G-/C-rich ssDNA-based Fe and Cu/Fe nanoclusters with peroxidase-like activity for intracellular ROS production and cytotoxicity applications.

Five G-/C-rich single-stranded DNA (ssDNA) with different sequences and lengths were templated to prepare the DNA-Cu, DNA-Fe, and bimetallic DNA-Cu/M nanoclusters (NCs). The peroxidase-like activities of these nanomaterials were studied using H2O2 and 3,3',5,5''-tetramethylbenzidine (TMB) as the reaction substrates in HAc-NaAc buffer. It was found that T30-G2-Fe NCs and T30-G2-Cu/Fe NCs, with a size of about 2 nm, exhibit similar and the strongest enzyme-like activity under optimal conditions. Both NCs possess a similarly high affinity to substrates, and the Michaelis-Menten constant (Km) values to TMB and H2O2 are about 11 and 2-3 times lower than those of natural horseradish peroxidase (HRP), respectively. The activity of both nanozymes decreases to about 70% after being kept for one week in pH 4.0 buffer at 4 °C, which is comparable with HRP. Hydroxyl radicals (•OH) are the main reactive oxygen species (ROS) produced in the catalytic reaction. Moreover, both NCs can facilitate in situ generation of ROS in HeLa cells using endogenous H2O2. MTT assays indicate that the T30-G2-Cu/Fe NCs exhibit the strong selective cytotoxicity to HeLa cells over HL-7702 cells. The cellular viability is about 70% and 50% after incubating with 0.6 M NCs for 24 h without or with 2 mM H2O2, respectively. The current study shows that the T30-G2-Cu/Fe NCs have the potential for chemical dynamic treatment (CDT).