Multiplexed electronic counting of scarce protein targets using nucleic acid nanoparticles

项目概要 这个跨学科项目整合了三个研究小组的专业知识，以进行原理验证 展示了一种准确、灵敏地检测稀有蛋白质生物标志物的新方法。关键 该方法的创新要素是使用线框状核酸纳米颗粒（NANP）来结合 直接或通过辅助抗体蛋白以高亲和力靶向蛋白质。蛋白质的结合 首先将样品与 NANP 混合物一起孵育，然后使用 固态膜中的纳米孔。蛋白质检测依赖于流过的离子电流的测量 纳米孔：当 NANP 为 某些类型进入纳米孔。重要的是，通过将 NANP 的物理尺寸与物理尺寸相匹配 纳米孔的尺寸，我们期望显着增加核酸的停留类型 纳米孔内的纳米颗粒，从而实现超灵敏（亚皮摩尔范围）的检测 蛋白质结合生物标志物。通过设计我们的 NANP 探针，在结合时产生不同的离子特征 他们的蛋白质目标，我们将使用与 以及通过将 NANP 探针和蛋白质生物标志物组装成一个生物标志物的组合检测 类似三明治的结构。该项目将由北卡罗来纳大学夏洛特分校的 Afonin 小组实施，该小组将设计 NANPs，东北大学的 Wanunu 小组将进行纳米孔检测实验，以及 Aksimentiev 小组将使用一系列建模技术来优化和改进检测 策略。我们的超灵敏、便携式、快速且潜在低成本的蛋白质定量技术 水平预计将广泛用于生物样品分析，最终提供灵敏、可靠、 以及疾病指示生物标志物的微创鉴定，这可能是重要的创新 癌症和其他疾病的早期诊断。

PROJECT SUMMARY This interdisciplinary project synergizes the expertise of three research groups for a proof-of-principle demonstration of a novel approach for accurate and sensitive detection of scarce protein biomarkers. The key innovative element of the approach is the use of wireframe-like nucleic acid nanoparticles (NANPs) to bind protein targets with high affinity either directly or by means of auxiliary antibody proteins. Binding of the protein targets is detected by first incubating the sample with a cocktail of NANPs and then examining them using a nanopore in a solid-state membrane. Protein detection relies on measurement of the ionic current flowing through the nanopore: the ionic current and particle dwell time decreases by a characteristic amount when a NANP of a certain type enters the nanopore. Importantly, by matching the physical dimensions of the NANP to the physical dimension of the nanopore, we expect to dramatically increase the residence type of the nucleic acid nanoparticles within the nanopore and thereby achieve ultra-sensitive (sub-picomolar range) detection of the protein-bound biomarkers. By designing our NANP probes to produce distinct ionic signatures when bound to their protein targets, we will achieve multiplex detection of several protein species using the same nanopore as well as combinatorial detection of biomarkers by assembling the NANP probes and protein biomarkers into a sandwich like structures. The project will be carried out by the Afonin group at UNC Charlotte that will design NANPs, the Wanunu group at Northeastern University that will perform the nanopore detection experiments, and the Aksimentiev group that will use an arsenal of modeling techniques to optimize and improve the detection strategy. Our ultra-sensitive, portable, rapid, and potentially low-cost technology for quantification of protein levels is expected to find broad use for the analysis of biological samples, eventually offering sensitive, reliable, and minimally invasive identification of disease-indicative biomarkers that could be important innovations for early-stage diagnostics of cancer and other diseases.