Peptide Fingerprinting of Single Protein Molecules Using In‐Plane Nanopore Sensors Integrated to an Extended Immobilized Nanoscale Enzymatic Reactor (eINER)

ABSTRACT We report a thermoplastic nanofluidic device that combines an extended immobilized nanoscale enzymatic reactor (eINER) with a dual in‐plane nanopore time‐of‐flight (DNP‐ToF) reader for label‐free peptide fingerprinting of single protein molecules. As opposed to traditional membrane‐based nanopores, the DNP‐ToF reader uses two sub‐10 nm pores placed in series and are made from plastic to allow securing the resistive pulse sensing (RPS) independent ToF variable as well as the traditional RPS variables. The device enables on‐chip proteolytic digestion with downstream identification of peptide fragments. Using amyloid‐β (Aβ) as a model system, we demonstrate efficient digestion under nanoscale confinement consistent with a reaction‐limited regime and convection‐dominated transport. The device achieved robust event detection with >95% successful pairing of dual nanopore RPS signals enabling extraction of five key features (ΔI 1 /I 0 , ΔI 2 /I 0 , t D1 , t D2 , and ToF) from individual peptide events. Machine learning provided high‐confidence peptide identification with a random forest (RF) classifier achieving 100% accuracy and a cross‐validated accuracy of 99.6% ± 0.9%. Digestion efficiency was tunable through occupancy‐controlled conditions with RF‐assigned intact Aβ events decreasing from ∼9.5% at 1 nM to ∼0.33% at 100 pM. These results establish a scalable, high‐sensitivity platform for integrated single molecule analysis to secure proteomic data from mass‐limited samples.