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供水，或者在被围困的城市的城墙上弹射携带瘟疫的身体。最近，生物和化学武器已成为无赖国家和恐怖组织的焦点。其中最引人注目的可能是2001年在美国发生的炭疽热袭击，造成5000人死亡。自1975年联合王国放弃使用生物武器以来，它加大了努力，以改进对这类生物武器制剂的检测。在这个项目中，我们的目标是开发一个简单快速的系统，能够更快、更有效地检测生物武器，改善英国的防御和安全。我们将开发的系统是我们在伯明翰首创的现有病原体检测系统的补充。现有的系统依赖于病毒来检测所选的病原体。这些病毒具有类似意大利面的结构，这意味着当含有它们的溶液被搅拌或强迫穿过狭窄的管子时，它们彼此对齐。我们已经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；当它们与病原体结合时，病毒不再能够比对。对这些病毒的修饰和它们在化验中的使用是一门名为合成生物学的新科学领域的核心部分。这一新学科的目标是使用生物材料来提高日常设备的性能。在这种情况下，我们使用合成生物学来增强生物武器的检测。检测包括检测病毒序列的变化，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构成了一个巨大的挑战，因为我们需要将来自病毒的信号(不到1米长的1,000,000)转换成可读的信号。为了实现这一点，我们使用了一种简单但未得到充分利用的方法(线性二色性光谱)，该方法能够检测病毒是否排列一致。如果它们对齐，则不存在病原体；如果它们不对齐，则存在病原体。进行化验的方法具有许多有用的特征，使其非常适合安全部门使用。该分析方法简单、快速，无需实验室设备即可进行。还可以配置该检测方法，以便在一次检测中可以检测到一种以上的病原体。这一点很重要，因为需要同时检测的生物制剂范围很广。然而，不幸的是，我们目前的方法只能使用现有的病原体检测模式中的一种，基于使用抗体来进行检测。在这个项目中，我们的目标是通过在我们的系统中增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鉴定生物制剂中的DNA。为了将这种方法添加到我们的系统中，我们必须将与生物杀菌剂中的DNA相匹配的小片段DNA添加到病毒表面。这些DNA片段专门粘在生物制剂中的DNA上，防止病毒排列，从而给我们提供信号。如果成功，我们还计划增加第二个步骤，使用一种酶来放大信号(采用一种名为聚合酶链式反应的方法)，这将使整个系统更加灵敏。在项目的最后阶段，我们将结合基于DNA的化验和基于抗体的化验，以产生一个比任何现有系统更有效、更灵活、更不容易出现错误结果的系统。通过这种方式，我们还将演示合成生物学在检测应用中的最早使用之一。

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.