A homogenous bimodal (immuno/PCR) pathogen detection system based on a bio-nanoparticle

自古以来，军队就试图使用生物武器来击败敌人。早期的例子包括用死动物污染水源，或者将瘟疫缠身的尸体弹射到被围困城市的城墙上。近年来，生化武器已成为流氓国家和恐怖组织关注的焦点。其中最引人注目的可能是2001年美国发生的导致5人死亡的炭疽袭击。自1975年英国宣布放弃使用生物武器以来，它加大了对此类生物战剂的探测力度。在这个项目中，我们的目标是开发一种简单的快速系统，可以更快、更有效地探测生物武器，从而改善英国的防务和安全。我们将开发的系统是对我们在伯明翰率先开发的现有病原体检测系统的补充。现有的系统依靠病毒来检测选择的病原体。这些病毒具有意大利面状结构，这意味着当含有它们的溶液被搅拌或强行通过狭窄的管道时，它们会相互排列。我们对这些病毒进行了改造，使它们也能够特异性地附着在病原体上；当与病原体结合时，病毒就不能再排列。这些病毒的修饰及其在实验中的应用是合成生物学这一新兴科学领域的核心部分。这门新学科旨在使用生物材料来提高日常设备的性能。在这种情况下，我们正在使用合成生物学来增强生物战探测。该试验包括检测病毒排列的变化，并将这些信息转化为士兵或安全人员可以读取的信号。合成生物学的这一领域提出了一个重大挑战，因为我们需要将来自病毒的信号（长度不到100万分之一米）转化为可读取的信号。为了实现这一点，我们使用了一种简单但尚未充分利用的方法（线性二色光谱），该方法能够检测病毒是否排列一致。如果它们排列整齐，那么就没有病原体，如果它们没有排列整齐，那么就有病原体。进行该化验的方法具有许多有用的特性，使其非常适合安全部门使用。该检测方法简单、快速，无需实验室设备即可进行检测。该分析还可以配置，以便在一次分析中可以检测到一种以上的病原体。这一点很重要，因为需要同时检测到各种各样的生物战剂。然而，不幸的是，我们目前的方法只能使用一种可用的病原体检测模式，基于使用抗体，以进行检测。在这个项目中，我们的目标是通过在我们的系统中添加第二种检测模式来解决这一不足。这种模式包括直接检测和识别来自生物战剂的DNA。为了将这种方法添加到我们的系统中，我们必须将与生物战剂相匹配的DNA片段添加到病毒表面。这些DNA片段特别粘附在生物战剂中的DNA上，防止病毒对齐，从而给我们一个信号。如果这是成功的，我们还打算增加第二步，使用一种酶来放大信号（在一种称为PCR的方法中），这将使整个系统更加敏感。在项目的最后阶段，我们将结合DNA为基础的分析与抗体为基础的分析，以产生一个系统，是更有效，更灵活，更不容易产生错误的结果比任何当前的系统。通过这种方式，我们还将展示合成生物学在检测应用中的第一个用途。

Since ancient times armies have attempted to employ biological weapons in order to defeat a foe. Early examples involved tainting water supplies with dead animals or catapulting plague ridden corpses over the walls of besieged cities. In more recent times biological and chemical weapons have become the focus of rogue states and terrorist organisations. Perhaps most high profile of these was the anthrax attacks that occurred in the US in 2001 killing 5 people. Since the UK renounced the use of biological weapons in 1975 it has increased efforts to improve detection of such biowarfare agents. In this project we aim to develop a simple rapid system that will detect biological weapons more quickly and effectively, improving the defence and security of the UK.The system that we will develop is an addition to an existing pathogen detection system that we have pioneered in Birmingham. The existing system relies on viruses to detect the pathogen of choice. These viruses have a spaghetti like structure which means they align with respect to one another when solutions containing them are stirred or forced through narrow tubes. We have modified these viruses so that they are also able to adhere specifically to pathogens; when bound to pathogens the viruses are no longer able to align. The modification of these viruses and their use in an assay is a core part of a new area of science called synthetic biology. This new discipline aims to use biological materials to enhance the performance of everyday devices. In this case we are using synthetic biology to enhance biowarfare detection.The assay involves detecting the changes in the alignment of the viruses and translating this information into a signal that can be read by a soldier or security operative. This area of synthetic biology poses a significant challenge as we need to translate a signal from the virus (which is less 1,000,000th of a metre long) into a signal that can be read. To achieve this we use a simple, yet underused, method (linear dichroism spectroscopy) that is able to detect whether the viruses are aligned or not. If they are aligned then there is no pathogen present, if they are not aligned then the pathogen is present. The method for carrying out the assay has a number of useful characteristics that makes it ideally suited for use by the security services. The assay is simple and very quick allowing the it to be carried out without the need for laboratory equipment. The assay can also be configured so that more than one pathogen can be detected in a single assay. This is important as there are a wide range of biowarfare agents that need to be detected at the same time. Unfortunately, however our current method is only able to use one of the available pathogen detection modes, based on using antibodies, in order to carry out detection.In this project we aim to address this insufficiency by adding a second detection mode to our system. This mode involves directly detecting and identify the DNA from the biowarfare agent. To add this method to our system we have to add small pieces of DNA that match those in biowarfare agent to the surface of the virus. These DNA fragments specifically stick to the DNA in the biowarfare agent preventing the virus from aligning and hence giving us a signal. If this is successful we also aim to add a second step that uses an enzyme to amplify the signal (in a method called PCR) which will make the whole system much more sensitive. In the final stage of the project we will combine the DNA based assay with the antibody based assay to produce a system that is more effective, more flexible and less prone to false results than any current system. In this way we will also demonstrate one of the first uses of synthetic biology in a detection application.