Development of ultrabright semiconducting polymer dots for ratiometric pH sensing

Abstract

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.

Figure 1

Characterization of fluorescein-functionalized Pdots. (A) UV-visible spectra of PPE Pdots (dashed plum line) and FITC (dashed black line) in water; emission spectra of PS-SH co PPE (solid red line), Pdot(A) Pdots (solid blue line), and FITC (solid green line) in pH 7 HEPES buffer when excited at 390 nm. The inset in the upper-right corner shows the photographs of solutions containing PS-SH co PPE (left) and Pdot(A) Pdot (right) under a 365 nm UV lamp. (B) Time resolved fluorescence decay of Pdots before (i.e. bare PS-SH co PPE Pdots; black circles) and after conjugation to fluorescein (i.e. Pdot(C); red circles) in pure water. (C) TEM image of Pdot(C) Pdots. The inset in the upper-left corner shows the enlarged view of a single Pdot(C). Scale bar represents 200 nm. (D) Size distribution of Pdot(C) Pdots as measured by TEM.

Figure 2

Fluorescence spectra of (A) Pdot(A), (B) Pdot(B), and (C) Pdot(C) at different pH, ranging from 5 to 8 (black line: pH=8, red line: pH=7.5, blue line: pH=7, green line: pH=6.5, gold line: pH=6, brown line: pH=5.5, pink line: pH=5). Excitation wavelength was 390 nm.

Figure 3

pH sensitivity and reversibility of the three fluorescein-conjugated Pdots. (A) Ratiometric pH calibration plot of the emission ratio (I_513nm/I_440nm) of Pdot(A) (●), Pdot(B) (■), and Pdot(C) (▲) as a function of pH. The blue, black, and red lines are linear fit to the data of Pdot(A) (R² = 0.995), Pdot(B) (R² = 0.991), and Pdot(C) (R² = 0.994), respectively. The slopes for Pdot(A), Pdot(B), and Pdot(C) are 0.37, 0.18, and 0.29, respectively. (B) The intensity ratios (I_513nm/I_440nm) of Pdot(A) (●), Pdot(B) (■), and Pdot(C) (▲) when the pH was toggled between 5.0 and 8.0 repeatedly, illustrating the reversibility and reproducibility of pH sensing.

Figure 4

Illustration of spectral overlap between the emission of donor (i.e. PPE) and the absorption of acceptor (i.e. FITC), and their calculated Förster distance, R_o. (A) Emission spectrum of PS-SH-FITC co PPE Pdots (dashed light green line) and excitation spectra of FITC at pH ranging from 5 to 8. The areas under the curves were colored for easier visualization of spectral overlap. (B) The corresponding Förster distance of PPE-FITC at different pH was plotted based on the overlap integral shown in (A).

Figure 5

Confocal microscopy images of HeLa cells labeled by (A–C) pure PPE Pdots and (E–G) Pdot(A) at λ_exc = 405 nm; their corresponding bright-field images are shown in (D) and (H), respectively. The blue fluorescence shown in (A) and (E) was produced by integrating the spectral region from 433–444 nm, while the green fluorescence shown in (B) and (F) was from 507–518 nm. The images (C) and (G) are the overlay of the blue and green fluorescence. The scale bars are 20 µm.

Figure 6

Confocal microscopy images of HeLa cells labeled by (A–C) Pdot(B) and (E–G) Pdot(C) at λ_exc = 405 nm; their corresponding bright-field images are shown in (D) and (H), respectively. The blue channel shown in (A) and (E) was produced by integrating the spectral region from 433–444 nm, while the green channel shown in (B) and (F) was from 507–518 nm. The images in (C) and (G) are the overlay of the blue and green channels. The insets show a magnified view of a single HeLa cell. The scale bars are 20 µm.

Scheme 1

Schematic showing three routes for the preparation of PPE Pdot-based pH sensors. (A) PS-SH co PPE Pdots in water were first prepared and then reacted with the isothiocyanate moieties on the FITC molecules. (B) FITC was first conjugated to PS-NH₂ polymers through the amine-isothiocyanate reaction, and the resulting fluorescein-labeled PS polymers were blended with PPE polymers to form the PS-NH₂-FITC co PPE Pdots. (C) PS-SH co PPE Pdots were first prepared in the same way as (A), but were subsequently coupled to fluorescein-5-maleimide. PPE: poly(2,5-di(3',7'-dimethyloctyl)phenylene-1,4-ethynylene; PS: polystyrene polymer; FITC: fluorescein isothiocyanate.