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 RNA 病毒导致全世界范围内的大量发病率和死亡率，从艾滋病毒到流感， 冠状病毒由于疾病的临床表现可能相似或无症状，因此准确且快速。 尽管如此，诊断对于监测疫情和实施有效治疗至关重要。 对快速（<30 分钟）、高度多重、紧凑和可靠的核酸扩增测试的需求尚未得到满足 在这个项目中，我们将使用 MEMS 技术进行微流体搅拌来加速固体。 相等温放大至少 10 倍。第一阶段提案的重点是开发 扩增反应室。 固相 (SP) 放大是一种众所周知的在单锅 INAA 中实现多重化的潜在解决方案。 SP-INAA，每个靶标的一个或两个引物固定在表面上，而其他试剂保留在 不幸的是，固相放大的效率明显低于液相反应， 因为模板需要扩散到引物位置才能扩增，因此 SP-INAA 速度较慢。 且 LOD 低于标准液相 NAAT。 我们的目标是证明 SP-INAA 比之前的实施速度至少快 10 倍，最多 100 倍 我们的模块将是一个独立的一体化放大模块，带有 INAA 主控器。 除了固定引物外，还需在室内混合冻干物，因此唯一需要的添加物是 如果成功的话，该项目将在核酸扩增方面取得突破。 通过消除速度、多路复用和设备复杂性之间的权衡来进行测试（NAAT）。