Ratiometric pH Sensing and Imaging in Living Cells with Dual-Emission Semiconductor Polymer Dots

Abstract

Polymer dots (Pdots) represent newly developed semiconductor polymer nanoparticles and exhibit excellent characteristics as fluorescent probes. To improve the sensitivity and biocompatibility of Pdots ratiometric pH biosensors, we synthesized 3 types of water-soluble Pdots: Pdots-PF, Pdots-PP, and Pdots-PPF by different combinations of fluorescent dyes poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO), poly[(9,9-dioctyl-fluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadazole)] (PFBT), and fluorescein isothiocyanate (FITC). We found that Pdots-PPF exhibits optimal performance on pH sensing. PFO and FITC in Pdots-PPF produce pH-insensitive (λ = 439 nm) and pH-sensitive (λ = 517 nm) fluorescence respectively upon a single excitation at 380 nm wavelength, which enables Pdots-PPF ratiometric pH sensing ability. Förster resonance energy transfer (FRET) together with the use of PFBT amplify the FITC signal, which enables Pdots-PPF robust sensitivity to pH. The emission intensity ratio (I₅₁₇/I₄₃₉) of Pdots-PPF changes linearly as a function of pH within the range of pH 3.0 to 8.0. Pdots-PPF also possesses desirable reversibility and stability in pH measurement. More importantly, Pdots-PPF was successfully used for cell imaging in Hela cells, exhibiting effective cellular uptake and low cytotoxicity. Our study suggests the promising potential of Pdots-PPF as an in vivo biomarker.

Keywords: FRET; Pdots; cell imaging; pH sensing; semiconductor polymer.

Scheme 1

A scheme showing the preparation and application of Pdots-PF and Pdots-PPF.

Figure 1

(a) Absorption of PFO (black dotted line), PFBT (red dotted line), and FITC (green dotted line), and emission spectra of PFO (black solid line), PFBT (red solid line), and FITC (green solid line) when excited at the optimal wavelength. (b) Emission spectra of PFO (black solid line), Pdots-PF (blue solid line), and FITC (green solid line) when excited at 380 nm. (c) TEM of Pdots-PF. Scale bar represents 50 nm. The inset shows the enlarged view of TEM of Pdots-PF. (d) The particle size distribution of Pdots-PF.

Figure 2

(a) Fluorescence emission spectra of FITC in pH 3.0–8.0 when excited at 488 nm, inset shows the linear correlation between the I₅₁₇ and the pH. (b) Fluorescence emission spectra of PFO Pdots in pH 3.0–8.0 when excited at 380 nm. (c) Fluorescence emission spectra of Pdots-PF in pH 3.0–8.0 when excited at 380 nm, inset shows the linear correlation between the I₅₁₇/I₄₃₉ and the pH. (d) Fluorescence emission spectra of Pdots-PP in pH 3.0–8.0 when excited at 380 nm.

Figure 3

(a) Fluorescence spectra of Pdots-PP (black solid line) and Pdots-PPF (red solid line) when excited at 380 nm. (b) Fluorescence spectra of Pdots-PF (blue solid line) and Pdots-PPF (red solid line) when excited at 380 nm. (c) TEM of Pdots-PPF. Scale bar represents 50 nm. The inset shows the enlarged view of TEM of Pdots-PPF. (d) The particle size distribution of Pdots-PPF.

Figure 4

(a) Fluorescence emission spectra of Pdots-PPF at pH 3.0–8.0 in 20.0 mM HEPES buffer solution when excited at 380 nm. (b) Plot of the relative fluorescence intensity ratios (I₅₁₇/I₄₃₉) of Pdots-PPF in response to pH values.

Figure 5

(a) Fluorescence intensity ratios (I₅₁₇/I₄₃₉) of Pdots-PPF upon repeatedly switching pH from 3.0 to 8.0. (b) Fluorescence intensity of λ₅₁₇ (a,c) and λ₄₃₉ (b,d) emission of Pdots-PPF in 20.0 mM HEPES buffer solution at pH 8.0 and 3.0. The inset: The corresponding time-dependent intensity ratios (I₅₁₇/I₄₃₉) of Pdots-PPF at pH = 8.0 (a) and 3.0 (b). (c) Selectivity of the Pdots-PPF.

Figure 6

MTT based cytotoxicity assay of Hela cells after 24 h incubation with different concentrations of Pdots-PPF (0, 0.3, 0.5, 1.5, 2.5, and 5.0 μg·mL⁻¹) (mean ± SD, n = 3).

Figure 7

(a) Bright-field and (a–c) fluorescent images of Hela cells incubated with Pdots-PPF. (d) merged image of (a), (b) and (c). Scale bar represents 25 μm.