(338f) N-Terminal Derivatization-Assisted Identification of Individual Amino Acids Using a Biological Nanopore

Nanopore technology holds remarkable promise for sequencing proteins and peptides. To achieve this, it is necessary to establish a characteristic profile for each individual amino acid through the statistical description of their translocation process. However, the subtle molecular differences among all twenty amino acids along with their unpredictable conformational changes at the nanopore sensing region result in very low distinguishability. Here we report the electrical sensing of individual amino acids using an α-hemolysin nanopore based on a derivatization strategy. Using derivatized amino acids as detection surrogates not only prolongs their interactions with the sensing region, but also improves their conformational variation. Furthermore, we show that distinct characteristics including current blockades and dwell time can be observed among all three classes of amino acids after 2,3-naphthalenedicarboxaldehyde (NDA)- and 2-naphthylisothiocyanate (NITC)-derivatization, respectively. These observable characteristics were applied towards the identification and differentiation of 9 of the 20 natural amino acids using their NITC derivatives. The method demonstrated herein will pave the way for the identification of all amino acids and further protein and peptide sequencing.

Figure (a) Schematic of NDA and NITC derivatization of 9 amino acids in 3 classes and the experimental setup (not to scale). An external voltage is applied to the trans side of the lipid bilayer while the cis side is grounded. (b) Representative fragment of ionic current recording before (open pore) and after (translocation blockades) NITC-Tyr derivatives were added. (c) Illustration of typical signal events caused by translocation blockade. (d) Histogram of events per bin of current blockade I/I₀ (left versus bottom axis) and scatter-plot of dwell time versus I/I₀ (right versus bottom axis) produced by NITC-Tyr in an α-HL nanopore. Inset depicts the histogram of events per bin of dwell time for NITC-Tyr.