Nanopore array force spectroscopy chip for rapid clinical genotyping

描述（由申请人提供）：在个性化医疗保健中利用基因型信息的主要障碍是现有基因分型过程的复杂性和所需的时间。由个体患者的遗传信息指导的快速临床干预需要在不到一小时的时间内确定每位患者的数十个突变。虽然目前存在能够在30-60分钟内分析单个突变的方法，但这极大地限制了基因型信息对依赖于单个突变的疾病和易感性的效用。用于分析数十至数百个突变的方法需要几个小时来进行，并且对于适当的临床反应来说太慢。同样，可以同时分析数千名患者的高通量基因分型技术不适合临床环境中所需的单个患者/快速反应。因此，在临床专家和行业的推动下，迫切需要能够在几分钟内分析一百个基因座数量级的简单快速基因分型技术。绝缘膜中的纳米孔代表了生物分子检测和分析的重要新模式。虽然基于纳米孔的单分子检测方案已经是高通量DMA测序的候选方案，但基因分型是临床上重要的近期应用，并且将为DMA测序社区带来益处。多纳米孔力谱技术有望成为一种快速、灵敏、无标记的核酸分析方法。在以前的工作中，我们证明了使用有机纳米孔力谱法以单碱基分辨率检测序列的能力。在我们以前工作的延续中，我们的目标是发展基于固态纳米孔的力谱，用于序列变化的快速电子检测。我们设想最终开发一种基于纳米多孔膜元件阵列的商业设备，每个元件都被设计为识别特定序列。在本授权申请中，我们提出了一种设备和方法的开发，以作为这种阵列的一个元素的概念验证。我们将构建一个原型元件，并在测试其灵敏度和特异性的同时进行制造和方法开发。

DESCRIPTION (provided by applicant): A major barrier to utilization of genotype information in personalized health care is the complexity and time required by existing genotyping processes. Rapid clinical intervention that is guided by genetic information from individual patients will require determination of tens of mutations per patient in less than one hour. Though methods currently exist that are capable of analyzing a single mutation in 30-60 minutes, this greatly limits the utility of genotype information to diseases and predispositions that depend on a single mutation. Methods for analyzing tens to hundreds of mutations require several hours to carry out, and are too slow for an appropriate clinical response. Likewise, high throughput genotyping technologies that can analyze thousands of patients simultaneously are inappropriate for the single-patient/rapid response required in a clinical setting. There is therefore a pressing need, driven by clinical experts and industry, for simple and rapid genotyping technologies capable of analyzing on the order of a hundred loci in a matter of minutes. Nanometer-sized pores in an insulating membrane represent an important new mode of detection and analysis of biomolecules. Though nanopore-based single-molecule detection schemes are already candidates for high throughput DMA sequencing, genotyping is a nearer-term application that is clinically important and will produce benefits to the DMA sequencing community. Multi-nanopore force spectroscopy promises to be a rapid, sensitive and label-free nucleic acid analysis scheme. In previous work we demonstrated the ability to detect sequence at single base resolution using organic nanopore force spectroscopy. In this continuation of our previous work, we aim at the development of solid-state nanopore- based force spectroscopy for rapid electronic detection of sequence variation. We envision the eventual development of a commercial device based on an array of nanoporous membrane elements, each designed to recognize a particular sequence. In this grant application, we propose the development of a device and methods to serve as a proof-of-concept of one element of such an array. We will construct a prototype element, and iterate on fabrication and methods development while testing its sensitivity, and specificity.