Reagents to Enhance Detection of Raw and Biologically Transformed Nanomaterials.

摘要

Engineered nanomaterials (eNMs) are widely exploited in many technology fields (e.g. medicine, energy, automotive, military) promising great benefits to mankind. They are also formulated into an ever expanding consumer product market. The increasing use of engineered nanomaterials in industrial and consumer applications has raised concerns about their environment health safety (EH&S). A particular concern is for occupational exposure to raw materials in the form of highly dispersed spherical and fiber-like shapes. Researchers seek to quantify nanomaterial interactions with biological systems and to develop predictive models of nanoparticle EH&S risk. However, detection of eNMs in the environment and in biological systems is challenged by limitations associated with commonly used analytical techniques. In this project we undertook an effort to overcome this challenge by developing a set of reagents (NProbes) that can be used to enhance the detection of eNMs in biological tissues. Phage display methodology was used to discover scFv antibodies that bind NPs dispersed in solution. The scFv antibodies were engineered with a peptide FLAG tag (DYKDDDDKL) to enable secondary detection/amplification of eNM presence in tissue sections using standard immunohistochemistry (IHC) staining with an enzymatic reporter. We successfully generated and characterized NProbes for detecting fluorescent quantum dots (QDs) and nonfluorescent titanium dioxide (TiO2) NPs in human skin using in vitro and ex vivo models. We investigated their cross reactivity binding to similar and dissimilar materials and proved that NProbes can detect QDs that could not be imaged above background tissue autofluorescence. Our proof-of-principle work as demonstrated that with continued development and refinement NProbes can be generated to detect NPs that vary in composition, size, and surface coating thereby comprising a powerful tool kit that can be used to advance nanotoxicology research and to develop a mechanistic understanding of the fate and transport of NPs in biological and environmental systems.