CAREER: Enhancing molecular recognition biosensing with nanopore force measurements

1150085德怀尔生物学功能出现在一个拥挤的、复杂的环境中，在这个环境中，分子内和分子间的转化是由相互作用的物种之间的能量交换精心设计的。分子识别利用高度进化的分子对这种相互作用能量的敏感性，使生物系统能够选择性地对非正常分子背景中的低水平分子信号作出反应。该提案的目标是开发一种可以探测这些相互作用能的技术，从而实现对分子构象的基本原子水平的理解及其在高灵敏度和选择性生物传感中的直接应用。该提案的核心是纳米孔力光谱（NFS）的发展和应用，这是一种能够在单分子水平上探测分子相互作用的技术。纳米孔？绝缘膜上的分子级孔洞使单分子传感没有单分子光学技术的成本或复杂性。虽然敏感，但纳米孔缺乏强大的天然化学选择性。适体分子识别剂将与NFS结合以克服这一缺陷。此外，NFS将被开发为探测和利用广泛类别的受体-配体相互作用的工具。拟议的研究将从基于NFS的基本适体性质的测量进展，例如在没有靶标的情况下的构象稳定性，以表征利用独特的分子约束和化学可调NFS传感环境的适体性能增强。在这些初始对照实验之后，将使用适体靶相互作用的NFS测量来优化实验条件，然后探索适体-NFS灵敏度和选择性的极限。由约束nanoporeenvironment提出的独特的机会，然后将探索通过nonlocalized表面功能化，其中表面chargeis变化。这将为随后的开发和表征纳米孔内部的位点选择性表面功能化提供基础。与约束环境中的化学有关的见解将对纳米纤维界普遍有用。本研究的主要学术价值体现在以下几点：（1）纳米孔力谱作为一种探索和利用受体-配体相互作用的通用方法，其发展和应用将（i）为力测量工具箱引入一种新的工具？一个具有独特的可调纳米级性能和（ii）显着地扩展nanopore方法的效用。与其他力的方法，如原子力显微镜（AFM），NFS约束分子在一个明确的，分子尺度的环境，允许调整的nanopore分子相互作用，如在酶结合口袋的性质。(2)化学选择性纳米孔传感的发展？人工抗体？适体分子识别剂与NFS的结合将丰富纳米孔生物传感能力，NFS本身可以通过提供对适体-靶标相互作用的实时、直接测量来扩展适体传感的能力和实用性。弱的，非特异性的相互作用，否则导致假阳性可以被拒绝。(3)受定点诱变性能的启发，位点选择性固态纳米孔功能化方法的开发将允许将关于蛋白质纳米孔功能化的大量先前工作转移到更稳健、可调的固态纳米孔。(4)成功的NFS测量DNA结合的小分子和蛋白质之间的相互作用将作为NFS用于直接药物筛选的可行性的原理验证实验，拟议研究的更广泛的影响包括：（1）扩展力显微镜工具包，包括一个强大的，廉价的，易于使用的单分子纳米孔方法在一个适合在实验室外部署的平台。(2)培养科学和工程研究人员成为熟练的科学和工程传播者。积极拓展，提高公？的理解和欣赏的科学和工程将针对：a。通过各种媒体和社群活动向公众宣传，B.公共关系专业的学生，其职业可能包括科学和工程传播，c. K-12学生在一个多样化的，城市学校通过非正式的互动和动手活动，d。大学本科生，他们将学习工程技术，因为他们制造廉价的诊断设备，e。研究人员本身通过强调统一科学和工程活动以满足人类需求的重要性。

1150085DwyerBiological function emerges from a crowded, complex environment in which the exquisiteprogression of intra- and intermolecular transformations is choreographed by the energy landscapebetween interacting species. Molecular recognition exploits highly evolved molecular sensitivity to thisinteraction energy to allow biological systems to respond selectively to low-level molecular signals in anoisy molecular background. The goal of this proposal is to develop a technique that can probe theseinteraction energies, enabling both the fundamental atomic-level understanding of molecular recognitionand its direct application for highly sensitive and selective biosensing. The core of this proposal is thedevelopment and application of nanopore force spectroscopy (NFS), a technique that enables molecularinteractions to be probed on the single molecule level. Nanopores?molecular-scale holes in insulatingmembranes?enable single molecule sensing without the cost or complexity of single molecular opticaltechniques. While sensitive, nanopores lack robust native chemical selectivity. Aptamer molecularrecognition agents will be combined with NFS to overcome this deficiency. In addition, NFS will bedeveloped as a tool to probe and exploit broad classes of receptor-ligand interactions.The proposed research will progress from NFS-based measurements of fundamental aptamerproperties such as conformational stability in the absence of target to characterizing aptamerperformance enhancements that leverage the unique molecularly-constrained and chemically tunableNFS sensing environment. Following these initial control experiments, NFS measurements of aptamertargetinteractions will be used to optimize experimental conditions and then to explore the limits ofaptamer-NFS sensitivity and selectivity. The unique opportunities presented by the constrained nanoporeenvironment will then be explored through nonlocalized surface functionalization in which surface chargeis varied. This will provide the basis for the subsequent development and characterization of site-selectivesurface functionalization in the nanopore interior. The insights that will emerge relating to chemistry inconstrained environments will be generally useful to the nanofabrication community. The effect of localsurface chemical modification of the solid-state nanopores on aptamer-NFS will then be explored before amodel drug-screening assay will be tested.The intellectual merit of the proposed research is captured in the following points:(1) The development and demonstration of nanopore force spectroscopy as a general method forexploring and exploiting receptor-ligand interactions will (i) introduce a new tool to the forcemeasurement toolbox?one with unique tunable nanoscale properties and (ii) dramatically extendthe utility of nanopore methods. In contrast to other force methods such as atomic force microscopy(AFM), NFS constrains molecules in a well-defined, molecular-scale environment allowing tuning ofthe nanopore-molecule interactions as done by nature in an enzyme binding pocket.(2) The development of chemically selective nanopore sensing by using ?artificial antibody? aptamermolecular recognition agents in combination with NFS will enrich nanopore biosensing capabilities.NFS, itself, can extend the power and utility of aptamer sensing by providing a real-time, directmeasurement of aptamer-target interactions. Weak, nonspecific interactions otherwise leading tofalse positives can be rejected.(3) Inspired by the performance of site-directed mutagenesis, the development of a site-selective solidstate nanopore functionalization method will allow the extensive prior body of work on proteinnanopore functionalization to be transferred to more robust, tunable solid-state nanopores.(4) The successful NFS measurement of the interaction between a DNA-bound small molecule and aprotein will serve as the proof-of-principle experiment that NFS is feasible for straightforward drugscreening.The broader impacts of the proposed research include:(1) The expansion of the force microscopy toolkit to include a robust, inexpensive, easy-to-use singlemolecule nanopore method in a platform suitable for deployment outside the laboratory.(2) The training of science and engineering researchers to become skilled science and engineeringcommunicators. Active outreach to increase the public?s understanding and appreciation of scienceand engineering will target:a. the general public through various media and community activities,b. public relations students whose careers may include science and engineering communication,c. K-12 students in a diverse, urban school through informal interactions and hands-on activities,d. university undergraduates who will learn engineering skills as they fabricate inexpensivediagnostic devices,e. the researchers, themselves, through the emphasis on the importance of unifying scientific andengineering activities to address human needs.