Fluorometric Tetrazolium Salt/Formazan Assays for Histochemistry and Biomedical Applications

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.

We have been working towards the development of a novel fluorescent method to track the uptake of redox dyes by a representative range of plankton organisms with different cell membranes to quantify how different cells take it up, which has important implications in the quantification of respiration and therefore CO2 production in the oceans. However, our current method does not allow to track the full process occurring in the biological systems, reducing the possible applications.
In the proposed Follow-On project, we will build upon our results in order to develop a fully fluorometric assay that can be applied not only to marine science but also to other areas of research, in particular biomedical research and drug discover, increasing the value of our approach and opening the possibility of commercialisation in several areas. Results from the Pathfinder Market Research and Proof of Concept will allow full exploitation of the technology with impact in the following areas:

1. Impact on Economy
Exploitation of the new assays in the commercial setting will open future possibilities of R&D investment, which will contribute to the UK's competitiveness. Results from this Pathfinder application will give us the crucial information needed in order to develop the Follow-On project towards commercialisation of our assays.
We envision developing commercial prototypes by the end of the Follow-On project influenced by the needs of the end-users, which will be fully identified by the market research.
Informed by the market research findings and in close collaboration with our IP department at UEA we will design the best strategy for commercialisation. We will consider commercial and intellectual property rights in order to file a patent of our compounds and assays and we will contemplate the creation of a spin-off company within UEA for the synthesis and manufacture of the fluorescent compounds to sell to other companies or end-users.

2. Impact on Society
The new fluorometric assays proposed in this project will have a high longterm impact on Society in the long run.
The results from our assays developed in the marine science setting will be of interest to policy makers interested in climate change, as the understanding of how different marine organisms contribute to the production of CO2 that is released to the atmosphere can have serious implications in our understanding of climate change.
Besides, our technology could be applied by commercial companies as a new method to differentiate active and dormant bacteria in water samples, which will have large implications for water quality monitoring, essential to our society.
As an add-on to the impact on environmental sciences, our assays have the potential to impact medical science. We envision that our fluorometric assays could replace current colorimetric commercial assays used for drug toxicity assessments and in the long term could influence drug discovery in collaboration with pharmaceutical companies, hospitals and medical research centres, which will therefore have a substantial impact in the treatment of relevant diseases in our Society.

3. Impact on people
In addition to direct commercial potential, this project will contribute to the development of current and new collaborations between groups at UEA and internationally, as well as further career development for academic staff and PDRAs.
The team involved will be exposed to the commercial aspects of the research. Therefore, this project will serve as a platform to transfer all the advanced knowledge and research skills from the academic setting to the market towards new and exciting adventures as entrepreneurs with benefits to the team involved, the University, the UK economy and Society.