A Platform Approach for the Electrical Detection of Protein Biomarkers using a Nanopore Device

使用纳米孔装置电检测蛋白质生物标志物的平台方法

• 批准号: 9350518
• 负责人: Trevor Justin Morin
• 金额: $ 3.25万
• 依托单位: TWO PORE GUYS, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-21 至 2017-09-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9350518
• 关键词: Amplifiers; Binding; Biological Assay; Biological Markers; Buffers; Capsid Proteins; Chemistry; Chimeric Proteins; Complex; DNA; DNA Binding Domain; Data; Detection; Devices; Diagnostic; Engineering; Event; Feasibility Studies; Glycoproteins; Hand; Health; Health Personnel; Healthcare Systems; Human; Immunologic Markers; Incubated; Individual; Location; Maleimides; Measures; Methods; Modeling; Nucleic Acids; Peptides; Performance; Pharmacy facility; Phase; Population; Proteins; Quality of life; Reagent; Reporting; Research; Sampling; Serum; Signal Transduction; Statistical Data Interpretation; Techniques; Technology; Tertiary Protein Structure; Test Result; Testing; Time; Viral Antibodies; aptamer; base; clinically relevant; cost; crosslink; design; ds-DNA; improved; laptop; molecular diagnostics; nanopore; nanoscale; point of care; protein biomarkers; protein complex; prototype; research study; scaffold; single molecule; solid state; specific biomarkers; targeted biomarker; tool; voltage

 DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to create a fully automated hand-held platform for nanopore-based biomarker detection and quantitation. Our single-molecule assay currently has a time-to- results of 5 minutes or less, achieves pM sensitivity without optimization, and costs $2 (materials and labor) per test by using a disposable test strip model. Test results can be encrypted and reported to a user via the cloud, resulting in a platform with tremendous potential for affordable, point of care molecular diagnostics. In a nanopore device, voltage force captures individual DNA from a fluidic chamber above the pore, and the measured current through the pore temporarily shifts to signal the presence and passing of each molecule. To achieve detection of specific biomarkers from among a population of other molecules, our method uses bi- functional fusion molecules; one end of the fusion binds the target biomarker, while the other end anchors the fusion/biomarker complex to a unique location on a dsDNA scaffold. When each scaffold/fusion passes through the pore, the occupancy status of the fusion is detected because the event signature is distinct when the biomarker is bound, providing a simple yes/no criterion for every captured molecule. Our assays are also designed so that background-molecule events are distinguishable from scaffold/fusion events. Building on preliminary data, the objectives of this Phase I effort are: 1) to advance the bimolecular engineering steps and analysis methods to optimize biomarker detection performance, and 2) to demonstrate detection of three clinically relevant protein biomarkers: a bacterial coat protein, a human glycoprotein, and an anti-viral antibody. Aim 1. Create each dsDNA/fusion molecule reagent that will bind each target biomarker in solution. Aim 2. Perform nanopore experiments to detect the presence of DNA/fusion/protein complexes, and thereby signal the presence of each biomarker. Also, implement tools to assign statistical significance to detection. Aim 3. Titrate biomarker concentration and establish the limit of biomarker detection. Also, perform nanopore experiments in titered amounts of serum as background, to emulate a realistic diagnostic scenario.

 描述（由适用提供）：拟议的研究的长期目标是为基于纳米孔的生物标志物检测和定量创建一个完全自动化的手持平台。我们的单分子测定目前的时间为5分钟或更短的时间，可实现PM灵敏度而无需优化，并且每次测试的费用为2美元（材料和人工），并使用一次性测试带模型。可以通过云加密测试结果并将其报告给用户，从而产生一个具有巨大负担得起的护理点分子诊断潜力的平台。在纳米孔装置中，电压力从孔上方的流体腔中捕获单个DNA，并通过孔临时移动进行测量的电流，以信号每个分子的存在和通过。为了从其他分子群中检测特定的生物标志物，我们的方法使用双功能融合分子。融合的一端结合了目标生物标志物，而另一端将融合/生物标志物复合物固定在dsDNA支架上的唯一位置。当每个脚手架/融合都通过孔时，会检测到融合的占用状态，因为当生物标志物绑定时事件签名是不同的，为每个捕获的分子提供了一个简单的是/否标准。我们的评估也设计为背景 - 分子事件与脚手架/融合事件可以区分。在初步数据的基础上，这一第一阶段的目标是：1） 为了推进优化生物标志物检测性能的生物分子工程步骤和分析方法，以及2）证明对三种临床相关蛋白质生物标志物的检测：一种细菌外套蛋白，人类糖蛋白和抗病毒抗体。 AIM 1。创建每个DSDNA/融合分子试剂，该试剂将在溶液中结合每个靶标生物标志物。 AIM 2。执行纳​​米孔实验以检测DNA/融合/蛋白质复合物的存在，从而发出每个生物标志物的存在。另外，实施工具以分配统计意义以进行检测。目标3。滴定生物标志物浓度并建立生物标志物检测的极限。此外，以滴定血清作为背景进行纳米孔实验，以模仿现实的诊断情况。