Peptide Sequencing With Single Acid Resolution Using a <i>Sub</i> ‐Nanometer Diameter Pore

Abstract The first step toward an accurate, early diagnosis of a disease or for monitoring the efficacy of treatment for it is to identify biomarkers in a clinical specimen. To that end, a tool that identifies a peptide by directly “reading” the sequence of amino acid (AA) residues that constitute its primary structure with extreme sensitivity is badly needed. A nanofluidic assay that uses a sub‐nanometer‐diameter pore spanning an amorphous silicon (a‐Si) membrane a few nanometers thick (i.e., a sub‐nanopore) fits the bill. When a denatured peptide isoform (i.e., a peptidoform) is impelled by a high electric field through a sub‐nanopore immersed in electrolyte, the ionic current through the pore is partially blocked. The AA sequence can be read by measuring the fluctuations in the blockade current. To call the acid sequence, molecular dynamics (MD) simulations of the pore current, including predictors such as the acid volume, mobility, charge, and nearby water molecules, are used to decipher the amplitudes and timing of the fluctuations. To prove out the prospects for sequencing peptidoforms, a fragment of amyloid‐beta (Aβ 1−42 ), along with site‐specific post‐translational modifications of it and point mutations in it, which are biomarkers for Alzheimer's disease (AD), are assayed this way.