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

• Applicant: Dr. Gang Wei
• Subject Area: Physical Chemistry of Solids and Surfaces, Material Characterisation ; Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 276102850

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.

• DFG Programme: Research Grants