Improved protein determination assays obtained after substitution of copper sulfate by copper oxide nanoparticles

Copper oxide nanoparticles (nano CuO) provide Cu²⁺ ions which can be easily harnessed for protein determination as an alternative to the use of copper sulfate (CuSO₄). In the present work, nano CuO of size <25 nm were substituted for CuSO₄ in two of the well-known protein assays viz. Lowry method and bicinchoninic acid (BCA) method. Use of nano CuO in the Lowry's assay had no effect on the assay time (30 min) but significantly lowered the limit of detection (LOD) from 0.01 to 0.001 μg/ml, while the BCA method when performed using nano CuO resulted in notable reduction of not only the assay time from 30 to 20 min but also the LOD from 0.1 to 0.001 μg/ml. Nano CuO based protein determination in the human serum and urd bean seeds extract produced reliable, reproducible and consistent results. Nano CuO also alleviated the inhibition of both the methods by common interfering substances such as ammonium sulfate, glucose, EDTA, SDS, Triton X-100, dithiothreitol and 2-mercaptoethanol. Hence, successful modification and improvement of Lowry and BCA methods by substitution of CuSO₄ with nano CuO for protein determination has been demonstrated.