Cell-Based Biosensor to Report DNA Damage in Micro- and Nanosystems

Abstract

Understanding how newly engineered micro- and nanoscale materials and systems that interact with cells impact cell physiology is crucial for the development and ultimate adoption of such technologies. Reports regarding the genotoxic impact of forces applied to cells in such systems that can both directly or indirectly damage DNA emphasize the need for developing facile methods to assess how materials and technologies affect cell physiology. To address this need we have developed a TurboRFP-based DNA damage reporter cell line in NIH-3T3 cells that fluoresce to report genotoxic stress caused by a wide variety of agents, from chemical genotoxic agents to UV-C radiation. Our biosensor was successfully implemented in reporting the genotoxic impact of nanomaterials, demonstrating the ability to assess size dependent geno- and cyto-toxicity. The biosensor cells can be assayed in a high throughput, noninvasive manner, with no need for overly sophisticated equipment or additional reagents. We believe that this open-source biosensor is an important resource for the community of micro- and nanomaterials and systems designers and users who wish to evaluate the impact of systems and materials on cell physiology.

Assessment of a panel of interleukin-8 reporter lung epithelial cell lines to monitor the pro-inflammatory response following zinc oxide nanoparticle exposure under different cell culture conditions

“…Although particularly fast-maturing and bright variants of fluorescent proteins (TurboGFP/RFP) were used, it seems likely that the accumulation time still exceeded the time needed to produce detectable amounts of luciferase since the fluorescent proteins lack the multiplication effect of Luc that is active as an enzyme. Consistent with this, assessment of fluorescent reporter expression is typically performed after exposure for 12 hours and longer [ 15 , 17 ]. Furthermore, the vector for luciferase differed from the ones used for the fluorescent proteins, which can also influence responsiveness, e.g.…”

“…Additional studies using reporter constructs, such as for markers of DNA damage and oxidative stress [ 15 , 16 ], were mostly validated indirectly, e.g. by comet assay or ROS measurements, rather than a specific assessment of the actual gene of interest by several endpoints.…”

“…Fluorescence-based reporter cells have also recently been used to detect additional biochemical endpoints, including oxidative stress and genotoxicity. For example, Fendyur and colleagues assessed the ability for Ag-NPs to induce reactive oxygen species (ROS)-associated DNA damage in NIH-3 T3 cells [ 15 ], whilst Karlsson et al [ 16 ] investigated the impact of copper (CuO), zinc (ZnO) and nickel oxide (NiO) NP exposure on mouse embryonic stem cells using green fluorescent protein (GFP) to quantify DNA damage and oxidative stress associated with metal oxide-induced cytotoxicity. Furthermore, the adaptability of fluorescence-based reporter cell lines has been highlighted in regards to their culture conditions.…”

High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system

“…Rider et al reported the first cell sensor that used B lymphocytes to recognize specific bacteria with the help of membrane-bound IgM antibodies32. Mammalian cell-based biosensors have a distinct advantage in reflecting cellular physiological action rather than just quantitative detection and therefore can provide insight into mechanism of action of analytes333435. More importantly, tests based on human cells rather than other cell types, are more likely to detect contaminants that can cause adverse effects in humans.…”

“…Though prior work has examined the gross effects (e.g., viability, proliferation) of analytes on cells313637, these gross assays do not reveal more subtle effects that mask or alter particular phenotypes of interest, such as the activation of signaling pathways. Recently, methods based on the reporting of analytes by fluorescence reaction in engineered cells offer a simple and nondestructive option33343538. Living cells used as biosensors are typically produced with a constructed plasmid, in which genes that code for the bio-reporter are placed under control of a promoter that recognizes the analyte of interest.…”