Rhodamine Pdots functionalized using the physical-blending method. (c, d) PFBT-PSMA-Rhodamine Pdots formed by the covalent cross-linking approach. The circled regions indicate the absorption peaks of Rhodamine, which clearly show significant dye leaching from PSMA/PSMA-Rhodamine Pdots prepared by physical blending, but no leaching in PFBT-PSMA-Rhodamine cross-linked Pdots.

Figure 2

Optical and physical properties of Pdots. (a) Absorption and (b) fluorescence spectra of physically blended PFBT-PSMA Pdots, cross-linked PFBT-NH-PSMA Pdots, and cross-linked PFBT-NH-PIMA Pdots in water. (c) Typical TEM image of cross-linked PFBT-NH-PIMA Pdots. Scale bar: 20 nm. (d) Histogram showing the hydrodynamic particle size of PFBT-NH-PIMA Pdots measured by DLS (average diameter = 8.7 nm).

Figure 3

Cellular labeling using cross-linked Pdots. (a, b) Flow cytometry measurements made using cross-linked PFBT-NH-PIMA Pdot-streptavidin. Blue curve in (b) shows the positive labeling and red curve shows the negative labeling in the control, where identical experimental conditions were followed but in the absence of primary biotinylated antibody. (c, d) Confocal fluorescence microscopy images of MCF-7 breast-cancer cells labeled with cross-linked PFBT- NH-PIMA Pdot-streptavidin. Panel c shows positive labeling and panel d shows negative labeling performed under the same condition but in the absence of biotinylated primary antibody. Images from left to right: blue fluorescence from the nucleus stain Hoechst 34580; green fluorescence images from Pdot. (e) Confocal fluorescence microscopy image of microtubules in HeLa cells labeled with cross-linked Pdot-streptavidin. The blue channel was from the nucleus stain, while the green channel showed emission from PFBT-NH-PIMA Pdots. The HeLa cells were incubated with both biotinylated anti-α-tubulin and PFBT-NH-PIMA Pdot-streptavidin. All the scale bars represent 20 μm for (c), (d) and (e).

Scheme 1

Schematic outlining the preparation of small cross-linked PFBT-NH-PIMA Pdots conjugated to streptadvidin for cellular imaging.