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（由申请人提供）：拟议研究的长期目标是创建一个用于基于纳米孔的生物标志物检测和定量的全自动手持平台，我们的单分子测定目前的结果时间为 5。分钟或更短的时间，无需优化即可实现 PM 灵敏度，使用一次性测试条模型每次测试成本为 2 美元（材料和人工）。测试结果可以加密并通过云报告给用户，从而形成一个具有巨大潜力的平台。价格实惠，在纳米孔装置中，电压从孔上方的流体室捕获单个 DNA，并且通过孔的测量电流暂时变化以发出每个分子的存在和通过的信号，从而实现对特定生物标记物的检测。在一群其他分子中，我们的方法使用双功能融合分子；当每个支架/融合物时，融合物的一端结合目标生物标志物，而另一端将融合物/生物标志物复合物锚定到dsDNA支架上的独特位置。通过孔，检测融合的占用状态，因为当生物标记物结合时事件特征是不同的，为每个捕获的分子提供简单的是/否标准我们的测定也被设计为使得背景分子事件是可区分的。根据支架/融合事件，基于初步数据，第一阶段工作的目标是：1) 推进双分子工程步骤和分析方法以优化生物标志物检测性能，以及 2) 演示三种临床相关蛋白质生物标志物的检测：细菌外壳蛋白、人类糖蛋白和抗病毒抗体 目标 1. 创建每个 dsDNA。 /融合分子试剂将结合溶液中的每个目标生物标志物。 进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信号。此外，使用工具进行统计分配。目标 3. 滴定生物标志物浓度并确定生物标志物检测的限度。此外，以滴定量的血清作为背景进行纳米孔实验，以模拟现实的诊断场景。