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

摘要 RNA病毒在世界范围内造成了大量的发病率和死亡率，从艾滋病毒到流感，再到 冠状病毒。由于疾病的临床表现可能类似或无症状，因此准确而迅速。 诊断对于监测疫情和实施有效治疗至关重要。尽管如此，仍有 对快速(30分钟)、高度多样化、紧凑和 性价比高。在这个项目中，我们将使用用于微流控搅拌的MEMS技术来加速固体 相位等温放大至少10倍。这项第一阶段建议的重点是发展 放大反应室。 固相(SP)放大是一种在单盆中子活化分析中实现多路复用的众所周知的潜在解决方案。在……里面 SP-INAA，每个靶的一个或两个引物固定在一个表面上，而其他试剂留在 解决方案。不幸的是，固相放大的效率远远低于液相反应， 因为模板需要扩散到引物位置才能被扩增。因此，SP-INAA速度较慢 并且比标准的液态NaAT具有更低的LOD。 我们的目标是演示SP-INAA比以前的实施至少快10倍，高达100倍 SP-扩增产物。我们的模块将是一个独立的、一体式放大模块，带有一个INAA主机 除了固定化的引发剂外，还要在小室内混合冷冻干燥的物质，因此唯一需要添加的是 样品准备好放大了。如果成功，这个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，测试(NAAT)。

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.