Single-molecule DNA biosensors for rapid microbial detection

人类、动物和植物群体的健康由于受到诸如细菌和病毒的大量病原微生物的感染而不断受到威胁。病原体能够迅速传播并危害人类，这一事实也导致了各种生物武器的发展，这些武器不仅威胁到战场上的士兵，而且甚至威胁到地铁站或机场等良好公共环境中的平民。对平民的威胁还包括来自非恶意来源的威胁，如医疗保健（医院获得性感染，如超级细菌MRSA）或来自食品工业（致病性大肠杆菌污染的食品）。coli）。快速诊断此类感染的能力将极大地帮助患者生存和预后，并防止疾病的进一步传播。在我们的提案中，我们描述了一种基于“单分子”荧光的诊断解决方案，用于快速识别多种病原体。我们已经建立了一个针对特定细菌菌株的DNA存在的基本测试，并旨在以此为基础开发一系列基于布尔逻辑和信号放大的“智能”生物传感器。本质上，这样的传感器将能够在给定目标输入的组合（例如，如果（病原体1和病原体2）但（不是病原体3））的情况下提供关于病原体威胁水平的是/否回答。我们的传感器的目标是在10-15分钟的时间内产生结果，而目前的技术由于样品扩增的要求（无论是细菌培养还是DNA扩增）而在数小时到数天之间变化。同时，我们建议进一步开发一种紧凑且经济实惠的单分子荧光显微镜来执行此类测试。目前单分子显微镜在尺寸和成本方面都令人望而却步;我们的目标是生产一种占地面积约为30 cmx 50 cmx 20 cm（适合台式操作）的精简版，其成本仅为全尺寸显微镜的一小部分。

The health of human, animal and plant populations is under constant threat due to infections by a large variety of pathogenic microorganisms, such as bacteria and viruses. The fact that pathogens are able to spread rapidly and harm human populations has also led to the development of a variety of biological weapons which threaten not only soldiers in the field of operations, but even civilian populations in benign public environments such as a tube stations or airports. Threats to civilians also include those from non malicious sources such as within healthcare (hospital acquired infections, e.g. the superbug MRSA) or from the food industry (contaminated foods with pathogenic E. coli). The ability to diagnose such infections rapidly would dramatically aid patient survival and outcome and prevent further spreading of the disease.In our proposal we describe a diagnostic solution based on 'single-molecule' fluorescence for the rapid identification of multiple pathogens. We have already established a basic test for the presence of DNA specific to a particular bacterial strain and aim to build on this to develop a range of 'intelligent' biosensors based on Boolean logic and signal amplification. Essentially, such sensors will be able to provide a yes/no answer on pathogen threat level given a combination of target inputs e.g. if (pathogen 1 AND pathogen 2) but (NOT pathogen 3). Our sensors aim to produce a time-to-result on the order of 10-15 min, compared with current technologies that vary between hours and days due to the requirement of sample amplification - either bacterial culturing or DNA amplification.In parallel, we propose to further develop a compact and affordable single-molecule fluorescence microscope to perform such tests. Currently single-molecule microscopes are prohibitive in terms of size and cost; we aim to produce a cut-down version with a footprint of approximately 30cmx50cmx20cm (suitable for benchtop operation) and a small fraction of the cost of the full-size microscopes.