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

基于原子力显微镜(AFM)的单分子力谱(SMFS)已经发展成为一种常规使用的技术，用于探测大约10pN的分子相互作用，这相当于断裂单氢键所需的力。由于其相对易于使用和高灵敏度，SMFS具有巨大的潜力，可以作为一种中央工具来检测特定(生物)分析物的存在，而不需要额外的标记分子的帮助，正如我们最近在初步交流中证明的那样。在这个项目中，我们想要探索SMFS作为一种无标记生物传感技术的能力和局限性，遵循三种不同的策略，所有这些策略都基于先前AFM尖端与特定生物分子连接物(DNA适配子或寡肽)的共价功能化。第一种策略依赖于在没有或存在与适配子有很大亲和力的分析物的情况下，检测适体功能化的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。第二种策略依赖于检测适体和它的互补序列之间测量的力的变化，这些序列嫁接在金底物上。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切割结合到AFM尖端的寡肽，以及另一侧的被动表面(通过亲和素/生物素桥)，由蛋白酶酶切割。在将寡肽置于张力下的强制夹持实验中，应通过监测连接物断裂前的特征时间来检测是否存在蛋白酶。前两种策略将应用于对污染金属离子(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传感。所有这三种策略都有望使我们能够检测到浓度低至10到100 PM的分析物除了开发这些策略外，我们的项目还旨在定义使传感选择性最大化和检测极限最小化的条件，并探索生物传感过程自动化的可能性。我们还期望该项目的成果将有助于从根本上了解DNA适配子(包括自由和结合到特定靶标的DNA适配子)与石墨/水界面的相互作用。最后，我们设想将这些技术扩展到广泛的传感应用中，用于无标记和高效地检测受污染的水中的有毒物质、药物分子、酶和病毒。

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.