Single-Molecule Monitoring of Formaldehyde-Lysine Residue Chemistry using an Engineered MspA Nanopore

Formaldehyde (HCHO) is a reactive electrophile that covalently modifies diverse amino acid residues in proteins. Although ensemble studies have characterized reactions between HCHO and amino acids, the reaction dynamics at individual residue sites have yet to be resolved at the single-molecule level. Here, we engineer a Mycobacterium smegmatis porin A (MspA) nanopore by positioning a single lysine residue at its constriction, enabling real-time single-molecule monitoring of HCHO-lysine chemistry. Distinct current blockade states and reversible transitions between them are observed, consistent with multistep HCHO-lysine reactions involving monoadducts, bisadducts, and Schiff bases under nanoconfinement. The reactions exhibited concentration- and pH-dependent kinetics and generated characteristic electrical signatures that differentiate HCHO from potential interferents, supporting label-free detection of airborne HCHO via aqueous trapping, as well as identification of HCHO in food matrices. This work establishes a residue-confined nanopore platform for real-time probing of HCHO-amino acid chemistry and selective detection of HCHO.