MEMS-enhanced solid-phase isothermal amplification for rapid, multiplexed molecular diagnostics

MEMS 增强固相等温放大，用于快速、多重分子诊断

• 批准号: 10484147
• 负责人: Jay Kenneth Fisher
• 金额: $ 30.43万
• 依托单位: REDBUD LABS, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10484147
• 关键词: Acids; Address; Agitation; Automobile Driving; Biological Assay; Clinical; Communities; Coronavirus; Data; Deposition; Development; Devices; Diagnostic; Diffuse; Diffusion; Disease; Disease Outbreaks; Exposure to; Freeze Drying; HIV; Hybrids; Immobilization; Influenza; Laboratories; Letters; Liquid substance; Location; Measures; Methods; Microfluidics; Molds; Molecular; Monitor; Morbidity - disease rate; North Carolina; Nucleic Acid Amplification Tests; Nucleic Acids; Phase; Printing; Pump; RNA Viruses; Reaction; Reagent; Sampling; Signal Transduction; Silicones; Solid; Speed; Surface; System; Technology; Testing; Time; Universities; accurate diagnosis; base; cost effective; density; design; detection limit; detection platform; effective therapy; improved; isothermal amplification; magnetic beads; magnetic field; microfluidic technology; molecular diagnostics; mortality; multiplex assay; point of care; rapid diagnosis; rapid test; success

ABSTRACT RNA viruses are responsible for substantial morbidity and mortality worldwide, from HIV to Influenza, to Coronavirus. As the clinical presentation for disease may be similar or asymptomatic, accurate and rapid diagnosis is essential for monitoring outbreaks and administering of effective therapy. Despite this, there remains an unmet need for nucleic acid amplification tests that are rapid (<30 minutes), highly multiplexed, compact, and cost-effective. In this project, we will use a MEMS technology for microfluidic agitation to accelerate solid phase isothermal amplification by at least 10x. The focus of this Phase I proposal is the development of the amplification reaction chamber. Solid-phase (SP) amplification is a well-known potential solution to achieve multiplexing in single-pot INAA. In SP-INAA, one or both primers for each target is immobilized on a surface, while the other reagents remain in solution. Unfortunately, solid-phase amplification is dramatically less efficient than liquid-phase reactions, because template needs to diffuse to the primer location in order to be amplified. SP-INAA is therefore slower and has a lower LOD than standard liquid-phase NAATs. We aim to demonstrate SP-INAA that is at least 10x—and as much as 100x—faster than prior implementations of SP-amplification. Our module will be a self-contained, all-in-one amplification module, with an INAA master mix lyophilized inside the chamber in addition to immobilized primers, so the only addition required will be a sample ready for amplification. In success, this project will deliver a breakthrough in nucleic acid amplification testing (NAATs) by eliminating the tradeoff between speed, multiplexing, and device complexity.

抽象的 从艾滋病毒到流感，RNA病毒负责全球范围内的大量发病率和死亡率 新冠病毒。由于疾病的临床表现可能相似或不对称，准确和快速 诊断对于监测暴发和进行有效治疗至关重要。尽管如此，仍然存在 对快速（<30分钟），高度多重，紧凑和 成本效益。在这个项目中，我们将使用MEMS技术进行微流体搅动来加速固体 相位温度至少10倍。该阶段我提出的重点是发展 放大反应室。 固相（SP）扩增是在单盘INAA中实现多路复用的众所周知的潜在解决方案。在 SP-INAA，每个靶标的一个或两个引物被固定在表面上，而其他试剂仍保留在 解决方案。不幸的是，固相扩增比液相反应的效率大大降低， 因为模板需要扩散到底漆位置才能放大。因此，sp-inaa较慢 并且比标准液相NAAT的LOD低。 我们的目的是证明至少比先前的实现的SP-INAA至少10倍，最多100倍。 SP扩增。我们的模块将是一个独立的，多合一的放大模块 除了固定的引物外，还将冻干的冻干混合在腔室内，因此唯一需要的添加是 样品准备放大。在成功中，该项目将在核酸扩增方面取得突破 测试（NAAT）通过消除速度，多路复用和设备复杂性之间的权衡。