Enzymatic nanolithography of FRET peptide layer using V8 protease-immobilized AFM probe

In our study, a method based on Enzymatic nanolithography was successfully performed in a buffered solution using Staphylococcal serine V8 protease and AFM. To estimate the lithographing activity of the protease immobilized on the AFM tip to peptides immobilized on a substrate, we designed fluorescence resonance energy transfer (FRET) peptides as reporter peptides that showed enzymatic action specific to the V8 protease. When the protease digested the reporter peptide a quencher residue was released from the peptide and resulted in the appearance of fluorescence. In the designed 9-mer peptides, TAMRA functioned as a good quencher for FAM. When the fluorescence resonance energy transfer peptides immobilized on a glass substrate were hydrolyzed by V8 protease at the C-terminal of glutamic acid, fluorescence of a reporter dye was observed because of the release of a quencher from the substrate. After contacting and lateral scanning of the protease-immobilized AFM tip to the reporter peptide layer, a fluorescent area was observed by imaging using total internal refection fluorescence microscopy (TIRFM). The increment of fluorescence intensity of the digested peptide indicates the performance of lithography. Lithographing rates increased in inverse relation to scanning rates of the probe. The maximum limit of the scanning rate, i.e., that was too fast to permit cutting of the peptide on the substrate, and the lithographing performance are discussed in this study.