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

用于基因组鉴定的无标记实时单分子检测平台

• 批准号: 9010919
• 负责人: Kenneth L Shepard
• 金额: $ 45.31万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9010919
• 关键词: Address; Affinity; Algorithms; Antibiotics; Behavior; Binding; Bioinformatics; Biological Assay; Carbon Nanotubes; Characteristics; Charge; Clinic; Clinical; Clinical Microbiology; Communicable Diseases; Communities; Complementary DNA; Complex; Complex Mixtures; Computer software; DNA Microarray Chip; DNA Probes; Data; Data Analyses; Detection; Development; Device or Instrument Development; Devices; Diagnosis; Diagnostic; Differential Diagnosis; Disease; Electrolytes; Electronics; Electrons; Encephalitis; Event; Genomics; Health Care Costs; Health Personnel; Hour; Hybridization Array; Immobilization; Infection; Infectious Agent; Intervention; Kinetics; Label; Measurement; Measures; Medicine; Microarray Analysis; Monitor; Morbidity - disease rate; Nanotubes; Noise; Nucleic Acids; Nucleotides; Organism; Performance; Pneumonia; Polymerase Chain Reaction; Preparation; Printing; Probability; Process; Proteins; Reporter; Research; Research Infrastructure; Risk; Sampling; Semiconductors; Signal Transduction; Site; Staging; Surface; Symptoms; System; Techniques; Technology; Temperature; Testing; Time; Transistors; Validation; Work; accurate diagnosis; base; cost; diagnostic assay; economic cost; effective intervention; gastrointestinal; genomic platform; improved; markov model; metal oxide; mortality; nanoscale; next generation sequencing; point of care; prototype; sensor; single molecule; social; software development; success; technique development

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.

临床表现，尤其是在疾病的早期，很少对特定感染剂感染感染。结果，诊断很复杂，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，并且成功的可能性随着时间的流逝而减少，因此能够快速，有效的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。聚合酶链反应（PCR）不太适合高度多重的微生物分析，因为引物相互作用可以降低敏感性，并且报告基因系统的曲目通常限制为10至20个目标。 DNA微阵列允许大量的多路复用，但现有测定比特定于特定的PCR敏感，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电位。但是，当前平台需要数小时到几天才能进行样本处理和生物信息学分析，并且对于大多数护理点应用程序来说太复杂了。在此项目中，我们将开发单分子现场效应晶体管（SMFET）诊断测定平台。该应用借鉴了我们最近的工作，在其中我们表明，具有单个共价DNA探针分子的碳纳米管的电导率对辅助DNA链杂交导致的电荷的增加非常敏感。 SMFET阵列上的活性互补金属 - 氧化物 - 氧化导体（CMOS）底物将允许将基因组材料分析到接近1 FM（或600个分子每毫升分子）的浓度（或与QPCR相当），但在允许将多路复用与微阵列相当的同时。我们将专门将该技术应用于基因组诊断平台，该平台将允许对传染病的有效，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到低至1 FM的目标浓度，并通过分析结合动力学来区分不匹配的方法；将这些设备整合到CMOS测量基板上，进一步改善电子性能并允许并行多路复用；用临床样品测试平台，该策略是从最小生物内生化的核定酸模板开始的，继续与潜在的感染材料合作生物内生化。将设备的外形尺寸减少到便携式USB棒的外形；并建立软件和生物信息学基础架构，以支持该平台在现场和诊所中进行部署。