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

在过去的十年里，英国性传播疾病的流行有了显著的增长，例如梅毒、衣原体和淋病分别增加了1800%、150%和42%，导致全国各地的泌尿生殖系统(GUM)诊所进行了200多万次检测。提供一种快速、成本效益高和强大的检测方法，以检测所有这些和其他性传播感染，可在护理时使用，这不仅将加强病人护理服务的提供，还将有助于减少感染的进一步传播。目前还不存在这样的检测方法，目前的方法学提供的双靶感染检测充其量不能满足基于实验室的检测的敏感性和特异性。因此，挑战是开发具有成本效益的、自动化的、信息丰富的测试，这些测试与实验室中进行的测试一样准确，并且可以在初级和二级保健环境中使用。因此，该项目的目标是通过整合两个现有的互补工艺(Hull的DNA提取和PCR扩增芯片以及Randox的DNA杂交阵列和基于化学发光的检测器)来实现这一未解决的挑战，以创造适当的技术。Randox-Hull财团不仅拥有开发这种技术的技术和工程能力，而且通过Randox在该领域的商业存在，也为其提供了一条通往市场的明确途径。基于最近资助的研究(EPSRC EP/H007385/1、EP/D040930和BBSRC BBE0027221)，该项目将开发一种单发诊断芯片，该芯片将预装所有必要的试剂和废物遏制要求，以便与专门设计的便携式低功率控制和数据处理仪器一起运行。在实践中，用户只需将尿液或拭子样本直接添加到芯片上样品引入腔中的吸附剂中。这种水平的样品处理和芯片处理是创造技术的关键，这种技术实际上将减少样品在实验室环境外使用时可能受到的感染和交叉污染。手动关闭样品室将启动DNA从样品中的释放，方法是与预加载的盐酸胍混合，DNA被捕获在硅胶整体柱上，从那里它将被洗脱到包含预加载试剂的聚合酶链式反应小室中。扩增后，生物素标记的DNA靶标将被电泳化，以避免体积变化，进入检测室，在那里杂交反应将它们拴在一系列特定的互补核苷酸序列上。然后，控制箱中的致动器将流体动力地输送试剂和芯片上的洗涤液，以产生化学发光信号，用于阵列上存在的对照和目标分析物的任何阳性杂交，该信号将使用CCD相机进行检测。该芯片将放置在一个控制仪器中，该仪器将容纳所需的电源、压力执行器、电阻加热风冷热循环器和CCD探测器。将开发软件，以控制拟议仪器的测序和数据采集，并提供适当的用户界面，以便在临床医生看病人之前向他们展示信息。

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.