研究目的
To establish a method for manipulating individual biomolecules inside zero mode waveguides (ZMWs) with an AFM cantilever, enabling simultaneous measurements of force response and fluorescence at high labeled substrate concentrations.
研究成果
The study demonstrates a method for routinely manipulating individual biomolecules inside ZMWs with an AFM cantilever, enabling simultaneous force and fluorescence measurements at high fluorophore concentrations. This approach opens new avenues for investigating force-mediated biochemical pathways and mechanosensing.
研究不足
The method is limited by the aspect ratio of the cantilever tip, which restricts the size of ZMWs and the concentration of fluorophores that can be used. High aspect ratio cantilevers are needed for smaller ZMW diameters.
1：Experimental Design and Method Selection
The study employs a custom-built TIRF AFM hybrid for simultaneous three laser line excitation and fluorescence readout. A protein model system of streptavidin blocked with a biotin-tagged ligand is used to demonstrate the method.
2：Sample Selection and Data Sources
Monovalent streptavidin (mSA) blocked with a biotinylated ligand construct is used as a force-activatable system. The ligand is pulled out of streptavidin by an AFM cantilever, allowing reoccupation by fluorescently labeled biotin.
3：List of Experimental Equipment and Materials
AFM cantilevers (BioLever mini, Olympus Corporation), ZMW chips fabricated in-house, Cy5-labeled biotin, TROLOX, pyranose oxidase, and catalase.
4：Experimental Procedures and Operational Workflow
The cantilever tip is localized and aligned to ZMWs for probing. The AFM cantilever is used to mechanically manipulate single molecules inside ZMWs, with fluorescence and force measurements recorded synchronously.
5：Data Analysis Methods
Fluorescence and force data are analyzed to study rebinding rates and mechanical unblocking events.
独家科研数据包，助您复现前沿成果，加速创新突破
获取完整内容
