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

提案标题：捕获阵列芯片实现快速、自动化和便携式抗生素耐药性筛查项目目标简述：将开发一种微型系统，能够在15分钟内快速、多路复用和便携式护理点鉴定抗生素耐药性细菌。非技术摘要：抗生素耐药性的快速增长被认为是美国和全球公共卫生面临的最严重威胁之一，越来越多的耐药细菌导致临床结果不佳和死亡率上升，同时消耗更多的医疗资源。在护理点或附近快速鉴定多种细菌并评估其抗生素敏感性的能力将大大提高临床医生通过基于特定感染开出适当药物处方或通过隔离高风险患者以防止进一步传播来优化治疗的能力。在这个项目中，我们将开发一种新技术，使用一次性芯片实验室设备，能够将患者样本分离到1000多个单独的反应室中，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，允许在15分钟内高效筛选多个潜在靶点。由此产生的技术被设想为一个简单而廉价的诊断平台，在护理点。在这项工作中开发的芯片实验室技术也将有助于推进其他诊断平台，这些平台需要简单的操作和最少的设备，用于缺乏中心实验室资源的临床环境。技术摘要：抗生素耐药性是对公共卫生的一个主要和日益增长的威胁，耐药病原体显着增加感染患者的发病率和死亡率。与抗微生物药物耐药性增加相关的一个主要挑战是缺乏用于在治疗的最早阶段鉴定致病病原体及其耐药性概况的快速测定。在这个项目中，我们将开发一种低成本和一次性热塑性微流体平台，采用一种新的陷阱阵列技术，有望打开大门，以常规的临床应用聚合酶链反应（PCR）的抗生素耐药性筛选在护理点。陷阱阵列平台将支持超过1000个同步PCR反应，使用多个抗药性基因靶标的引物，而不需要外部泵送，阀门，底物制备或试剂引入。由此产生的平台将专门用于开发多重耐药碳青霉烯酶产生细菌的快速多重检测，目前这在多种临床环境中提出了重大挑战。为了实现这些目标，该努力将导致新技术的开发，该新技术能够实现（1）将连续样品体积单步离散化为单个样品阱阵列，以实现高度平行的分析，（2）在芯片制造期间将所有所需的PCR试剂（包括用于多重分析的限定引物组）直接整合到样品阱阵列中，和（3）在一次性热塑性芯片中对所得扩增子进行快速PCR和高分辨率解链分析（HRMA），仅需要单个手动移液步骤进行操作，极大地简化了用于近患者环境的测定实施，而无需进入中心生物化学实验室。

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.