Developing label-free biosensing techniques with AFM-based single-molecule force spectroscopy

利用基于 AFM 的单分子力谱开发无标记生物传感技术

• 批准号: 276102850
• 负责人: Dr. Gang Wei
• 金额: --
• 依托单位: Conrad Naber Stiftungsprofessur Grenzflächen in der Bio-Nano-Werkstofftechnik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/276102850?language=en
• 关键词: Developing; label; free; biosensing; techniques

Single-Molecule Force-Spectroscopy (SMFS) based on atomic force microscopy (AFM) has evolved as a routinely used technique to probe molecular interactions in the range of about 10 pN, which corresponds to the force required to rupture single hydrogen bonds. Because of its relatively easiness of use and high sensitivity, SMFS has a great potential as a central tool to detect the presence of specific (bio)analytes without the help of additional label molecules, as we have recently proven in a preliminary communication. In this project we would like to explore the capabilities and limits of SMFS as a label-free biosensing technique following three different strategies, all based on the previous covalent functionalization of AFM tips with specific biomolecular linkers (DNA aptamers or oligopeptides). The first strategy relies on detecting a change of the average adsorption force between an aptamer-functionalized AFM tip and a passive solid substrate (typically, a graphite surface) in the absence or in the presence of analytes that bind with large affinity to the aptamer. The second strategy relies on detecting a change of force measured between the aptamer and its complementary sequence grafted on a gold substrate. Here, binding to the analyte is expected to hinder, or at least reduce, the hybridization between the two complementary strands. The third strategy relies on the enzymatic cleavage of an oligopeptide bound to the AFM tip on one side, and to a passive surface (via an avidin/biotin bridge) on the other side, by a protease enzyme. In force-clamping experiments where the oligopeptide is put under tension, the presence of proteases shall be sensed by monitoring the characteristic time before rupture of the linker. The first two strategies will be applied to the sensing of polluting metal ions (such as Hg2+, Co2+ or Pb2+) and drug molecules (such as adenosine or cocaine). The third strategy will be applied to the sensing of thrombin. All three strategies are expected to enable us to sense the analytes in concentrations as low as 10 to 100 pM. Beside developing these strategies, our project aims at defining the conditions that maximize the sensing selectivity and minimize the detection limit, and at exploring the possibility of automatizing the biosensing process. We also expect that the achievements in this project will be helpful for a fundamental understanding of the interactions between DNA aptamers (both free and bound to their specific target) and graphite/water interfaces. Finally, we envisage possible extensions of these techniques to a wide range of sensing applications for a label-free and highly effective detection of toxic substances in polluted water, drug molecules, enzymes, and viruses.

基于原子力显微镜（AFM）的单分子力谱（SMFS）已经发展为常规使用的技术，以探测约10 pN范围内的分子相互作用，其对应于断裂单个氢键所需的力。由于其相对易于使用和高灵敏度，SMFS具有很大的潜力，作为一个中心工具来检测特定的（生物）分析物的存在，而无需额外的标记分子的帮助，正如我们最近在初步沟通证明。在这个项目中，我们想探索的能力和限制SMFS作为一种无标记的生物传感技术以下三种不同的策略，所有基于先前的共价功能化的AFM提示与特定的生物分子连接器（DNA适配体或寡肽）。第一种策略依赖于检测在不存在或存在以大亲和力结合到适体的分析物的情况下适体官能化AFM针尖和被动固体基底（通常为石墨表面）之间的平均吸附力的变化。第二种策略依赖于检测适体与其接枝在金基底上的互补序列之间测量的力的变化。在此，预期与分析物的结合会阻碍或至少减少两条互补链之间的杂交。第三种策略依赖于结合到AFM针尖一侧的寡肽的酶促切割，并且通过蛋白酶结合到另一侧的被动表面（通过抗生物素蛋白/生物素桥）。在将寡肽置于张力下的力钳实验中，应通过监测接头断裂前的特征时间来感测蛋白酶的存在。前两种策略将应用于污染金属离子（如Hg 2+，Co 2+或Pb 2+）和药物分子（如腺苷或可卡因）的传感。第三种策略将应用于凝血酶的传感。所有这三种策略都有望使我们能够检测浓度低至10至100 pM的分析物。除了开发这些策略，我们的项目旨在定义最大化传感选择性和最小化检测限的条件，并探索自动化生物传感过程的可能性。我们还希望，在这个项目中的成就将有助于DNA适体（自由和结合到其特定的目标）和石墨/水界面之间的相互作用的基本理解。最后，我们设想这些技术可能扩展到广泛的传感应用，用于无标记和高效检测污染水，药物分子，酶和病毒中的有毒物质。