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）的潜在新药至关重要。此外，研究海洋生物的呼吸作用对于了解碳循环及其对气候的影响至关重要。有趣的是，在这两种应用中，目前使用的方法都涉及基于化学反应的比色分析，这种化学反应将无色、不可检测的化合物转化为可检测和量化的有色化合物。虽然比色测定法非常流行，但它们有几个缺点。最重要的一点是，最初的化合物是无色的，所以一旦加入到样品中，它们就无法被观察到。因此，假设是否所有海洋生物都能够利用它们，它们如何穿透细胞膜，或者在何处发生转化（细胞内或细胞外），在定量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品，这些产品通常是不溶性晶体，并且检测的固有终点性质使这些检测成为体外工具，但不适用于体内研究的预测性测试。我们设想，荧光测定法可以与之竞争，并且在完全开发后，将取代市场上的比色测定法，成为一种更敏感的分析方法，从而允许对体内分析进行本地化研究。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法，特别是对于不易增殖的细胞或代谢活性低的生物体，以减少测定的时间和成本。我们的目标是开发检测，其中荧光起始化合物可以可视化并与产品区分。检测起始化合物将允许研究它们被不同生物吸收，以及化学过程的精确定位。这将有助于理解当前比色法测定报告的差异。我们的方法将避免需要提取产物进行定量，因为两种化合物的不同荧光发射将允许鉴定和定量，而无需进一步修改样品，这将简化方案并减少测量误差。我们的方案将允许在不同的时间点对研究过程进行实时监测，以实现体内应用。我们已经在东英吉利大学化学学院和环境科学学院之间建立了合作关系，在不同领域的国际专家团队的支持下，合成新的荧光化合物来研究浮游生物的呼吸作用，以了解浮游生物中存在的不同生物体如何对海洋中的呼吸事件做出贡献。我们已经在合成一系列新的荧光化合物方面取得了重大进展。我们的最佳候选物在所有测试的水溶液条件下都能保持荧光，使其成为此类的第一个例子。这一点很重要，因为不同应用的检测需要不同的条件，所以我们的化合物可以用于拟议的应用（海洋科学和生物医学）和其他应用。然而，需要研究以改善该化合物的荧光和物理性质（溶解度）。这是该探路者申请的目标之一，并将通过向东安格利亚大学创新执行小组提交的概念验证申请来支持该申请。为了证明进一步开发分析方案是合理的，我们必须进行市场研究，以了解这类分析的需求水平、最终用户的多样性和真正的商业化潜力。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.