Highly Photostable Carbon Dots from Citric Acid for Bioimaging

Abstract

Bioimaging supported by nanoparticles requires low cost, highly emissive and photostable systems with low cytotoxicity. Carbon dots (C-dots) offer a possible solution, even if controlling their properties is not always straightforward, not to mention their potentially simple synthesis and the fact that they do not exhibit long-term photostability in general. In the present work, we synthesized two C-dots starting from citric acid and tris (hydroxymethyl)-aminomethane (tris) or arginine methyl ester dihydrochloride. Cellular uptake and bioimaging were tested in vitro using murine neuroblastoma and ovine fibroblast cells. The C-dots are highly biocompatible, and after 24 h of incubation with the cells, 100% viability was still observed. Furthermore, the C-dots synthesized using tris have an average dimension of 2 nm, a quantum yield of 37%, high photostability and a zeta potential (ζ) around -12 mV. These properties favor cellular uptake without damaging cells and allow for very effective bioimaging.

Keywords: bioimaging; carbon dots; cytotoxicity; photoluminescence.

Figure 1

Schematic representation of the synthesis of CATris and CAarg.

Figure 2

UV-Vis absorption spectra of CATris (left) and CAArg (right) in aqueous solution.

Figure 3

3D fluorescence spectra (excitation (y-scale), emission (x-scale) and intensity (false colour scale)) of CATris (left) and CAArg (right).

Figure 4

FTIR absorption spectra of CATris (left) and CAArg (right) samples. The spectra of citric acid, Tris and Arg methyl ester are shown for reference.

Figure 5

Photostability under UV exposure for CATris (left) and CAArg (right) carbon dots. The line in CATris represents the exponential decay fit and in CAArg it is a guide for eyes.

Figure 6

Dynamic light scattering analysis of CATris (red) and CAArg (blue) filtered samples.

Figure 7

Fibroblast (top) and Neuroblastoma cells (bottom) from MTT assays with different CATris and CAArg concentrations after 24 h incubation.

Figure 8

Fluorescence confocal microscopy images of fibroblast cells with internalized CATris, at a concentration of 1 and 3 mg mL⁻¹, after 4 and 24 h of incubation under green, blue and red channels. Bottom row: bright field, fluorescence and merged imaging of fibroblast cells with internalized CATris, at a concentration of 1 mg mL⁻¹, after 24 h of incubation. (λ_ex = 400 nm; size 385.5 µm). Some of the cells showing overall uptake of C-dots have been marked with orange arrows for a better understanding of the images.

Figure 9

Fluorescence confocal microscopy images of fibroblast cells with internalized CAArg at a concentration of 1 and 3 mg mL⁻¹ after 4 and 24 h of incubation under green, blue, and red channels. Few black spots as references are indicated with red circles. Bottom row: Bright field, fluorescence and merged imaging of fibroblasts cells with internalized CAArg, at a concentration of 1 mg mL⁻¹, after 24 h of incubation (λ_ex = 400 nm; size 385.5 µm). Some of the cells showing an overall C-dots uptake are marked with orange arrows for a better understanding of the images.

Figure 10

Fluorescence confocal microscopy images of neuroblastoma cells with internalized CATris (left) and CAArg (right), at a concentration of 1 mg mL⁻¹, after 24 h of incubation under merged of green and blue channels (CATris) and green channel (CAArg). (λ_ex = 400 nm; size f85.5 µm). Some of the cells showing an overall uptake of the C-dots are marked with red circles for a better understanding of the images.