Novel dual labelled nanoprobes for nanosafety studies: Quantification and imaging experiment of CuO nanoparticles in C. elegans

Metal oxide nanoparticles have been extensively studied for their toxicological impacts. However, accurate tracing/quantification of the nanomaterials and their biological responses are difficult to measure at low concentrations. To overcome the challenge, we developed a dual-labelling technique of CuO nanoparticles with a stable isotope of ⁶⁵Cu, and with rhodamine dye. In vivo experiments on C. elegans were performed using natural feeding of Rhodamine B isothiocyanate-(3 aminopropyl) triethoxysilane functionalized ⁶⁵CuO nanoprobes (RBITC-APTES@⁶⁵CuO) (size = 7.41 ± 1 nm) within the range of Predicted Environmental Concentration (PEC) of CuO nanoparticles in soil and sediments. Fluorescence emission (570 nm) was detected in the lumen of the intestine and the pharynx of C. elegans with no impact of nanoparticle exposure on the brood size and life span of worms. The ingested fluorescent labelled RBITC-APTES@⁶⁵CuO nanoprobes did not enter the reproductive system and were distributed in the alimentary canal of C. elegans. Strong fluorescent signals from the ingested RBITC-APTES@⁶⁵CuO nanoprobes were achieved even after 24 h of exposure demonstrating the high stability of these nanoprobes in vivo. The net accumulation measured of ⁶⁵Cu in C. elegans after background subtraction was 0.001 μg mg⁻¹ (3.52 %), 0.005 μg mg⁻¹ (1.76 %) and 0.024 μg mg⁻¹ (1.69 %) for an exposure concentration of 0.0284 μg mg⁻¹, 0.284 μg mg⁻¹, and 1.42 μg mg⁻¹ of ⁶⁵Cu, respectively. Using C. elegans as a model organism, we demonstrated that RBITC-APTES tagged ⁶⁵CuO nanoparticles acted as novel nanoprobes for measuring the uptake, accumulation, and biodistribution through quantification and imaging the nanoprobes at a very low exposure concentration (⁶⁵CuO concentration: 0.033 μg mg⁻¹).