A one-step and eco-friendly approach for the room-temperature synthesis of trypsin-mediated three-dimensional (3D) gold nanoflowers (AuNFs) with high colloidal stability is demonstrated. To prepare AuNFs, ascorbic acid (AA) was quickly added into the premixed solution of HAuCl_4 and trypsin at pH = 5.0. The results show that the molar ratio and feeding order of reactant agents, pH and reaction time play important roles in the formation of NFs. The growth mechanism of AuNFs is suggested as three steps: (1)immobilization of AuCl_4~-ions with a positively charged trypsin template, (2)spontaneous reduction of AuCl_4~-ions with AA insitu and capping Au~0 by 12 cysteines of trypsin, (3)reduction of more AuCl_4~-ions on the Au nuclei formed in the initial stages and anisotropic growth into AuNFs.
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