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

Abstract

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.

Figure 1

Transmission-electron-microscopy (left) and dynamic-light-scattering (right) measurements of Pdots with different density of side-chain carboxylic acid groups: (a) PFBT-C2; (b) PFBT-C14; (c) PFBT-C50. Scale bar: 100 nm.

Figure 2

Absorption and fluorescence spectra of Pdots with different density of side-chain carboxylic acid functional groups.

Figure 3

Single-particle fluorescence images of (a) PFBT-C2, (b) PFBT-C14, and (c) PFBT-C50 dots, obtained under identical excitation and detection conditions. Scale bars represent 10 μm. Histograms of intensity distribution of single-particle fluorescence for (d) PFBT-C2, (e) PFBT-C14, and (f) PFBT-C50 Pdots; the blue curves were obtained by fitting a lognormal distribution to the histogram, and gave 3350, 2960 and 610 mean CCD counts for the three types of Pdots, respectively.

Figure 4

Absorption and fluorescence spectra of tetraphenylporphyrin-doped PFBT dots with different density of side-chain carboxylic acid functional groups. The left panels show the absorption spectra of PFBT-C2 (black), PFBT-C14 (red), and PFBT-C50 (blue) aqueous solution of Pdots before (a) and after (b) centrifugal filtration; (c) shows the absorption spectra of the filtrate for the three types of Pdots. The right panels show the fluorescence spectra of PFBT-C2 (black), PFBT-C14 (red), and PFBT-C50 (blue) aqueous solutions before (d) and after (e) centrifugal filtration; (f) shows the fluorescence spectra of the filtrate for the three types of Pdots. The insert in (f) shows the fluorescence spectra obtained under the excitation wavelength of 450 nm for the filtrate of PFBT-C2, PFBT-C14, and PFBT-C50 aqueous solutions.

Figure 5

Flow-cytometry measurements of the intensity distributions of cancer cells (MCF-7) labeled via non-specific binding, for (a) control blank sample in the absence of Pdots, (b) PFBT-C2 dots, (c) PFBT-C14 dots, and (d) PFBT-C50 dots.

Figure 6

Fluorescence images of SK-BR-3 breast-cancer cells labeled with Pdot-streptavidin (SA) probes. (a) Positive labeling using PFBT-C2-SA probe. (b) Negative labeling carried out under the same condition as (a) but in the absence of EDC in the bioconjugation step. (c) Positive labeling using red emitting PFTBT/PFBT-C2-SA probe. Images from left to right: blue fluorescence from the nuclear stain Hoechst 34580; green or red fluorescence images from Pdots-SA probes; Nomarski (DIC) images; combined fluorescence images. Scale bars: 20 μm.

Figure 7

Fluorescence images of newly synthesized proteins in MCF-7 breast-cancer cells tagged with PFBT-C14A probes. (a) Positive labeling using PFBT-C14A probe. (b) Negative labeling carried out under the same condition as in (a) but in the absence of Cu(I) catalyst. From left to right: blue fluorescence from the nuclear stain Hoechst 34580; green fluorescence images from PFBT-C14A probes; Nomarski (DIC) images; and combined fluorescence images. Scale bars: 20 μm.

Scheme 1

Schematic illustration of covalently functionalized semiconducting polymer and Pdot-bioconjugates for specific cellular targeting.