Fig. 2. Highly efficient and biocompatible click reaction in living cells. (a) Effect of solution pH values on NCBT–Cys click reaction. Reaction conditions: 10 μM Cys and 200 μM NCBT in pure H₂O solution at 37 °C for 1 h; formic acid was used to regulate the solution pH values. (b) NCBT concentration optimization experiment. HepG2 cells (approximately 1 × 10⁵) were incubated with different concentrations of NCBT (50–800 μM) in serum-free culture medium at 37 °C for 1 h, and then, the cells were washed with PBS solution and lysed in 100 μL pure H₂O solution. (c) NCBT incubation time optimization experiment. HepG2 cells (approximately 1 × 10⁵) were incubated with 200 μM NCBT in serum-free culture medium at 37 °C for different times (15–120 min), and then, the cells were washed with PBS solution and lysed in 100 μL pure H₂O solution. (d) MTT assay of the effect of NCBT on HepG2 cells. The MTT proliferation assay estimated the cell viability (%) of HepG2 cells cultured in the presence of 200 μM NCBT at 37 °C for 30, 60, and 90 min. All error bars denote s.d.; n = 3.