II.IV. Dye Reduction Assay

The catalytic activity of CuNPs was determined by a modified protocol of the dye reduction assay technique¹² using methylene blue (MB) as a substrate and sodium borohydride (NaBH₄) as a reducing agent. The assay was performed at room temperature (approximately 25 °C). First, 1 mL of CuNP (from 0.134 mg/mL stock) to obtain a final working concentration of 0.027 mg/mL was mixed with various amounts of MQ water (4990, 4987.5, 4985, and 4980 μL, respectively) to adjust the total volume to 5 mL, after the addition of MB and NaBH₄. In the respective solutions, 10 μL of 5 mM MB from the stock was added and mixed so that the final concentration of MB in the working solution became 10 μM. Then, various freshly prepared concentrations of sodium borohydride (amounts: 0, 2.5, 5, and 10 μL, respectively, from the 10 mM stock) were added into the solution, and the final concentration became 0, 5, 10, and 20 μM respectively. The addition of sodium borohydride shortens the reaction time.¹³ Hence, in our case, the dye reduction took place immediately after the addition of sodium borohydride within 1–5 min. The spectral changes were recorded using a Parkin-Elmer spectrometer over the duration of 60 min in the presence of 10 μM methylene blue and 20 μM sodium borohydride, with 0.027 mg/mL CuNPs as the final concentration. In the other case, spectral changes were recorded by the consecutive addition of 2 μL of CuNPs from the 0.134 mg/mL stock using the same concentration of methylene blue (10 μM) and sodium borohydride (20 μM).