Development of a rapid multiplex Lab on a Chip system for detection of 10 STI pathogens using Biochip Array Technology

在过去的十年中，世界上性传播感染的流行率显著增长，例如英国的梅毒，衣原体和淋病分别增加了1800%，150%和42%，导致全国各地的生殖泌尿医学（GUM）诊所进行了超过200万次测试。如果能够提供一种快速、具有成本效益和强有力的检测方法来检测所有这些和其他性传播感染，并可在护理点使用，不仅会加强对病人的护理服务，而且还有助于减少感染的进一步传播。目前不存在这样的测试，目前的方法充其量只能提供双重靶感染测定，不能满足基于实验室的测试的灵敏度和特异性。因此，面临的挑战是开发具有成本效益的、信息丰富的自动化测试，这些测试与在实验室中进行的测试一样准确，并且可以在初级和二级护理环境中使用。因此，该项目的目的是通过整合两个现有的互补过程（来自船体的DNA提取和PCR扩增芯片以及来自Randox的DNA杂交阵列和基于化学发光的检测器）来实现这一未满足的挑战，以创建适当的技术。Randox -船体联合体不仅拥有开发此类技术的技术和工程能力，而且还通过Randox在该领域的商业存在为市场提供了明确的途径。基于最近资助的研究（EPSRC EP/H 007385/1，EP/D 040930和BBSRC BBE 0027221），该项目将开发一种单次诊断芯片，该芯片将预装所有必要的试剂和废物控制要求，以便与专门设计的便携式低功耗控制和数据处理仪器一起运行。在实践中，用户将仅需要将尿液或拭子样品直接添加到芯片上的样品引入室中的吸附剂。这种水平的样品处理和芯片处理是创建技术的关键，该技术将在实验室环境外使用时实际减少潜在的感染暴露和样品之间的交叉污染。样品室的手动关闭将通过与预加载的盐酸胍混合来开始从样品中释放DNA，其中DNA被捕获在二氧化硅整料上，从那里它将被洗脱到含有预加载试剂的PCR室中。扩增后，生物素标记的DNA靶标将被电泳运输，以避免体积变化，进入检测室，在检测室中杂交反应将它们连接到特定互补核苷酸序列的阵列。然后，控制盒中的致动器将流体动力学地输送保持在芯片上的试剂和洗涤溶液，以产生用于阵列上存在的对照和靶分析物的任何阳性杂交的化学发光信号，其将使用CCD相机检测。该芯片将位于控制仪器中，该仪器将容纳所需的电源、压力致动器、连续加热的空气冷却热循环仪和CCD检测器。将开发软件来控制所提议仪器的测序和数据采集，以及适当的用户界面，以便在临床医生看到患者之前向其提供信息。

Over the past decade the world has seen a significant growth in the prevalence of sexually transmitted infections STIs in the UK for example Syphilis, Chlamydia and Gonorrhoea have shown increases of 1800%, 150% and 42%, respectively, resulting in over 2 million tests being performed in genito-urinary medicine (GUM) clinics across the nation. The availability of a rapid, cost-effective and robust test for the detection of all of these and other STIs, which could be used at the point of care, would not only enhance the delivery of patient care services but would also aid in reducing the further spread of infection. At present no such test exists, with current methodology offering at best duplex target infection assays that fail to meet the sensitivity and specificity of laboratory based tests. The challenge is therefore to develop cost-effective, automated information rich tests that are as accurate as those conducted in the laboratory and that can be used in both primary and secondary care settings. The aim of this project is therefore to realise this unmet challenge through the integration of two existing and complimentary processes (the DNA extraction and PCR amplification chip from Hull and the DNA hybridisation array and chemiluminescence based detector from Randox), to create appropriate technology. The Randox - Hull consortium not only has the technical and engineering capability to develop such technology but also through Randox's commercial presence in the field a clear route to market. Based on recently funded research (EPSRC EP/H007385/1, EP/D040930 and BBSRC BBE0027221), this project will develop a single shot diagnostic chip, which will come pre-loaded with all the necessary reagents and waste containment requirements for it to operate in conjunction with a specifically designed portable low power control and data processing instrument. In practice the user will only need to add a urine or swab sample directly to an adsorbent in a sample introduction chamber on the chip. This level of sample handling and on-chip processing is key in creating technology that will practically reduce both potential exposure to infection and cross contamination between samples when used out of the laboratory environment. Manual closure of the sample chamber will initiate the release of DNA from the sample by mixing with pre-loaded guanidine hydrochloride with the DNA being trapped on a silica monolith from where it will be eluted into a PCR chamber containing pre-loaded reagents. Following amplification the biotin-labelled DNA targets will be transported electrophoretically, to avoid volumetric changes, into the detection chamber where a hybridisation reaction will tether them to an array of specific complimentary nucleotide sequences. Actuators in the control box will then hydrodynamically deliver reagent and wash solutions held on chip to generate a chemiluminescence signal for any positive hybridisation of controls and target analytes present on the array which will be detected using a CCD camera. The chip will be located in a control instrument which will house the required power supplies, pressure actuators, resistively heated air cooled thermocycler and the CCD detector. Software will be developed to control the sequencing and data acquisition of the proposed instrument together with a suitable user interface for presenting information to a clinician prior to them seeing a patient.