Label-free Real-time Single-molecule Assay Platform for Genomic Identification

临床表现，特别是在病程的早期，很少是由特定的感染源感染引起的。因此，诊断是复杂的，许多不同的生物会引起相似的症状。鉴于有效的干预需要准确的诊断，而且成功的可能性随着时间的推移而降低，能够快速、有效地进行鉴别诊断的测试有可能降低传染病的发病率、死亡率以及社会和经济成本。聚合酶链式反应(PCR)不太适合于高度多重的微生物分析，因为引物相互作用会降低灵敏度，而且报告系统的资料库通常限制在10到20个靶标。DNA微阵列允许广泛的多路复用，但现有的分析方法不如特定试剂的PCR灵敏，需要放大、荧光标记和几个小时的处理。下一代测序具有无限的多重潜力。然而，目前的平台需要数小时到数天的时间进行样本处理和生物信息分析，并且对于大多数医疗保健应用程序来说过于复杂。在这个项目中，我们将开发一个单分子场效应晶体管(SmFET)诊断分析平台。这一应用借鉴了我们最近的工作，在这些工作中，我们已经证明了具有单个共价连接的DNA探针分子的碳纳米管的电导对互补DNA链杂交引起的电荷增加非常敏感。在有源互补金属氧化物半导体(CMOS)衬底上的smFET阵列将使基因组材料的检测浓度接近1 fM(或每毫升600个分子)，与qPCR相当，但同时允许与微阵列相媲美的多路复用。我们将专门将这项技术应用于一个基因组诊断平台，该平台将允许对传染病进行高效、低成本的鉴别诊断。 我们的目标是优化和开发传感器，以检测低至1 fM的目标浓度，并开发通过结合动力学分析区分不匹配的方法；将这些设备集成到CMOS测量基板上，进一步提高电子性能并允许并行多路复用；以分阶段策略使用临床样本测试平台，该策略从最小限度地使用核酸模板进行生物遏制，然后开始使用生物遏制中的潜在传染病材料；将设备的外形因数降低到便携式USB棒的形状；以及构建软件和生物信息基础设施，以支持该平台在现场和临床部署。

Clinical presentation, particularly early in the course of disease, is only rarely pathognomonic of infection with a specific infectious agent. As a result, diagnosis is complex with many different organisms causing similar symptoms. Given that effective intervention requires accurate diagnosis and that the probability of success diminishes over time, tests that enable rapid, efficient differential diagnosis have potential to decrease morbidity, mortality, and social and economic costs of infectious diseases. Polymerase chain reaction (PCR) is not well suited to highly multiplexed microbiological analyses because primer interactions can reduce sensitivity and the repertoire of reporter systems is typically limited to 10 to 20 targets. DNA microarrays allow extensive multiplexing but existing assays are less sensitive than agent-specific PCR and require amplification, fluorescent labeling and several hours for processing. Next generation sequencing has unlimited multiplex potential. However, current platforms require hours to days for sample processing and bioinformatic analysis and are too complex for most point-of-care applications. In this project we will develop a single-molecule field-effect transistor (smFET) diagnostic assay platform. This application draws on our recent work, in which we have shown that the conductance of a carbon nanotube with a single covalently tethered DNA probe molecule is exquisitely sensitive to the increased charge that results from hybridization of a complementary DNA strand. smFET arrays on active complementary metal-oxide-semiconductor (CMOS) substrates will allow genomic materials to be assayed to concentrations approaching 1 fM (or 600 molecule per mL), comparable to qPCR, but while allowing multiplexing comparable to microarrays. We will specifically apply this technology to a genomic diagnostic platform that will allow efficient, low-cost differential diagnosis of infectious diseases. Our objectives we will be to optimize and develop the sensor to detect target concentration as low as 1 fM and develop approaches to distinguish mismatches through analysis of binding kinetics; integrate these devices onto CMOS measurement substrates, further improving electronic performance and allowing parallel multiplexing; test the platform with clinical samples in a staged strategy that begins in minimal biocontainment with nucleic acid templates, proceeds to work with potentially infectious materials in biocontainment; reduce the form factor for the device to that of a portable USB stick; and build software and bioinformatics infrastructure to support this platform for deployment in the field and clinics.