Thin-Film-Based Three-Chamber Microfluidic Chip Powered by Pyrococcus furiosus Argonaute for Sample-to-Answer Multiplex Genotyping of Tuberculosis Drug-Resistance Mutations

Rapid genotyping of rifampicin (RIF) and isoniazid (INH) resistance in Mycobacterium tuberculosis is constrained by complex specimen processing, contamination risk, and multiplex single-nucleotide mutation discrimination. We report an automated three-detection-chamber thin-film microfluidic chip compatible with sputum, tongue swabs, and cultured suspensions, with pretreatment and nucleic-acid processing on chip. The automated, sealed chip integrates alcohol-free lysis, silica-membrane purification, PCR amplification, and single-enzyme Pyrococcus furiosus Argonaute (pfAgo) detection under programmed fluid routing. Results are reported in ∼73 min (18 min extraction, 35 min PCR, 20 min pfAgo cleavage). Guide-directed, sequence-specific pfAgo cleavage enables multiplex SNP calling in a single-enzyme cleavage scheme. Fluorophore-labeled reporters provide four-plex fluorescence reporting per chamber, enabling parallel readout across three chambers with mutation-specific guide DNAs. To our knowledge, this is the first thin-film chip demonstrating single-enzyme multiplex pfAgo detection, reporting 11 targets in one run. The panel includes IS6110 and 10 high-frequency RIF/INH resistance mutations in rpoB, katG, and inhA. IS6110 was detected down to 0.01 copies/μL and resistance mutations to 1-10 copies/μL, with 103 CFU/mL on-chip sensitivity, as determined by the proposed readout method. In 48 clinical TB specimens, chip calls showed 100% concordance with Sanger sequencing. By adding katG and inhA, our assay reports additional INH resistance in contrast to RIF-focused tests that interrogate only rpoB (e.g., Xpert MTB/RIF), providing more informative genotyping for clinical decision-making. This closed, automated platform supports scalable panel expansion for decentralized TB drug-resistance genotyping.