New Point of Care Diagnostics for Clinical Enzyme Biomarkers

对于护理点诊断和药物开发应用，迫切需要开发测量生物分子相互作用的分析方法。能够快速、高灵敏度地监测酶活性的检测方法具有极大的重要性。从历史上看，酶的检测需要耗时、费力的分离方法来监测底物物理结构的变化，或者使用放射性标记的底物。使用荧光检测技术的不那么麻烦的光学方法已经被开发出来。虽然非常流行，但这些方法通常涉及生物底物与荧光团的衍生化，这可以改变分子相互作用。在此，我们描述了我们团队开发的一种强大的检测方法，用于检测特定类别的酶，磷脂酶（PLs），我们将根据产品在选定患者群体中的商业前演示和临床前验证来优化它，以评估性能和最终用户反馈，并在完全商业开发之前完善演示产品的性能。该项目将由伦敦帝国理工学院（ICL）、帝国创新、哈默史密斯医院和Mologic有限公司合作完成。此前，Stevens教授的团队已经开发了一个强大的平台，用于检测生物医学酶（PLs），该平台基于金纳米颗粒（NPs）与合成肽的功能化。PLs失调是一些危及生命的疾病的特征，如胰腺炎、动脉粥样硬化、急性败血症、关节炎和某些癌症。由于胰腺炎非常难以发现，并且通常需要昂贵的影像学检查，因此我们将重点关注这种疾病，作为关键的初始市场目标。我们希望开发用于揭示PL活性的原型是基于金NPs聚集的比色测定法。金纳米粒子有一个有趣的特性，当它们分散时，会形成一种鲜红色的溶液，当它们聚集时，会变成深蓝色。PL活性是通过脂质体的降解来确定的，脂质体由天然底物脂质形成，填充有NP交联剂（合成肽）。交联剂的释放聚集了金NPs（与互补肽功能化），诱导颜色变化，可以以快速，定量的方式与PL的活性相关。我们的分析的商业化潜力存在于一些明确的卖点中。首先，这种方法使用的底物完全没有经过自然状态的修饰。这种能力不同于任何其他当前的分析，使我们的系统相当灵活，更具生物学相关性。其次，这种测定法测量酶活性而不仅仅是浓度，这意味着它适用于药物筛选应用。最后，在我们的分析中使用的成分是稳定的存储超过四个月。本项目的目标是将我们开发并获得专利的技术扩展到横向流动装置（LFD），并通过模拟实验室和实际生理样品研究LFD的性能。该设备将由Mologic在其工厂设计和制造，性能/优化研究将在ICL进行。这些数据随后将用于重新优化设备的性能，以在使用患者样本的注册前临床评估中满足最终用户的要求。由于目前没有能够检测酶的LFD，该项目将涉及一种新型设备的早期设计。在药物开发和药物筛选领域使用这些技术也存在着重大的商业机会。

For both point of care diagnostic and drug development applications, there is a critical need to develop assays that measure biomolecular interactions. Immense importance is given to assays that enable rapid, high-sensitivity monitoring of enzyme activity. Historically, the detection of enzymes has required time-intensive, laborious separation methods to monitor alterations in the physical structure of a substrate, or the use of radiolabelled substrates. Less cumbersome optical methods have been developed that utilise fluorescent detection technology. Although very popular, these methods usually involve the derivatisation of the biological substrate with a fluorophore, which can alter the molecular interactions. Herein, we describe a powerful assay developed by our group for the detection of a specific class of enzymes, phospholipases(PLs), and we will optimise it in view of a pre-commercial demonstration and a pre-clinical validation of the product in selected patient groups to assess performance and end-user feedback and refine the demonstration product performance ahead of full commercial development.This project will be a collaboration between Imperial College London (ICL), Imperial Innovations, Hammersmith Hospital and Mologic Ltd. Previously, the group of Prof. Stevens has developed a powerful platform for the detection of biomedical enzymes (PLs) based on the functionalisation of gold nanoparticles (NPs) with synthetic peptides. Dysregulation of PLs is a feature of several life-threatening diseases such as pancreatitis, atherosclerosis, acute sepsis, arthritis and some cancers. Since pancreatitis is very difficult to detect, and often requires expensive imaging tests, we will focus on this disease as a key initial market goal. The prototype we wish to develop for revealing PL activity is a colorimetric assay based on the aggregation of gold NPs. Gold NPs have the interesting property that when they are dispersed they form a bright red solution and when they aggregate become deep blue. The PL activity is determined via the degradation of liposomesformed from the natural substrate lipids and filled with a NP crosslinking agent (synthetic peptide). Release of the crosslinker aggregates the gold NPs (functionalised with a complimentary peptide), inducing colour change that can be correlated to the activity of PL in a rapid, quantitative manner.The potential for commercialisation of our assay resides in some clear selling points. Firstly, this method uses substrates that are entirely unmodified from the natural state. This capability is unlike any other current assay and makes our system considerably more flexible and more biologically relevant. Secondly, this assay measures enzyme activity rather than just concentration, meaning that it is appropriate for drug-screening applications. Finally, the components used in our assay are stable in storage for more than four months.The goals of the present project are to scale up the technology developed and patented by us into a lateral flow device (LFD) and to study the performance of the LFD with both simulated laboratory and actual physiological samples. The device will be designed and manufactured by Mologic at their facility and the performance/optimisation study will be carried out at ICL. These data will subsequently be used to re-optimise the performance of the device to meet the end-user requirements in a pre-registration clinical evaluation using patient samples.Since there is currently no LFD capable of detecting enzymes, the project will involve the early stage design of a new type of device. There also exists a significant business opportunity to use these technologies in the field of drug development and drug screening.