A nucleic acid nanostructure built through on-electrode ligation for electrochemical detection of proteins, peptides, and small molecules

通过电极上连接构建的核酸纳米结构，用于蛋白质、肽和小分子的电化学检测

• 批准号: 10458097
• 负责人: Christopher J Easley
• 金额: $ 29.92万
• 依托单位: AUBURN UNIVERSITY AT AUBURN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-18 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10458097
• 关键词: Affinity; Amino Acid Substitution; Antibodies; Antibody Affinity; Architecture; Binding; Biological Assay; Biological Markers; Biosensor; Biotin; Cells; Clinical; Complex; Custom; DNA; DNA Modification Process; Data; Detection; Development; Diabetes Mellitus; Diagnosis; Diffusion; Digoxigenin; Disease; Disease Marker; Drops; Drug Monitoring; Electrochemistry; Electrodes; Epitopes; Exhibits; Fab Immunoglobulins; Funding; Future; Glucose; Health Status; Heart Diseases; Human; Hydrocortisone; Immunosuppression; Immunosuppressive Agents; Label; Laboratories; Lead; Ligation; Measurement; Medical; Methodology; Methods; Microfluidics; Modification; Molecular; Monitor; Nanostructures; Nucleic Acids; Organic Chemistry; Oxidation-Reduction; Peptides; Pharmaceutical Preparations; Physicians; Preparation; Proteins; Reporting; Research; Scheme; Signal Transduction; Streptavidin; Stress; Structure; Surface; Tacrolimus; Techniques; Technology; Temperature; Therapeutic; Time; Work; analog; base; clinically relevant; cost; design; exenatide; experience; feasibility research; health application; improved; innovation; instrumentation; interest; method development; novel; point of care; response; sensor; small molecule; success; temporal measurement; theories; user-friendly

Diagnosis and treatment of medical conditions could be revolutionized by technology capable of rapid and specific quantification of an arbitrary analyte in real time, over a wide concentration range. To quantify wide ranging clinically relevant targets—small molecules, nucleic acids, or proteins—most method development has drifted towards being target-focused and has lacked generalizability. Currently, the toolbox for potential point-of- care (POC) analysis is a conglomerate of methods or specially targeted probes, and measurement of many targets remains inaccessible to anything other than a large clinical laboratory. There is a pressing, unmet need to develop a platform amenable to rapid, quantitative readout of multiple classes of clinically relevant targets. Electrochemical (EC) sensors have attracted renewed interest for biomarker and drug quantification due to low cost and adaptability to the POC. Still, current approaches (aptasensors, steric hindrance assays) are lacking in generalizability or have complex, noncovalent structures that are not amenable to simple, drop-and-read workflow. In this proposal, we describe our recent development of an innovative nucleic acid nanostructure that exhibits unprecedented generalizability. Strong preliminary data shows this same nanostructure capable of quantifying proteins and antibodies (streptavidin, anti-digoxigenin, anti-exendin-4), peptides (exendin-4), and small molecules (biotin, digoxigenin, tacrolimus). The immunosuppressant drug, tacrolimus, can already be quantified in its therapeutic range. Our objective in this funding period is not only to further develop this new and promising technique, but also to develop a fully surface-confined version that allows true drop-and-read assay workflow that is ideal for POC or real-time clinical measurements. In Aim 1, we will expand the utility of the DNA nanostructure, and modification schemes will be adapted to the most efficient means of detecting proteins, peptides, and small molecules. Nine targeted analytes are relevant to stress/heart disease, immunosuppression, and diabetes monitoring. In Aim 2, we will use organic chemistry to make structural modifications to small molecules or peptides appended to anchor-DNA to fine-tune antibody binding equilibria and improve competitive assays for drop-and-read quantification. In Aim 3, we will develop a fully surface-confined sensor architecture for drop-and-read workflow and real-time measurements. Antibody-DNA or Fab-fragment-DNA conjugates will be used for tethering anchor molecules to the surface alongside DNA nanostructures. Finally, Aim 4 studies will develop instrumentation for improved sensitivity and user experience with the assay. The rationale for this research is to enable measurement of clinically relevant analytes previously inaccessible to EC, while providing a generalizable framework for many other future analytes. The proposed work is significant as a first-of-its-kind assay platform, which we expect to lead to an expanded list of future analytes, previously inaccessible to EC. This proposal is thus innovative in both its technological approach and in its human health applications. Preliminary evidence strongly supports feasibility, and the research team has a proven track-record of success.

能够快速、准确地诊断和治疗疾病的技术可以彻底改变疾病的诊断和治疗。 在很宽的浓度范围内实时对任意分析物进行特定定量。广泛量化 范围广泛的临床相关目标——小分子、核酸或蛋白质——大多数方法开发都已 偏向以目标为中心，缺乏普遍性。目前，潜在点的工具箱 护理（POC）分析是方法或专门针对探针的组合，并且测量许多 除了大型临床实验室之外，其他任何东西仍然无法访问目标。有一个紧迫的、未满足的需求 开发一个能够快速、定量读取多类临床相关目标的平台。 电化学 (EC) 传感器由于其低功耗而重新引起了人们对生物标志物和药物定量的兴趣。 成本和对 POC 的适应性。尽管如此，目前的方法（适体传感器、空间位阻测定）仍缺乏 普遍性或具有复杂的非共价结构，不适合简单的即放即读 工作流程。在本提案中，我们描述了我们最近开发的一种创新核酸纳米结构，该结构 表现出前所未有的普遍性。强有力的初步数据表明，同样的纳米结构能够 定量蛋白质和抗体（链霉亲和素、抗地高辛、抗 exendin-4）、肽（exendin-4）和 小分子（生物素、地高辛、他克莫司）。免疫抑制剂他克莫司已经可以 量化其治疗范围。我们在本次资助期间的目标不仅是进一步开发这一新的和 有前途的技术，而且还开发出一种完全表面限制的版本，允许真正的掉落和读取测定 非常适合 POC 或实时临床测量的工作流程。在目标 1 中，我们将扩大 DNA 的效用 纳米结构和修饰方案将适应最有效的蛋白质检测方法， 肽和小分子。九种目标分析物与压力/心脏病、免疫抑制、 和糖尿病监测。在目标 2 中，我们将使用有机化学对小分子进行结构修饰。 附加到锚定 DNA 上的分子或肽，以微调抗体结合平衡并提高竞争性 滴读定量分析。在目标 3 中，我们将开发一种完全表面限制的传感器架构 用于拖放式工作流程和实时测量。抗体-DNA 或 Fab 片段-DNA 缀合物将 可用于将锚分子与 DNA 纳米结构一起束缚在表面。最后，目标 4 研究将 开发仪器以提高检测的灵敏度和用户体验。这样做的理由 研究的目的是能够测量以前 EC 无法获得的临床相关分析物，同时提供 许多其他未来分析的通用框架。拟议的工作作为同类的首创具有重要意义 分析平台，我们预计该平台将扩大未来分析物的列表，这是 EC 以前无法访问的。 因此，该提案在技术方法和人类健康应用方面都是创新的。 初步证据有力地支持了可行性，并且研究团队拥有良好的成功记录。