A Cascaded RPA-SDA Amplification Strategy on a Sliding Microfluidic Chip for the Ultrasensitive and Rapid Detection...

In this study, a sliding microfluidic biosensor integrating RPA-SDA cascaded amplification was developed for the rapid, visual detection of Shigella. A novel RPA primer targeting the specific ipaH gene was designed to include a 5'-end G-quadruplex (G4) sequence and the complementary sequence of an Nt.BstNBI endonuclease recognition site. The RPA product templates a subsequent SDA reaction, generating abundant G4 structures that form peroxidase-mimicking DNAzymes with hemin, catalyzing a TMB reaction that produces a distinct blue color for visual readout (on-chip detection at OD₃₇₀, distinct from conventional tube assays at OD₄₅₀). The core on-chip detection process was completed within 13 min (10 min for SDA and 3 min for color development), achieving a limit of detection of 3.5 × 10^-4 ng/μL for Shigella genomic DNA. This timing explicitly excludes the preceding, off-chip steps of nucleic acid extraction and RPA amplification. Validation using spiked lettuce samples confirmed the platform's high specificity and sensitivity. This work establishes a proof-of-concept for a portable screening tool, highlighting its potential for on-site food safety applications. However, further validation in diverse food matrices and under real-world field conditions is required to fully establish its practical utility.