br Experimental br Synthesis of sulfur doped carbon microsph
2.1. Synthesis of sulfur-doped carbon microspheres (S–C)
Sulfur-doped carbon microspheres were synthesized as follows: 1 g of glucose was dissolved in 50 mL of Milli-Q water, and the resulting solution was sonicated for 30 min. Then, 0.25 g of thiourea was dis-solved in 20 mL of Milli-Q water. The resulting solution was also so-nicated for 30 min. The solution containing thiourea was added to the glucose solution and continually stirred for 4 h. After that, the solution was transferred to 100 mL Teflon-sealed autoclaves and maintained at 180 °C for 8 h. After cooling to room temperature, the black precipitate was washed several times with water/ethanol and then dried at 60 °C for 24 h. The S–C was annealed at 800 °C under N2 atmosphere for 3 h with step raising temperature of 2 °C/min.
2.2. Dressing of [email protected]–C microspheres and electrode fabrication
Bump mapping of the S–C microspheres by 10-nm Ru proceeded as follows: i) 50 mg of S–C was dispersed in 50 mL of 0.1 M citric Vaborbactam by ultrasonication for 5 h. RuNPs were synthesized through the reduction of ruthenium trichloride in the presence of reducing agents and stabi-lizing agents; ii) 5 mg of RuCl3 was dissolved in 10 mL of DI-water with continuous stirring and then poured to the dispersed S–C; iii) Freshly prepared 10 mL of 0.1 M NaBH4 was added dropwise. The precipitate was washed with water and ethanol several times. Working materials were prepared by dispersing 5 mg of [email protected]–C in 1 mL of distilled H2O (5 mg/mL). The glassy carbon electrode (GCE, diameter 4.0 mm) was Sensors & Actuators: B. Chemical 284 (2019) 456–467
successively polished to a mirror with 0.05 μM alumina slurry and diamond slurry with washing by deionized water at every sweep. The polished GCE was sonicated in acetone and double-distilled water and then dried at room temperature. An electrode was fabricated by drop casting of 20 μL of [email protected]–C onto the surface of the GCE and then drying at room temperature.
2.3. Cell culture and in vitro study
The PC12 cell line was obtained from PC12 (ATCC® CRL1721™) and was cultured by incubation under 5% CO2 at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 10% heat-inactivated serum. Cells were passaged every five days and the medium was changed from 2 to 3 times a week throughout the lifetime of all cultures.
For cells visualization experiments using ([email protected]), cells were seeded in 6 well-plates under 5 × 106 Cells/mL. The desired amount of [email protected] (20 μg/ml) was added to each well, incubated in a humid chamber for additional 30 min under 5% CO2 at 37 °C followed by thoroughly washing using PBS. Nuclear counterstaining was carried out using 0.1 μg/ml of 4′, 6-diamidino-2-phenylindole (DAPI) in PBS for 10 min, and F-actine staining by pethidine. Finally, the images of the cells were immediately captured at ambient temperature on a confocal laser scanning microscopy using Leica TCS SPE5 X machine
3. Results and discussion
3.1. Wrinkled spheres of [email protected]–C constructions
The facile construction of carbon spheres was facilitated by the successive polymerization of D-glucose (carbon source). The ploy glu-cose was aromatized and carbonized at 180 °C and under hydrothermal treatment. This temperature is higher than the normal glycosidation temperature [31,34]. Carbon chains were actively doped by sulfur in the presence of thiourea as S-source under hydrothermal conditions, as shown in Scheme 1. The D-glucose was polymerized after successive condensations and spherical growths under temperatures higher than 140 °C (normal glycosidation temperature). Continuous aromatization and formation of carbon microspheres were also observed. The pre-sence of thiourea (the source of S) during the polymerization and spherical carbon growth led to the formation of hybrid carbon chains with S atoms. The microporous construction of S–C microspheres was established after annealing at 800 °C under N2 flow at a temperature increment of 2 °C/min. Wrinkled micrometric spheres with 10 nm Ru bumps formed after the decoration of Ru nanoparticles at the sulfur-doped carbon microspheres. The S–C solution was dispersed in 0.1 M citric acid (capping agent) and the resulting solution was vigorously stirred. The dressing process occurred after the reduction of Ru (III) in the presence of sodium borohydride (NaBH4) as a strong reducing agent. Finally, the [email protected]–C was synthesized and further used as an electrocatalyst for H2O2 reduction.