One-pot synthesis of robust dendritic sulfur quantum dots for two-photon fluorescence imaging and “off–on” detection of hydroxyl radicals and ascorbic acid

The straightforward preparation of fluorescent sulfur quantum dots (SQDs) with good photostability and biocompatibility and multifunction remains a challenge. Herein, a simple method to improve the performance of SQDs is reported, that is, using hyperbranched polyglycerol (HPG) as a ligand to direct the synthesis of dendritic HPG-SQD nanocomposites from cheap elemental sulfur. Thanks to the protection of HPG, the HPG-SQDs show much better biocompatibility and photostability as compared with the widely reported polyethylene glycol (PEG) ligand-capped SQDs (PEG-SQDs). In addition, the HPG-SQDs also present excellent aqueous solubility, stable fluorescence against environmental variation, good cell uptake capability, and strong single- and two-photon fluorescence. Moreover, the HPG-SQDs display sensitive and selective fluorescence “off–on” behavior to hydroxyl radicals (˙OH) and ascorbic acid (AA), respectively, and thereby hold potential as a fluorescent switch to detect ˙OH and AA. For the first time, the utilization of two-photon fluorescence of HPG-SQDs to monitor ˙OH and AA in cells is demonstrated in this study.


SQDs)
Fluorescent HPG-SQDs can be easily synthesized by one-pot reaction under pure oxygen atmosphere. In a typical procedure, sublimed sulfur (1.6 g), HPG (1.0 g) and NaOH (4.0 g) were added to 50 mL of deionized water in a 100 mL flask. The mixture was stirred at 95 o C in a pure oxygen atmosphere. After stirring for 24 h, the resulting mixture was dialyzed against water with a dialysis bag (molecular weight cutoff = 5000 Da), and then freeze-dried to afford purified HPG-SQDs.

Sensing of hydroxyl radical (•OH)
The •OH was formed by Fenton reaction between H 2 O 2 and Fe 2+ . For sensing of •OH, typically, 980 μL of aqueous solution of HPG-SQDs (50 μg/mL) and 20 μL of •OH with different concentrations (0-1.2 mM) were mixed in a cuvette by shaking at room temperature for 2 min. Fluorescent spectra of the mixture were determined on a Varian Cary 100 fluorescence spectrophotometer at 25 o C with excitation at 330 nm and 420 nm, respectively. Correspondingly, the fluorescence intensity at the wavelength of 434 nm and 501 nm were recorded.

Sensing of ascorbic acid (AA)
For detection of AA, the mixture of HPG-SQDs and •OH was employed as a sensing system. In a typical procedure, 900 μL of the mixture of HPG-SQDs (50 μ g/mL) and •OH (24 μM) was added to a quartz cuvette. Subsequently, 100 μL of AA solution with known concentration (0−16 mM) was added to the mixture of HPG-SQDs/•OH. After shaking the mixture for 3 min, the emission spectra of mixture were collected with excitation at 330 nm and 420 nm, respectively.

Cytotoxicity evaluation
The cytotoxicity of HPG-SQDs and PEG-SQDs was assessed using the standard MTT assay. Firstly, HeLa cervical cancer cells were seeded in 96-well plates at a density of 1×10 4 cells/mL. After 24 h of incubation, the culture medium was replaced by the aqueous solution of HPG-SQDs (or PEG-SQDs) with concentrations of 200, 100, 50, 25, and 0 μg/mL and further incubated for 24 h. Subsequently, the wells were washed thrice with PBS buffer, and 100 μL of freshly prepared MTT (0.5 mg/mL) solution in culture medium was added to each well. The MTT medium solution was carefully removed after 3 h of incubation. Dimethyl sulfoxide (100 μL) was then added into each well, and the plate was gently shaken for 20 min. The absorbance of MTT at 570 nm was recorded by the microplate reader. Cell viability was expressed by the ratio of absorbance of the cells incubated with HPG-SQDs (or PEG-SQDs) to that of the cells incubated with culture medium only.

Single-photon fluorescence imaging
Typically, HeLa cells were cultured in Dulbecco's modified Eagle's medium at 37 °C for 12 h under 5% CO 2 . After removing the growth medium, medium containing HPG-SQDs (50 µg/mL) was added to incubate with the HeLa cells for 2 h at 37 °C.
Subsequently, the cells were rinsed with phosphate buffer to remove free HPG-SQDs.
After washing with PBS buffer for two times, imaging of HeLa cells was conducted on a confocal laser-scanning microscopy (CLSM, Olympus FV3000).

Two-photon fluorescence imaging
HeLa cells in chamber were cultured at 37 o C for 24 h. When the cells density reaches 80% of the culture plate, the cells were incubated with culture medium and HPG-SQDs (50 μg/mL) for 2 h. After that, the cells were washed twice with phosphate buffer to remove the unbound HPG-SQDs. In order to monitor •OH and AA in cells, the HPG-SQDs-stained HeLa cells were further treated with •OH (30 μM) and AA (1.5 mM). Subsequently, the two-photon fluorescence images of cells were recorded by TCS SP8 DIVE multiphoton fluorescence microscope (Leica, Germany) with femtosecond laser pulse.