First, 2-nm Au-TIOP NPs were synthesized using a previously reported method (25). Typically, 6 ml of methanol, 1 ml of acetic acid, and 5 ml of 1% HAuCl4 were mixed and kept stirring for 5 min, and 64 mg of tiopronin powder was added under vigorous stirring. After the solution changed color to nearly colorless, NaBH4 solution (100 mg in 500 μl of ice-cold water) was added into the above mixture under vigorous stirring. The reaction was terminated after 3 hours, and the resulting mixture was dialyzed (dialysis membrane, Solarbio; molecular weight cutoff, 8000 to 14,000 Da) against Milli-Q water for 72 hours, which was changed every 8 hours. Two-nanometer Au-POY2T NPs were obtained by replacing the tiopronin on the surface of Au-TIOP NPs with thiol-modified oligonucleotides (SH-POY2T: SH-C6-AAAAAATGGGT GGGTGGTTTGTTTTTGGG) through a ligand exchange method (26). SH-POY2T sequence was first treated by 5 mM Tris-(2-carboxyethyl)-phosphine (TCEP) for 30 min in phosphate-buffered saline (PBS) buffer. Then, 2-nm Au-TIOP NPs were added and kept stirring at room temperature for 24 hours. The exchange efficiency was determined by gel electrophoresis, and the intensity of the band indicates that ~50% POY2T were conjugated to the 2-nm Au NPs.

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