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

基于原子力显微镜（AFM）的单分子力光谱（SMFS）已经发展成为一种常规技术，用于探测大约10 pN范围内的分子相互作用，这对应于破坏单氢键所需的力。由于其相对容易使用和高灵敏度，SMFS具有很大的潜力，可以作为检测特定（生物）分析物存在的中心工具，而无需额外的标签分子的帮助，正如我们最近在初步通信中所证明的那样。在这个项目中，我们将探索SMFS作为一种无标记生物传感技术的能力和局限性，采用三种不同的策略，所有这些策略都基于先前的AFM尖端与特定生物分子连接体（DNA适体或寡肽）的共价功能化。第一种策略依赖于检测适配体功能化的AFM尖端和被动固体底物（通常是石墨表面）之间的平均吸附力的变化，无论是否存在与适配体有很大亲和力的分析物。第二种策略依赖于检测接枝在金衬底上的适配体及其互补序列之间测量的力的变化。在这里，与分析物的结合预计会阻碍或至少减少两条互补链之间的杂交。第三种策略依赖于酶促裂解，将一侧与AFM尖端结合的寡肽和另一侧的被动表面（通过亲和素/生物素桥接）通过蛋白酶裂解。在将寡肽置于张力下的夹紧力实验中，应通过监测连接体破裂前的特征时间来检测蛋白酶的存在。前两种策略将应用于污染金属离子（如Hg2+， Co2+或Pb2+）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检测。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。除了开发这些策略外，我们的项目旨在确定最大化传感选择性和最小化检测限制的条件，并探索自动化生物传感过程的可能性。我们还期望本项目的成果将有助于从根本上理解DNA适体（包括自由的和与特定靶标结合的）与石墨/水界面之间的相互作用。最后，我们设想将这些技术扩展到广泛的传感应用中，以实现对污染水中有毒物质、药物分子、酶和病毒的无标签和高效检测。

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.