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

医疗条件的诊断和治疗可以通过能够快速和 在很宽的浓度范围内，实时对任意分析物进行特定的定量。量化广度 范围临床相关的靶点--小分子、核酸或蛋白质--大多数方法的发展 倾向于以目标为重点，缺乏概括性。目前，潜在接入点的工具箱 CARE(POC)分析是一种方法或特殊目标探针的集合，并测量了许多 除大型临床实验室外，其他任何机构都无法接触到目标。有一种迫切的、未得到满足的需求 开发一个平台，能够快速、定量地读出多种临床相关靶点。 电化学传感器在生物标志物和药物定量方面因其低成本而重新引起人们的兴趣 成本和对PoC的适应性。尽管如此，目前的方法(适配子传感器、空间位阻分析)在 通用性或具有复杂的非共价结构，不适合简单的丢弃和阅读 工作流程。在这项提案中，我们描述了我们最近开发的一种创新的核酸纳米结构，该结构 表现出前所未有的普适性。强有力的初步数据显示，这种相同的纳米结构能够 定量蛋白质和抗体(链霉亲和素、抗地高辛、抗exendin-4)、多肽(exendin-4)和 小分子(生物素、地高辛、他克莫司)。免疫抑制药物他克莫司已经可以 在其治疗范围内进行量化。我们在这一资金阶段的目标不仅是进一步开发这一新的和 前景看好的技术，但也开发了一种完全表面受限的版本，允许真正的滴读分析 非常适合POC或实时临床测量的工作流程。在目标1中，我们将扩展DNA的用途 纳米结构和修饰方案将适用于检测蛋白质的最有效手段， 多肽和小分子。九种靶向分析物与应激/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 附加在锚定DNA上的分子或多肽，以微调抗体结合平衡并提高竞争性 滴读定量分析方法。在目标3中，我们将开发一种完全表面受限的传感器体系结构。 用于拖放和读取工作流程和实时测量。抗体-DNA或Fab-片段-DNA偶联物 用于将锚定分子与DNA纳米结构捆绑在一起。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。这样做的理由是 研究是为了能够测量以前EC无法接触到的临床相关分析物，同时提供 一个适用于许多其他未来分析物的通用框架。这项拟议的工作具有重大意义，因为这是此类工作的第一次。 分析平台，我们预计这将导致未来分析物的扩大列表，以前欧共体无法访问。 因此，这项提议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，研究小组已经证明了成功的记录。

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.