Trap Array Chips Enabling Rapid, Automated, and Portable Antibiotic Resistance Screening

提案标题：陷阱阵列芯片实现快速、自动化和便携式抗生素耐药性筛查项目目标简介：将开发一种微型系统，能够在15分钟内快速、多路和便携式地识别抗生素耐药性细菌。非技术摘要：抗生素耐药性的快速增长被认为是美国和世界范围内对公共健康最严重的威胁之一，耐药性细菌的数量不断增加导致临床结果不佳和死亡率上升，同时消耗更多的卫生保健资源。能够快速识别多种细菌并在护理地点或附近评估它们的抗生素敏感性，将极大地提高临床医生的能力，通过根据特定感染开出适当的药物处方，或通过隔离高危患者以防止进一步传播。在这个项目中，我们将开发一种使用一次性芯片实验室设备的新技术，该设备能够将患者样本分离到1000多个单独的反应室，在这些反应室中可以快速分析与抗生素耐药性相关的不同微生物基因片段，从而在15分钟内高效筛选多个潜在目标。由此产生的技术被设想为一种简单且廉价的诊断平台，用于在护理点使用。在这项工作中开发的基础芯片上实验室技术也将有助于推动其他诊断平台的发展，这些平台需要简单的操作和最少的设备，用于缺乏中央实验室资源的临床环境。技术摘要：抗生素耐药性是对公众健康的一个重大且日益严重的威胁，耐药病原体显著增加了感染患者的发病率和死亡率。与抗菌素耐药性增加相关的一个主要挑战是，在治疗的最早阶段，缺乏快速分析来确定致病病原体及其耐药性图谱。在这个项目中，我们将开发一个低成本和一次性的热塑性微流控平台，采用一种新的陷阱阵列技术，有望打开聚合酶链式反应(PCR)在护理点进行抗生素耐药性筛查的常规临床应用的大门。TRAP阵列平台将支持1000多个同时进行的PCR反应，使用针对多个抗生素耐药基因靶点的引物，而不需要外部泵送、瓣膜、底物准备或试剂引入。产生的平台将专门应用于开发一种快速多重分析耐多药碳青霉烯酶产生菌，这目前在多个临床环境中是一个重大挑战。为了实现这些目标，这项工作将导致开发新技术，从而能够(1)将连续样本量一步到位地离散成单个样本陷阱阵列，以实现高度并行分析；(2)在芯片制造过程中将所有所需的聚合酶链式反应试剂(包括用于多重分析的已定义引物集)直接集成到样本陷阱阵列中；以及(3)在一次性热塑性芯片中对所得扩增产物进行快速聚合酶链式反应和高分辨率熔融分析(HRMA)，只需一个手动进样步骤即可进行操作，从而极大地简化了无需进入中心生化实验室即可在接近患者的环境下使用的分析方法。

Proposal Title: Trap Array Chips Enabling Rapid, Automated, and Portable Antibiotic Resistance Screening Brief description of project Goals: A miniature system enabling rapid, multiplexed, and portable point-of-care identification of antibiotic-resistant bacteria in under 15 minutes will be developed.Nontechnical Abstract:The rapid growth of antibiotic resistance is recognized as one of the most serious threats to public health in the U.S. and worldwide, with increasing numbers of drug-resistant bacteria leading to poor clinical outcomes and higher death rates, while consuming greater health-care resources. The ability to rapidly identify multiple bacteria and evaluate their antibiotic susceptibility at or near the point of care would greatly enhance the ability of clinicians to optimize treatment through the prescription of appropriate drugs based on the specific infections, or through the isolation of high risk patients to prevent further transmission. In this project we will develop a new technology using disposable lab-on-a-chip devices capable of segregating a patient sample into over 1000 individual reaction chambers where different microbial gene fragments associated with antibiotic resistance may be rapidly analyzed, allowing highly effective screening of multiple potential targets in under 15 min. The resulting technology is envisioned as a simple and inexpensive diagnostic platform for use at the point of care. The underlying lab-on-a-chip technologies developed in this work will also serve to advance other diagnostic platforms requiring simple operation and minimal equipment for use in clinical settings lacking central laboratory resources. Technical Abstract:Antibiotic resistance represents a major and growing threat to public health, with drug-resistant pathogens significantly increasing rates of morbidity and mortality for infected patients. A major challenge associated with the increase in antimicrobial drug resistance is the lack of rapid assays for identifying causative pathogens and their drug resistance profiles during the earliest stages of treatment. In this project we will develop a low-cost and disposable thermoplastic microfluidic platform employing a novel trap array technology expected to open the door to routine clinical application of polymerase chain reaction (PCR) for antibiotic resistance screening at the point of care. The trap array platform will support over 1000 simultaneous PCR reactions using primers for multiple antibiotic-resistance gene targets, without the need for external pumping, valving, substrate preparation, or reagent introduction. The resulting platform will specifically be applied to the development of a rapid multiplexed assay for multi-drug resistant carbapenemase-producing bacteria, which currently presents a significant challenge across multiple clinical environments. To achieve these goals, the effort will result in the development of new technologies enabling (1) single-step discretization of a continuous sample volume into an array of individual sample traps to enable highly parallel analysis, (2) integration of all required PCR reagents, including defined primer sets for multiplexed analysis, directly into the array of sample traps during chip manufacture, and (3) rapid PCR and high resolution melt analysis (HRMA) of the resulting amplicons in a disposable thermoplastic chip requiring only a single manual pipetting step for operation, vastly simplifying assay implementation for use in near-patient settings without access to a central biochemical laboratory.