In-Line Tapered Microfiber Sensors for Label-Free Simultaneous Detection of Dual Genes via Enzymatic Recombinase Amplification

As a sensitive and efficient isothermal amplification method, enzymatic recombinase amplification (ERA) holds strong potential for nucleic-acid-based disease diagnostics. However, current multiplexed ERA assays rely on distinct fluorescent labels to separate detection signals, increasing cost and optical complexity. Herein, we present a label-free dual-gene detection platform that integrates ERA with refractive-index-based sensing using an in-line tapered microfiber transducer. Gene-specific primer pairs are covalently immobilized on spatially separated microfiber segments via click chemistry, enabling site-selective initiation of ERA and inducing measurable spectral shifts. Fast Fourier transform and frequency-selective filtering of the composite optical signal allow real-time, parallel monitoring of amplification for each target without fluorescent labeling. Using hepatitis B virus and hepatitis C virus genes as examples, the system achieves simultaneous detection and discrimination within 20 min, covering a range from 10 to 10⁵ copies/μL with a detection limit of 13 copies/μL per target, using only ∼15 μL of the sample. The sensing result was comparable to that of the standard fluorescent ERA assay. Spatial selectivity was confirmed using nontarget sequences, with each region responding exclusively to its corresponding target. Moreover, the sensor demonstrated applicability in detecting viral nucleic acids extracted from clinical serum samples and validated against fluorescence-based ERA assays, confirming accuracy in real-world diagnostics, highlighting its potential for practical diagnostic applications. The flexible layout and remote sensing capability of optical fibers may further reduce onsite instrumentation needs, offering promise for deployment in outbreak or resource-limited settings.