Bridging the Gap in the Development of Gold Nanoparticle-based Enzyme Biosensing

定量测量酶活性是一个巨大的挑战，因为这种活性的产物通常仅通过单个分子键改变。目前的许多方法依赖于荧光类似物的产生，这些荧光类似物在被酶修饰时表现出亮度的增加或减少。然而，这种方法是有问题的，因为添加庞大的荧光基团会改变分子相互作用。此外，为新的应用程序重新设计这些测试通常是一个缓慢而乏味的过程。相比之下，我们已经开发了一系列基于金纳米颗粒独特光学特性的检测方法，这些方法没有这些缺点。通过选择性地对金纳米颗粒表面进行功能化，我们能够使它们响应于酶的激活而聚集或分散。然后，这种物理状态的变化表现为溶液的显着颜色变化。这种性质自然适用于酶活性的定性和定量测量。在过去的几年里，我们已经开发和测试了许多基于这一基本概念的检测方法。迄今为止最成功的两种是蛋白酶测定和脂肪酶测定，两者都具有很大的临床相关性。我们已经探索了使用蛋白酶检测来检测HIV生物标志物，由于蛋白酶参与了许多病毒和寄生虫感染，我们预计将有更多的应用。脂肪酶在医学上也很重要，因为它们在体内的水平可能由于关节炎，急性败血症和某些类型的癌症而变得过高。由于这种广泛的参与，制药行业目前正在努力开发针对脂肪酶的药物。我们的技术的一个巨大优势是，除了诊断疾病，它还可能有助于寻找治疗这些疾病的新药。与简单测量酶浓度的方法相比，我们的方法实际上测量酶活性。我们刚刚开始利用这一能力，通过使用我们的分析来帮助筛选化学库中的新候选药物。在这个后续基金项目的过程中，我们将解决从台式到床边过渡所涉及的一些实际问题。特别是，我们将研究可扩展性、制造和存储，并确定商业前景和未来路线。这些问题通常不会在基础研究层面得到解决，但对于创建实用产品非常重要。在这项工作结束时，我们将处于有利地位，立即将这项技术商业化，为更广泛的医疗保健市场带来好处。

Quantitatively measuring enzyme activity is a great challenge since the product of this activity is often altered by only a single molecular bond. Many current methods rely on the creation of fluorescent analogues that exhibit an increase or decrease in brightness when modified by enzymes. This approach is problematic, though, because the addition of a bulky fluorescent group can change the molecular interaction. Furthermore, it is often a slow and tedious process to redesign these tests for new applications. In contrast, we have developed an array of assays based on the unique optical properties of gold nanoparticles, which do not suffer from these drawbacks.By selectively functionalising the surface of gold nanoparticles, we are able to make them either aggregate or disperse in response to enzyme activation. This change in physical state then manifests itself as a dramatic colour change of the solution. This property naturally lends itself to both qualitative and quantitative measurements of enzyme activity. Over the past several years, we have developed and tested a number of assays based on this basic concept. The two most successful to date have been a protease assay and a lipase assay, both of which are of great clinical relevance. We have explored using the protease assay to test for HIV biomarkers and, since proteases are involved in a number of viral and parasitic infections, we expect that there will be many more applications to come. Lipases are also medically important, as their level in the body can become too high due to arthritis, acute sepsis and certain types of cancer. Because of this wide-ranging involvement, efforts are currently underway in the pharmaceutical industry to develop drugs that will target lipases.One of the great advantages of our technology is the fact that, in addition to diagnosing disease, it can also potentially help in the search for new drugs to treat those diseases. In contrast to methods that simply measure enzyme concentration, our method actually measures enzyme activity. We are just now starting to take advantage of this capability by using our assays to help screen chemical libraries for new drug candidates.In the course of this Follow-on fund project, we will address some of the practical questions involved in the transition from benchtop to bedside. In particular, we will look at scalability, manufacturing and storage and determine the commercial landscape and route forwards. These are concerns that are typically not addressed at the level of fundamental research but are important for the creation of a practical product. At the conclusion of this work, we will be in a strong position to immediately commercialise this technology, bringing its benefits to the broader healthcare market.