Multiplexed electronic counting of scarce protein targets using nucleic acid nanoparticles

项目总结 这个跨学科的项目结合了三个研究小组的专业知识，以进行原则证明 展示了一种准确而灵敏地检测稀缺蛋白质生物标志物的新方法。钥匙 该方法的创新元素是使用线框状核酸纳米颗粒(NANP)结合 直接或通过辅助抗体蛋白具有高亲和力的蛋白质靶标。蛋白质的结合 检测目标的方法是首先将样本与NANP的鸡尾酒孵化，然后使用 固态膜中的纳米孔。蛋白质的检测依赖于流经的离子电流的测量 纳米孔：当NANP的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 纳米孔的尺寸，我们预计将显著增加核酸的驻留类型 纳米孔内的纳米颗粒，从而实现超灵敏(亚皮摩尔范围)对 蛋白质结合的生物标记物。通过将我们的NANP探针设计为在结合到 他们的蛋白质靶标，我们将使用相同的纳米孔实现对几种蛋白质物种的多重检测 以及通过将NANP探针和蛋白质生物标记组装成 三明治状的结构。该项目将由北卡罗来纳大学夏洛特分校的阿弗宁小组实施，该小组将设计 东北大学的Wanunu小组将进行纳米孔探测实验，以及 Aksimentiev小组将使用大量建模技术来优化和改进检测 策略。我们的超灵敏、便携、快速、潜在低成本的蛋白质定量技术 预计Level将广泛用于生物样本的分析，最终提供灵敏、可靠、 以及微创识别疾病指示生物标记物，这可能是 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.