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(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化手持平台。我们的单分子分析目前的结果时间不超过5分钟，无需优化即可实现PM灵敏度，使用一次性试纸模型，每次测试成本为2美元(材料和人力)。测试结果可以被加密并通过云报告给用户，从而产生了一个具有巨大潜力的平台，可以进行负担得起的护理点分子诊断。在纳米孔设备中，电压力从孔上方的流体室捕获单个DNA，通过孔的测量电流暂时移动，以发出每个分子的存在和通过的信号。为了从其他分子中检测特定的生物标记物，我们的方法使用双功能融合分子；融合的一端结合目标生物标记物，而另一端将融合/生物标记物复合体锚定到dsDNA支架上的唯一位置。当每个支架/融合通过孔时，融合的占用状态被检测到，因为当结合生物标志物时，事件特征是不同的，为每个捕获的分子提供了一个简单的是/否标准。我们的分析也是这样设计的，以便背景分子事件与支架/融合事件区分开来。在初步数据的基础上，这一阶段工作的目标是：1) 推进双分子工程步骤和分析方法，以优化生物标记物的检测性能；2)展示三种临床相关蛋白质生物标记物的检测：细菌外壳蛋白、人糖蛋白和抗病毒抗体。目的1.创建每一种dsDNA/融合分子试剂，该试剂将结合溶液中的每个靶生物标记物。目的2.进行纳米孔实验以检测DNA/融合/蛋白质复合体的存在，从而发出每个生物标记物的存在的信号。此外，还应实施工具来为检测分配统计意义。目的3.测定生物标记物浓度，建立生物标记物检测限值。此外，在滴定数量的血清中进行纳米孔实验作为背景，以模拟现实的诊断场景。