CAREER: Enhancing molecular recognition biosensing with nanopore force measurements

生物功能从拥挤、复杂的环境中出现，在这个环境中，分子内和分子间转换的精细进展由相互作用的物种之间的能量景观编排。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阳离子分子背景中的低水平分子信号做出反应。这项提议的目标是开发一种能够探测这些相互作用能量的技术，使人们能够在原子水平上理解分子识别，并将其直接应用于高灵敏和选择性的生物传感。这一提议的核心是纳米孔力光谱的开发和应用，这是一种使分子相互作用能够在单分子水平上被探测的技术。纳米孔--绝缘膜上的分子尺度小孔--能够实现单分子传感，而不需要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。适体分子识别试剂将与非离子表面活性剂结合来克服这一缺陷。此外，NFS将被开发为一种工具来探测和利用广泛类别的受体-配体相互作用。拟议的研究将从基于NFS的基本适配子属性的测量，如在没有靶子的情况下的构象稳定性，到表征利用独特的分子约束和化学可调的NFS传感环境的适配子性能增强。在这些初步的对照实验之后，将使用适体靶标相互作用的非对称测量来优化实验条件，然后探索适体-非对称相互作用的灵敏度和选择性的极限。然后将通过表面电荷变化的非局域表面功能化来探索受限纳米金属环境所提供的独特机会。这将为后续纳米孔内部位置选择表面功能化的开发和表征提供基础。将出现的关于化学受限环境的见解将通常对纳米制造社区有用。在模型药物筛选分析之前，我们将探索固态纳米孔的局部表面化学修饰对适体-NFSs的影响。所提出的研究的智力优势体现在以下几点：(1)纳米孔力光谱的开发和演示作为研究和利用受体-配体相互作用的一般方法，将(I)为测力工具箱引入一个新的工具--具有独特的可调纳米尺度性质的工具，以及(Ii)显著扩展纳米孔方法的应用。与原子力显微镜(AFM)等其他作用力方法不同，NFS将分子约束在定义明确的分子尺度环境中，允许调节纳米孔与分子的相互作用，就像在酶结合口袋中自然完成的那样。(2)利用人工抗体？人工抗体？核酸适体分子识别试剂与核糖核酸的结合将丰富纳米孔的生物传感能力。核糖核酸本身可以通过实时、直接地测量适配子与靶标的相互作用来扩展适配子传感的能力和用途。(3)受定点突变性能的启发，一种定点选择的固态纳米孔功能化方法的开发将允许将先前关于蛋白质纳米孔功能化的大量工作转移到更健壮、可调的固态纳米孔上。(4)对DNA结合的小分子与蛋白质之间相互作用的成功的NFS测量将作为用于直接药物筛选的NFS是可行的原理证明实验。所提议的研究的更广泛的影响包括：(1)力显微镜工具包的扩展，包括一个健壮、廉价、易于使用的单分子纳米孔方法在适合在实验室外部署的平台中。(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。积极拓展活动以增加公众对科学和工程的理解和欣赏？S将针对：a.通过各种媒体和社区活动，向普通公众宣传科学和工程；b.公关专业的学生，他们的职业生涯可能包括科学和工程交流；c.通过非正式互动和动手活动，让K-12学生在多元化的城市学校学习工程技能；d.大学本科生，通过制造廉价的诊断设备，学习工程技能；E.研究人员，他们自己，通过强调统一科学和工程活动来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.