Detection of singlet oxygen in vitro

The EPR spectra were recorded on a Bruker EMX-8/2.7 spectrometer operating at 9.873 GHz (microwave power: 20 mW; modulation frequency: 100 kHz; modulation amplitude: 0.5 G; receiver gain: 4 × 10⁵). The mixture of NaCl (100 mM), H₂O₂ (100 μM) and SCNGs or free CPO (including 8 U CPO) with the ¹O₂ trapper (TEMP) was rapidly transferred to a standard quartz capillary and placed into the EPR spectrometer. The EPR spectrum was then recorded every 4 min. Another assay was performed as following: NaCl (100 mM), XO (0.26 U), X (10 mM) and free SOD (60 U), free CPO (40 U) or free SOD/CPO (including 60 U SOD and 40 U CPO) was rapidly mixed with the ¹O₂ trapper (TEMP) to monitor the EPR spectrometer immediately.

SOSG was used as the probe molecule to detect the generation efficiency of ¹O₂ in SCNGs. Typically, 10 μL of SOSG (50 μM) was added into the solution composed of NaCl (100 μL, 1 M), H₂O₂ (10 μL, 10 mM) and SCNGs or free SOD/CPO (including 10 U SOD and 8 U CPO) to form a 1 mL solution in the cuvette. The fluorescence intensity was then recorded on an F-7000 fluorescence spectrophotometer (Hitachi, Japan) at different time intervals. ¹O₂ detection was conducted by recording the fluorescence emission spectra of SOSG (Ex/Em = 504/525 nm) with the excitation wavelength fixed at 488 nm.

HepG2 and HL-7702 cells were obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences (Shanghai, China). All cells were tested negative for mycoplasma contamination. To study the production of ¹O₂ in cancer cells, the HepG2 cells were seeded in confocal dish with a density of 5 × 10⁴ per well. After incubation for 24 h, the medium was replaced with fresh culture medium. Then, PBS, NGs (300 µg mL⁻¹) or SCNGs (300 µg mL⁻¹) was injected to the well. After further incubation for 2 h, the cells were treated with SOSG probe (5 µM). Subsequently, the fluorescence emission spectrum of oxidized SOSG (E_x/E_m = 504/525 nm) was immediately recorded on a confocal fluorescence microscope (Carl Zeiss AG, Jena, Germany). Measurements were made for 140-second periods for 20 pictures, with 40-second intervals between each measurement. 10 group of 3D-CLSM pictures were captured automatically using 30 min (3 min/group). For fully showing the living cell fluorescence intensity, the photomicrograph at 18 min with YZ and XZ planes were captured.