Multiplexed Small-Molecule Detection Using Sequence-Defined DNA Reporters in an Argonaute−Nanopore System

Abstract

Multiplexed small-molecule detection remains challenging because chemically diverse analytes lack universal amplification and encoding mechanisms. Here, we present SMART-pore, a programmable molecular encoding-decoding platform, that converts target recognition events into sequence-defined DNA reporters and resolves them through machine-learning-assisted nanopore fingerprinting. A modular reporter architecture comprising a cobalamin (Cbl) anchor, an encoding domain, and an electrophoretic guiding segment was developed to generate orthogonal nanopore signatures. Target-induced release of guide DNAs activates PfAgo-mediated cleavage, producing sequence-defined DNA fragments that can be readily transformed into nanopore-readable reporters through a simple Cbl-capping reaction. Four representative mycotoxins (AFB1, OTA, PAT, and ZEN) were simultaneously detected with picomolar sensitivity, while an expanded six-reporter library demonstrated highly accurate machine-learning-assisted classification. The SMART-pore strategy establishes a scalable route for transforming molecular recognition into programmable nanopore-readable codes, providing a general framework for highly multiplexed small-molecule analysis.