Deciphering the catalytic mechanism of superoxide dismutase activity of carbon dot nanozyme

Nanozymes with superoxide dismutase (SOD)-like activity have attracted increasing interest due to their ability to scavenge superoxide anion, the origin of most reactive oxygen species in vivo. However, SOD nanozymes reported thus far have yet to approach the activity of natural enzymes. Here, we report a carbon dot (C-dot) SOD nanozyme with a catalytic activity of over 10,000 U/mg, comparable to that of natural enzymes. Through selected chemical modifications and theoretical calculations, we show that the SOD-like activity of C-dots relies on the hydroxyl and carboxyl groups for binding superoxide anions and the carbonyl groups conjugated with the π-system for electron transfer. Moreover, C-dot SOD nanozymes exhibit intrinsic targeting ability to oxidation-damaged cells and effectively protect neuron cells in the ischemic stroke male mice model. Together, our study sheds light on the structure-activity relationship of C-dot SOD nanozymes, and demonstrates their potential for treating of oxidation stress related diseases.


Supplementary Methods
Synthesis Synthesis of C-dots-HCl: C-dots (5 mg) was added to HCl solution (0.1 M, 10 mL) and then refluxed for 12 h. The resulting solution was neutralized with NaHCO3 and then dialyzed for 3 d.
Synthesis of C-dots-Cy5.5: 5 mg of C-dots and 2-3 mL of thionyl chloride (SOCl2) were added to 6-8 mL of acetonitrile. The mixture was heated at 80~100 °C until all of C-dots solid powder dispersed. The C-dots acyl chloride (solid) was obtained and redispersed in 6-8 mL of anhydrous acetonitrile after removing excess SOCl2 and acetonitrile by distillation. Then, 167 µL of Sulfo-Cyanine5.5 amine (3 mg/mL in DMF) was added to the solution of C-dots acyl chloride. After continue stirring for 30 min at room temperature in dark, the solvent was removed by rotary evaporation. The resulting product was redispersed in water and purified by agarose gel electrophoresis (1%).

The CAT-like activity of C-dots
The CAT-like activity of C-dots was detected by monitoring the elimination of H2O2 and generation of oxygen.
The concentration of H2O2 was monitored in time-drive mode at 240 nm by using a UV-Vis spectrophotometer. The reaction solutions contained 10 mM H2O2 and C-dots with a concentration of 10 μg/mL in 500 µL of PBS (25 mM PBS, pH 7.4). The absorbance peak at 240 nm of the solution was monitored by using a UV-vis spectroscopy. The oxygen generation was measured by using a specific oxygen electrode on Multi-Parameter Analyzer (JPSJ-605F, Leica China). In a typical test, 60 μL of 30% H2O2 solution was added to 14.94 mL of water, and then C-dots (final concentration of 10 μg/mL) was added. The generated oxygen solubility (unit: mg/L) was recorded from 0 to 60 s.

The POD/OXD-like activity of C-dots
The POD-like activity assays of C-dots (10 μg/mL) were carried out using TMB (1 mM) as the substrate in the presence of 1 M H2O2 in citric acid-NaAc buffer solution. The absorbance (at 652 nm for ox-TMB) of the reaction was recorded using a UV-vis spectroscopy. The OXD-like activity of C-dots were tested under the same condition in the absence of H2O2.

Measurement of ESR
The free radicals were quantitatively estimated by the ESR signal intensity of the free radical spin adduct using the peak-to-peak height of the line of the ESR spectrum. Superoxide radicals were produced by 13 mM

Theoretical calculation
All geometries, including local minima and transition states (TS), were fully optimized by employing the B3LYP density functional 1 and the 6-31G(d,p) basis set 2 . In order to identify whether the stationary point is a local minimum or a transition state and obtain the Gibbs free energy, the harmonic frequency analysis was calculated for each structure. In our calculations, the PCM solvation model was used to model the water environment. All the calculations were carried out using the Gaussian 09 package (Gaussian, Inc. Wallingford In this work, we performed a series of chemical modifications to transform the functional groups, especially hydroxyl and carboxyl groups, which involved changes in FT-IR. In order to accurately identify the absorption O-H, we carried out strict drying treatment on the samples, so that the O-H on the surface of the C-dots can be determined by the absorption band around 3400 cm -1 . The broadband around 2560 cm -1 could be attributed 7 to the stretching vibration of the hydrogen bond of associating carboxyl group, which enabled one to distinguish a carboxylic acid from all the other carbonyl compounds 3 . For verifying the existence of carboxyl groups on the surface of C-dots, the sample was acidified or alkalified by HCl or NH4OH before FT-IR measurement. As shown in Supplementary Figure 2, the peaks at 3400, 2560, 1720, and 1240 cm -1 in the FT-IR spectrum of C-dots-HCl increased significantly. After reacted with NH4OH, the peaks at 2560, 1720, and 1240 cm -1 disappeared. Two strong peaks at 1600 and 1400 cm -1 appeared, which according to asymmetric and symmetric stretching vibrations of −COO − , respectively, because the carboxyl group (−COOH) was converted to carboxylate anion (−COO − ). Therefore, we can attribute the peaks at 1720, 1240, and 2560 cm -1 to the C=O, C−O, and associated hydrogen bond, respectively, of carboxylic acid. While, the peak at 1620 cm -1 is more likely to be attributed to the acid-insensitive C=C.