Single-Molecule Discrimination of Labeled DNAs and Polypeptides Using Photoluminescent-Free TiO₂ Nanopores

Multicolor fluorescence substantially expands the sensing capabilities of nanopores by complementing or substituting the resistive pulsing signals. However, to date single-fluorophore detection in multiple color channels has proven to be challenging primarily due to high photoluminescence (PL) emanating from the silicon nitride (SiN_x) membrane. We hypothesize that the high bandgap of titanium oxide (TiO₂) would eliminate the PL background when used as a substrate for a nanopore, and hence enable individual fluorophore sensing during the fast passage of biomolecules through the pore. Herein, we introduce a method for fabricating locally supported, free-standing, TiO₂ membranes, in which solid-state nanopores can be readily drilled. These devices produce essentially no PL in the blue-to-red visible spectral range, even when excited by multiple lasers simultaneously. At the same time, the TiO₂ nanopores exhibit low electrical noise comparable with standard SiN_x devices. Importantly, the optical signal-to-background ratio (SBR) in single-molecule sensing is improved by an order of magnitude, enabling the differentiation among labeled DNA molecules of similar length based solely on their labeling scheme. Finally, the increased SBR of the TiO₂ devices allows detection of single fluorophores conjugated to the lysine or cysteine residues of short polypeptides, thus introducing the possibility for optical based peptide/protein discrimination in nanopores.