Fluorometric Tetrazolium Salt/Formazan Assays for Histochemistry and Biomedical Applications

评估新化合物的毒性对于确定治疗现有疾病（如癌症）和新疾病（如流感大流行）的新的潜在药物至关重要。此外，研究海洋生物的呼吸作用对于了解碳循环及其如何影响气候至关重要。有趣的是，在这两种应用中，目前使用的方法涉及基于化学反应的比色测定，该化学反应将无色、不可检测的化合物转化为可以检测和定量的有色化合物。尽管比色测定非常流行，但它们具有若干缺点。最重要的一点是，起始化合物是无色的，因此一旦加入样品中，它们就无法可视化。因此，假设是否所有海洋生物都能够使用它们，它们如何穿透细胞膜，或者转化发生在哪里（细胞内或细胞外），在量化有色产品的生产时可能会导致错误的结果。此外，在分析之前需要溶解有色产物（其通常是不溶性晶体），以及测定的固有终点性质使这些测定成为体外工具，但不能用作体内研究的预测性测试。将取代市场上的比色测定法，作为一种更灵敏的分析方法，允许对体内测定进行定位研究。因此，我们的目标是开发具有更高灵敏度和动态范围的荧光测定，特别是对于不易增殖的细胞或具有低代谢活性的生物体，以减少测定的时间和成本。我们的目标是开发荧光起始化合物可以可视化并与产品区分开的检测方法。检测起始化合物将允许研究它们被不同生物体的吸收，以及化学过程的精确定位。这将有助于理解当前比色测定报告的差异。我们的方法将避免提取用于定量的产物的需要，因为两种化合物的不同荧光发射将允许鉴定和定量而无需进一步的样品修改，这将简化方案并减少测量中的误差。我们的方案将允许在真实的时间内对研究的不同时间点的过程进行监测，以实现体内应用。我们已经在化学学院和环境科学学院之间建立了合作，由不同领域的国际专家团队支持，致力于合成新的荧光化合物来研究浮游生物呼吸，以了解浮游生物中存在的不同生物如何促进海洋中的呼吸事件。我们已经在一系列新的荧光化合物的合成方面取得了重大进展。我们最好的候选物在所有测试的水性条件下都保持荧光，使其成为此类的第一个例子。这一点很重要，因为不同应用的测定需要不同的条件，因此我们的化合物可用于拟议的应用（海洋科学和生物医学）和其他应用。然而，需要进行研究，以改善该化合物的荧光和物理性质（溶解度）。这是本探路者应用程序的目标之一，并将通过概念验证应用程序提交给UEA创新执行小组在此应用程序的同时支持。为了证明进一步开发检测方案的合理性，我们必须进行市场研究，以了解对此类检测的需求水平、最终用户的多样性和商业化的真正潜力。这是这个Pathfinder应用程序的主要目标。

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.