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、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。在较宽的浓度范围内对任意分析物进行实时特异性定量。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目前，潜在目标点的工具箱已转向以目标为中心，但缺乏通用性。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，目标仍然无法实现，这是一个紧迫的、未得到满足的需求。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。尽管如此，目前的方法（适体传感器、空间位阻测定）仍然缺乏。普遍性或具有复杂的非共价结构，不适合简单的即放即读在本提案中，我们描述了我们最近开发的一种创新的核酸纳米结构。强有力的初步数据表明，这种纳米结构具有前所未有的普适性。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）已经可以作为免疫抑制剂。我们在本次资助期间的目标不仅是进一步开发这种新的和治疗范围。技术，而且还开发一种有前途的完全表面限制版本，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，我们将扩展 DNA 的实用性。纳米结构和修饰方案将适应最有效的蛋白质检测方法，九种目标分析物与压力/心脏病、免疫抑制、在目标2中，我们将利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。用于滴读工作流程和实时测量抗体-DNA 或 Fab 片段-DNA 缀合物。最后，Aim 4 研究将用于将锚分子与 DNA 纳米结构一起束缚在表面。开发仪器以提高检测的灵敏度和用户体验。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，同时提供所提出的工作作为同类中的第一个工作具有重要意义。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。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Building a Nucleic Acid Nanostructure with DNA-Epitope Conjugates for a Versatile Approach to Electrochemical Protein Detection.

使用 DNA-表位缀合物构建核酸纳米结构，用于电化学蛋白质检测的多功能方法。

DOI：
发表时间：
2023-12-12
期刊：
影响因子：
7.4
作者：
Gurukandure, Asanka;Somasundaram, Subramaniam;Kurian, Amanda S N;Khuda, Niamat;Easley, Christopher J
通讯作者：
Easley, Christopher J

Ionic Strength and Hybridization Position near Gold Electrodes Can Significantly Improve Kinetics in DNA-Based Electrochemical Sensors.

金电极附近的离子强度和杂交位置可以显着改善基于 DNA 的电化学传感器的动力学。