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

摘要 RNA病毒是世界范围内从HIV到流感， 冠状病毒。由于疾病的临床表现可能相似或无症状，因此准确和快速 诊断对于监测疾病爆发和实施有效治疗至关重要。尽管如此， 对快速（<30分钟）、高度多重、紧凑和 性价比在这个项目中，我们将使用MEMS技术进行微流体搅拌， 相等温扩增至少10倍。第一阶段提案的重点是开发 扩增反应室 固相（SP）扩增是一个众所周知的潜在的解决方案，以实现在单锅INAA多重。在 在SP-INAA中，每个靶标的一种或两种引物固定在表面上，而其他试剂保留在表面上。 溶液不幸的是，固相扩增的效率明显低于液相反应， 因为模板需要扩散到引物位置以便被扩增。因此，SP-INAA较慢 并且具有比标准液相NAAT更低的LOD。 我们的目标是证明SP-INAA至少比以前的实现快10倍，甚至快100倍 SP扩增。我们的模块将是一个独立的，所有功能于一身的放大模块，与INAA主 除了固定的引物外，还可以在室内混合冻干，因此仅需要添加 样品准备扩增。如果成功，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，实现了NAAT测试。

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.