Fluorogenic biosensor immobilisation within surface modified fluoropolymer microdevices for rapid smartphone antibiotic susceptibility testing

抗生素耐药性被广泛认为是全球健康的重大挑战。有限的诊断技术导致了这一问题，因为抗生素通常是在患者样本进行测试以确定其是否对抗生素敏感或耐药之前开的。这是因为目前使用非常古老的技术来检测和测量微生物和细菌，这是有效的，但非常缓慢，并且使用只有在中央实验室才能找到的大型设备。一个重要的例子是尿路感染（UTI）。尽管我们熟悉“水感染”，这些似乎很常见，但都是小问题，但事实上，它们是全国最昂贵的感染之一，因为在某些患者中，感染会变得更严重，要么是因为早期感染没有得到抗生素治疗，要么是因为感染对所选抗生素具有耐药性。理想情况下，患者的尿液样本将进行测试，看看哪种抗生素将有效地治疗感染。但不幸的是，检测结果需要好几天才能出来，所以全科医生通常不会费心去检测，要么根据当前的指南选择一种“最有可能”的抗生素，要么根本不治疗。微型实验室系统--称为“芯片实验室”或微流控技术--最近被证明非常适合更小、更便携、可能更快的微生物检测。虽然这些最初的概念验证研究（其中之一最近由PI Edwards的研究小组发表）显示了微生物检测克服快速微生物检测挑战的潜力，我们自己的EPSRC第一个赠款资助的概念验证研究使用了申请人爱德华兹博士小组发明的一种新技术（阅读）与合作者Reis博士（巴斯）合作，允许非常低成本的微流体设备从一种称为“微毛细管膜”的新材料中批量生产。这些都是非常透明的，允许敏感的生物测试进行，和设备的几何形状是理想的阅读测试结果使用移动的手机相机，平板扫描仪，或数码相机。这种低成本的制造方法加上简单的数字记录，为革命性的数字微生物学工具打开了大门，这些工具可以将抗生素耐药性测试从临床实验室转移到患者身边。我们的第一项研究表明，使用这些新型低成本设备进行抗生素耐药性测试是可能的。该项目的重点不仅仅是原理验证，而是对临床微生物学微流体设备的几个重要组件进行了详细的改进和研究。一个主要的重点是开发有效的化学物质，使我们能够添加明亮的荧光染料，检测微毛细管内的细菌。我们的含氟聚合物设备受益于这些“特氟隆”塑料的不寻常的材料特性，但不幸的是，这种材料的“不粘”性质使得更难对设备进行化学改性。在这个项目中，我们将使用特定的化学改性方法与这种不粘表面反应，使其对细菌检测更有用。同时，我们将研究使用几种不同的染料进行细菌检测的速度和灵敏度的基础科学，这些染料在细菌存在时会改变颜色，从而找出快速检测细菌的最佳方法，即使它们在患者尿液样本中非常稀释。我们检测细菌的速度越快，我们就能越快地判断它们是否被抗生素杀死，因此病人就能越快地得到有效的治疗。最终，这项合成化学研究与细菌检测设备的工程科学相结合，将为我们提供加快诊断临床微生物学所需的工具和技术，确保细菌感染患者得到更快更有效的治疗。

Antimicrobial resistance is widely acknowledged to be a global health grand challenge. Limited diagnostic technology contributes to this problem because antibiotics are usually prescribed before the patient sample has been tested to see if it is sensitive or resistant to an antibiotic. This is because very old technology is currently used detect and measure microbes and bacteria, which is effective but very slow and uses large equipment only found in centralised labs.One important example is urinary tract infection (UTI). Although we are familiar with "water infections" which seem common but minor problems, in fact they are one of the most expensive infections nationally, because in some patients the infection gets worse, either because early infection is not treated with antibiotics, or the infection is resistant to the chosen antibiotic. Ideally, the patient urine sample will be tested to see which antibiotic will effectively treat the infection. But unfortunately it takes several days to receive the test result, so GPs often don't bother testing and either select a ''most likely" antibiotic based on current guidelines, or they don't treat at all.Miniaturised lab systems- termed "lab-on-a-chip" or microfluidic technology have recently been shown to be ideally suited to smaller, more portable, and potentially faster microbial testing. Whilst these initial proof-of-concept studies (one of which was recently published by PI Edwards' research group) show the potential for miniaturisation to overcome the challenge of rapid microbiology testing, significant technical barriers must now be overcome to make sure this exciting technology fulfils its potential.Our own EPSRC first grant funded proof-of-concept study used a novel technology invented by applicant Dr Edwards' group (Reading) with collaborator Dr Reis (Bath) that allows very low cost microfluidic devices to be mass-produced from a novel material called "microcapillary film". These are very transparent allowing sensitive biological tests to be performed, and the device geometry is ideal for reading test results using a mobile phone camera, flatbed scanner, or digital camera. This low cost manufacturing method coupled to simple digital recording, opens the door to revolutionary digital microbiology tools that can transport antibiotic resistance testing out of the clinical lab and near to the patient. Our first study showed that it is possible to perform antibiotic resistant tests using these novel low-cost devices.The focus of this project goes beyond proof-of-principle and refines and studies in great detail several important components of microfluidic devices for clinical microbiology. One major focus is on developing effective chemistry that allows us to add brightly fluorescent dyes that detect bacteria inside microcapillaries. Our fluoropolymer devices benefit from unusual material properties of these "Teflon" plastics, but unfortunately the 'non-stick' nature of this material makes it harder to chemically modify the devices. We will in this project use specific chemical modification methods to react with this non-stick surface and make it more useful for bacterial detection. In parallel, we will study the underlying science of speed and sensitivity of bacterial detection using several different dyes that change colour in the presence of bacteria, and thus work out the best way to very rapidly detect bacteria, even when they are very dilute in patient urine samples. The faster we can detect bacteria, the quicker we can tell if they are killed by antibiotics, and therefore the sooner that the patient can be given effective treatment.Ultimately, this synthetic chemistry research combined with the engineering science of miniaturised bacterial testing devices, will give us the tools and technology needed to speed up diagnostic clinical microbiology, and ensure patients with bacterial infections are treated faster and more effectively.