Fluorometric Tetrazolium Salt/Formazan Assays for Histochemistry and Biomedical Applications

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Assessing the toxicity of new compounds is essential in order to identify new potential drugs to treat existing (e.g. cancer) and new diseases (e.g. influenza pandemic). In addition, the study of respiration of marine organisms is crucial to understand the carbon cycle and how it affects the climate. Interestingly, in both applications, the methods currently used involve colorimetric assays based on a chemical reaction that will transform a colourless, non-detectable compound into a coloured one that can be detected and quantified. Although colorimetric assays are very popular, they suffer from several drawbacks. The most important one is that the starting compounds are colourless, so they cannot be visualised once added to the sample. Therefore, assumptions on whether all marine organisms are able to use them, how they penetrate through the cell membrane, or where the transformation takes place (inside or outside the cell), can lead to erroneous results when quantifying the production of the coloured products. Besides, the need to solubilise the coloured products, which are generally insoluble crystals, prior to analysis, and the inherent endpoint nature of the assay makes these assays an in vitro tool but are not useful as predictive tests for in vivo studies.We envision that fluorometric assays can compete and when fully developed, will replace colorimetric assays in the market as a more sensitive method for analysis that will allow localisation studies towards in vivo assays. Therefore, our aim is to develop fluorometric assays with higher sensitivities and dynamic range, particularly with cells that do not readily proliferate or organisms with low metabolic activity, to reduce the time and cost of the assay. We aim to develop assays where the fluorescent starting compounds could be visualised and distinguished from the product. Detecting the starting compounds will allow the study of their uptake by different organisms, as well as the exact localisation of the chemical process. This will help to understand the discrepancies reported for current colorimetric assays. Our method will avoid the need to extract the products for quantification, as the different fluorescence emissions of the two compounds will allow identification and quantification without further sample modification, which will simplify the protocols and reduce error in the measurements. Our protocol will allow monitoring of the process at different timepoints for the study in real time towards in vivo applications. We have already established collaboration between the School of Chemistry and the School of Environmental Sciences at UEA supported by an international team of experts in different areas towards the synthesis of new fluorescent compounds to study plankton respiration to understand how different organisms present in the plankton contribute to respiration events in the ocean. We have already made significant progress in the synthesis of a series of new fluorescent compounds. Our best candidate retains fluorescence under all tested aqueous conditions, making it the first example of this kind. This is important, as assays for different applications need different conditions, so our compound could be used in both proposed applications (marine science and biomedicine) and others. Research is required, however, to improve on the fluorescence and physical properties (solubility) of the compound. This is one of the objectives of this Pathfinder application and will be supported by a Proof of Concept application submitted to the UEA Innovation Executive panel in parallel to this application. In order to justify further development of the assay protocol, we must undertake market research to understand levels of demand for this type of assay, the diversity of end-users and the true potential for commercialisation. This is the main objective of this Pathfinder application.