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

基本信息

批准号：
10671646
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
项目类别：
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

项目摘要

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.

能够快速和在较大浓度范围内实时的任意分析物的特定数量。量化宽范围与临床相关的靶标 - 小分子，核酸或蛋白质 - 最大的方法发展具有倾向于以目标为中心，缺乏普遍性。目前，潜在点的工具箱护理（POC）分析是方法或特殊目标问题的集合，并且测量了许多方法除大型临床实验室以外的任何事物都无法访问目标。有一个紧迫的，未满足的需求为了开发一个适合多种类别相关目标的快速，定量读数的平台。电化学（EC）传感器引起了对生物标志物和药物定量的新兴趣对POC的成本和适应性。尽管如此，还缺乏当前的方法（Aptasensors，空间障碍分析）可推广性或具有复杂的，非共价结构，这些结构不适合简单，滴读工作流程。在该提案中，我们描述了我们最近对创新核酸纳米结构的发展表现出前所未有的概括性。强的初步数据显示了同样的纳米结构量化蛋白质和抗体（链霉亲和素，抗二戈氧基蛋白，抗抑制蛋白4），肽（Exendin-4）和小分子（生物素，二高氧素，他克莫司）。免疫抑制药物克莫司可以是在其治疗范围内进行量化。我们在这个资金期间的目标不仅是为了进一步发展这个新的和有希望的技术，但也要开发一个完全表面上的版本，该版本允许真正的滴定测定法适用于POC或实时临床测量的工作流程。在AIM 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检测蛋白质的方法，肽和小分子。九个针对性分析物与压力/心脏病，免疫抑制有关，和糖尿病监测。在AIM 2中，我们将使用有机化学对小型化学进行结构修饰分子或胡椒粉附加以锚定DNA以微调抗体结合并改善竞争力滴定数量的测定。在AIM 3中，我们将开发一个完全表面上的传感器架构用于掉落工作流和实时测量。抗体-DNA或Fab-Fragment-DNA结合物将与DNA纳米结构一起将锚固分子绑在表面上。最后，AIM 4研究将开发仪器可提高敏感性和用户体验，并具有测定法。理由研究是为了启用以前无法访问的临床相关分析物的测量，同时提供许多其他未来分析物的可推广框架。拟议的工作是首先的工作测定平台，我们希望这会导致扩展的未来分析物，以前无法访问EC。因此，该建议在技术方法和人类健康应用中都是创新的。初步证据强烈支持可行性，研究团队获得了成功的成功纪录。