Multiplexed AKI biomarker detection with a single molecule biosensor

这项工作计划的关键目标是改善急性肾损伤（AKI）患者的临床结果。AKI是一种无声杀手，发生在急性患者中，并与不良结局相关，包括住院时间延长、死亡率增加和长期不良结局，包括慢性肾脏疾病和心血管死亡。多个报告也描述了AKI与COVID-19的关联。最近的一份出版物发现，入院的COVID-19患者中有36.6%患有AKI， 14.3%需要透析，35%的患者死亡。AKI发生早期，与呼吸衰竭有时间关联。在英国，在COVID-19大流行的第一波期间，重症监护病房患者的透析供应被推到了极限。我们提出的解决方案是一种全新的诊断设备，能够检测与AKI相关的两种生物标志物，c反应蛋白（CRP）和中性粒细胞明胶酶脂钙蛋白（NGAL）。这些生物标志物可以在几个小时内响应个体AKI疾病状态的变化而增加100倍以上，使其成为AKI早期诊断的重要候选物。该设备采用纳米孔技术，使用手持设备在几分钟内检测生物液体中这些生物标志物的存在。纳米孔技术允许一次计数一个生物标记物，因为它对通过纳米孔的单个分子很敏感，这是与竞争技术相比的一个关键优势，竞争技术通常需要在处理信号之前检测数万亿的分析物。使用纳米孔技术检测生物标记物仍然具有挑战性，因为该技术无论分析的生物标记物是什么都能提供相同的信号，这在血液等生物液体中进行测试时是一个特别的问题。该项目是基于我们最近的发现，该发现允许对单个生物标记物的信号进行指纹识别，同时提高了在血液等生物流体中的测试性能。我们的发现采用DNA折纸作为载体，能够结合感兴趣的生物标志物，并提高其检测与纳米孔平台。DNA折纸是一种可以将长DNA分子折叠成坚固的几何形状的技术，类似于日本的折纸艺术。我们设计了一种形状像相框的DNA折纸，能够捕获空洞内的AKI生物标志物（CRP）。这种特殊的设计产生了一个独特的纳米孔信号，使我们能够计算每个DNA折纸捕获了多少生物标记物。这种方法使我们能够在几分钟内测量生物流体中生物标志物的浓度。在这项工作计划中，我们将进一步完善这项技术，使其能够同时检测两种对AKI早期诊断重要的生物标志物（CRP和NGAL）。我们还将扩大可以用我们的技术分析的生物标志物的范围，以便将来可以用于治疗其他疾病。我们将与一家领先的仪器制造商合作，提供一种原型仪器，该仪器将与NHS专家合作在临床环境中进行测试。除了技术开发工作外，我们还将通过定义该技术的临床用例来促进AKI的早期检测，从而展示这种新颖且高度创新的方法的临床相关性。同时，我们将开发一个早期的经济模型来证明我们提出的诊断设备的临床和经济后果。

The key objective of this programme of work is to improve the clinical outcomes for patients affected by Acute Kidney Injure (AKI). AKI is a silent killer, which occurs in acutely ill patients and is associated with poor outcomes, which include increased length of hospital stays, increased mortality, and long-term adverse outcomes including chronic kidney disease and cardiovascular mortality. Multiple reports have also described the association of AKI with COVID-19. A recent publication identified that 36.6% of patients admitted to hospital with COVID-19 developed AKI, with 14.3% requiring dialysis and 35% of patients dying. AKI occurs early and in temporal association with respiratory failure. In the UK, during the first wave of the COVID-19 pandemic, the provision of dialysis to patients on the ICUs was pushed to the limit.Our proposed solution is a radically new diagnostics device able to detect two biomarkers linked to AKI, C-reactive protein (CRP) and Neutrophil Gelatinase Lipocalin (NGAL). These biomarkers can rise by more than 100-fold within a matter of hours in response to changes in an individual's AKI disease state, making them very important candidates for early diagnosis of AKI. The device employs nanopore technology to detect the presence of these biomarkers in biological fluids within minutes using a hand-held device. Nanopore technology allows the counting of biomarkers one at the time as it is sensitive to just a single molecule passing through the nanopore which is a key advantage over competing technologies that often require the detection of trillions of analytes before a signal can be processed.The detection of biomarkers using nanopore technology is still challenging because the technology provides the same signal regardless of the biomarker analysed which is a particular problem when the test is carried out in biological fluids like blood.This project is based on our recent discovery that allowed to fingerprint the signal of individual biomarkers while enhancing the performance of the test in biological fluids such as blood. Our discovery employed DNA origami as carriers able to bind the biomarker of interest and improve its detection with the nanopore platform. DNA origami is a technique that allows to fold long DNA molecules into a robust geometry of choice, similar to the Japanese art of paper origami. We have designed DNA origami shaped like a picture frame able to capture AKI biomarkers (CRP) inside the void. This particular design generates a unique nanopore signal allowing us to count how many biomarkers were captured per DNA origami. This approach allowed us to measure the concentration of biomarkers in biological fluids in a matter of minutes.Within this programme of work we will further refine this technology to allow the simultaneous detection of two biomarkers important for the early diagnosis of AKI (CRP and NGAL). We will also expand the range of biomarkers that can be analysed with our technology so that it could be use to tackle other diseases in the future. We will have partnered with a leading instrument manufacturer to deliver a prototype instrument that will be tested in clinical settings in collaboration with NHS experts. Alongside the technology development work, we will also demonstrate the clinical relevance of this novel and highly innovative approach by defining a clinical use case for the technology to facilitate the early detection of AKI. In parallel, we will develop an early economic model to demonstrate the clinical and economic consequences of our proposed diagnostic device.