A Nucleic Acid-Encoded Universal Array Enables Multiplex Pathogen Detection Through Programmable Cascade Amplification

Respiratory infections have high mortality, exacerbated by frequent co-infections and antimicrobial resistance, posing challenges for timely, accurate diagnostics. Arrays offer multiplex pathogen detection, but remain limited by complex instrumentation, complex probe design, and poor universality. Here, a programmable and universally applicable array platform, termed the universal cascade amplification molecular signal conversion system (UCSC), is established with a core based on flap endonuclease 1 (FEN1)-mediated linear amplification reaction (LIAR) coupled with hybridization chain reaction (HCR). Target-independent flap sequences encoded in the primers serve as universal mediators, decoupling target recognition from signal output. Upon recognition, the flap initiates LIAR to amplify the universal sequence, which subsequently triggers HCR-driven isothermal selfassembly of DNA nanowires on the array for direct visual readout. This cascade amplification strategy establishes precise correspondence between recognition, amplification, and signal output, overcoming the "application-specific" limitation of traditional arrays. The platform enables multiplexed detection of nine respiratory pathogens with high sensitivity (1.13-1.25 copies & sdot;mu L- 1) and specificity, completing the workflow within 70 min without sophisticated equipment. Unlike qPCR requiring thermocycling or most isothermal methods needing target-specific redesign of both recognition and amplification primers, UCSC interrogates new targets by replacing only the recognition primers, while downstream amplification modules and arrayed probes remain fixed, enabling a single chip to perform versatile, multiplexed diagnostics across pathogens and molecular analyses, including serotyping, antibiotic resistance, methylation, and single-nucleotide polymorphisms, establishing a broadly deployable toolkit that operates without reliance on dedicated laboratory instruments and supports smartphone-based readout, offering strong potential for pathogen detection in resource-limited and epidemiological surveillance.