Detection and Characterization of Individual Proteins in Solution Using Solid State Nanopores

Amit Meller, Biomedical Engineering, Technion, Haifa, Israel

Nanopores are single molecule sensors, which utilize an electrophoretic focusing and transport of biomolecules through a nanoscale pore formed in a thin membrane. Nanopores fabricated in solid-state substrates were proven to be among the most sensitive nucleic acids biosensors. But the detection of individual small proteins has been challenging so far, primarily due to the poor signal to noise ratio that these molecules produce during their passage through the pore. Here we show that fine adjustments of the buffer’s acidity close to the analytes’ isoelectric point can be used to slow down the translocation speed of the analytes, hence permitting sensing and characterization of small globular proteins. Applying our method to the detection of mono and poly-Ub molecules, we show that we can discriminate among two equal molecular weight di-Ub conformers, which only posses a slightly different 3D structure due to a different Lysine linkage location. Our method opens up a novel approach for analyzing proteins at unprecedented detail using solid-state nanopores. It serves as a proof of concept for approaching nanopore detection of sub 10 kDa proteins and its ability to differentiate among native individual proteins of the same amino acid sequence.