Bioluminescence Sensing in 3D Spherical Microtissues for Multiple Bioactivity Analysis of Environmental Samples

Abstract

The development of predictive in vitro sensing tools able to provide rapid information on the different bioactivities of a sample is of pivotal importance, not only to monitor environmental toxicants, but also to understand their mechanisms of action on diverse molecular pathways. This mechanistic understanding is highly important for the characterization of toxicological hazards, and for the risk assessment of chemicals and environmental samples such as surface waters and effluents. Prompted by this need, we developed and optimized a straightforward bioluminescent multiplexed assay which enables the measurement of four bioactivities, selected for their relevance from a toxicological perspective, in bioluminescent microtissues. The assay was developed to monitor inflammatory, antioxidant, and toxic activity, and the presence of heavy metals, and was successfully applied to the analysis of river water samples, showing potential applicability for environmental analyses. The assay, which does not require advanced equipment, can be easily implemented in general laboratories equipped with basic cell culture facilities and a luminometer.

A Luciferase Mutant with Improved Brightness and Stability for Whole-Cell Bioluminescent Biosensors and In Vitro Biosensing

“…Dose-response curves for TNFα were obtained with both monolayer cultures ( Figure 5 a) and spheroids ( Figure 5 b), obtaining EC 50 values of 10.1 ng/mL and 35.2 ng/mL, respectively. In agreement with previous reports by others [ 48 ] and us [ 38 , 46 ] a higher NF-kB basal activation (about 3 fold) was found in 3D spheroids than the 2D format; this is probably due to the presence of an intra-spheroid cytokine signaling that induces JNK and NFkB pathways. The results were compared with that obtained with the Promega commercial plasmid reporter (EC 50 values of 15.3 and 60 ng/mL for 2D cultures and spheroids, respectively) confirming that the BgLuc enzyme has great potential as a BL reporter and in imaging applications.…”

“…No change in red-shifting in emission spectra was observed with the BgLuc luciferase (λmax = 560 nm), confirming its suitability for dual luciferase applications. On the contrary, as previously reported by us, Luc2 shows a marked red-shifting using D-luciferin (λmax = 623 nm), less pronounced (λmax = 608 nm) with the lysing Bright-Glo™ commercial substrate [ 46 ].…”

Illuminating Progress: The Contribution of Bioluminescence to Sustainable Development Goal 6—Clean Water and Sanitation—Of the United Nations 2030 Agenda

“…Recently, a multiplexed assay based on BL 3D spherical microtissues was developed for monitoring the presence of heavy metals and for evaluating the inflammatory, antioxidant, and toxic activities of environmental samples. The assay was successfully applied to the analysis of superficial and transitional water samples, allowing us to obtain measurements of four bioactivities in a single assay, albeit still based on mammalian cell models grown in laboratory settings equipped with cell culture facilities [ 76 ].…”

Special Issue “Feature Papers in Biosensors Section 2022”

“…3D-printing leads to low-cost biosensors, such as an immunosensor for the rapid detection of Escherichia coli bacteria [ 2 ]. A multiplexed bioluminescent assay based on 3D microtissues was performed for monitoring inflammatory, antioxidant bioactivities, presence of heavy metals and toxicity [ 9 ]. After an overview of the important biomarkers for several common children cancers, the developed biosensors for early detection of pediatric cancer are enumerated [ 10 ].…”