CAREER: Enhancing molecular recognition biosensing with nanopore force measurements

[1150085]生物功能产生于拥挤复杂的环境中，在这种环境中，相互作用的物种之间的能量景观精心设计了分子内和分子间转化的精细进展。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，允许生物系统在嘈杂的分子背景下选择性地响应低水平分子信号。本提案的目标是开发一种可以探测这些相互作用能量的技术，使分子识别的基本原子水平理解和其直接应用于高灵敏度和选择性生物传感。该提案的核心是纳米孔力谱（NFS）的开发和应用，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？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。适配体分子识别剂将与NFS结合以克服这一缺陷。此外，NFS将被开发为一种工具来探测和利用广泛的受体-配体相互作用。拟议的研究将从基于nfs的基本适配体特性测量（如缺乏目标的构象稳定性）进展到利用独特的分子约束和化学可调的lenfs传感环境来表征适配体性能增强。在这些初始控制实验之后，NFS对适体与靶标相互作用的测量将用于优化实验条件，然后探索适体与NFS敏感性和选择性的限制。受约束的纳米环境所提供的独特机会将通过表面电荷变化的非局部表面功能化来探索。这将为纳米孔内部选择性表面功能化的后续开发和表征提供基础。与化学无约束环境相关的见解将对纳米制造社区普遍有用。在进行模型药物筛选试验之前，将探索固态纳米孔局部表面化学修饰对适体- nfs的影响。提出的研究的智力价值体现在以下几点：(1)纳米孔力光谱作为探索和利用受体-配体相互作用的一般方法的发展和示范将(i)为力测量工具箱引入新的工具？一是具有独特的可调纳米尺度特性，二是极大地扩展了纳米孔方法的实用性。与原子力显微镜（AFM）等其他作用力方法相比，NFS将分子限制在定义良好的分子尺度环境中，允许调整纳米孔分子相互作用，就像在酶结合口袋中自然完成的那样。(2)利用？人工抗体?适配体分子识别剂与NFS的结合将丰富纳米孔生物传感能力。NFS本身可以通过提供适配体-目标相互作用的实时、直接测量来扩展适配体感知的功能和效用。弱的，非特异性的相互作用，否则会导致假阳性，可以拒绝。(3)受位点定向诱变性能的启发，位点选择性固体纳米孔功能化方法的发展将允许广泛的先前蛋白质纳米孔功能化工作转移到更健壮、可调的固体纳米孔上。(4) NFS对dna结合小分子与蛋白质之间相互作用的成功测量将作为NFS用于直接药物筛选的可行性的原理验证实验。拟议研究的更广泛影响包括：(1)力显微镜工具包的扩展，包括一个强大的，廉价的，易于使用的单分子纳米孔方法，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。积极外展以增加公众？学生对科学和工程的理解和欣赏将针对：公众通过各种媒体和社区活动；公共关系专业的学生，其职业可能包括科学和工程通信，c。K-12学生在一个多元化的城市学校通过非正式的互动和实践活动。大学本科生将在制造廉价诊断设备的过程中学习工程技能。这些研究人员通过强调统一科学和工程活动来解决人类需求的重要性。

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.