Nanoapertures for AFM-based single-molecule force spectroscopy

Simultaneous single–molecule force spectroscopy and microfluorescence binding measurements are often hampered by background fluorescence from the bulk. Zero–Mode Waveguides (ZMW) restrict the excited volume but require a special design, which allows the tip of the force probing cantilever to protrude into the nanoaperture. Here, we describe the design and fabrication of such ZMW and report the first experiments where binding of fluorescent adenosine triphosphate to the force–activated enzyme titin kinase was measured while the enzyme was subjected to mechanical forces.