Spider-Inspired S-Loop Amplification Repurposes an Electron Cage for Photoelectrochemical–Fluorescence Detection of Perfluorooctanoic Acid

Abstract Per- and polyfluoroalkyl substances require analytical methods that combine sensitivity with internal result checking. Here, we report an S-Loop CRISPR dual-mode assay (SCDA) for perfluorooctanoic acid (PFOA). PFOA-induced aptamer displacement initiates an AMPLON-derived isothermal S-Loop reaction mediated by an eight-arm poly(ethylene glycol) spider-inspired primer (SP), and the resulting amplicons activate Cas12a trans-cleavage. A FAM–quencher reporter provides the fluorescence (FL) output, whereas cleavage-induced removal of CdS from a CdS/gold nanoparticle/ZnIn2S4 photoelectrode releases an electron cage produced by type-I carrier confinement and visible-light shielding of the underlying photoactive layer, thereby restoring the photoelectrochemical (PEC) signal. At target concentrations of 10–13, 10–10, and 10–7 M, Cas12a reporter cleavage was linear with time over the first 10 min, and PEG20000-SP produced the largest amplification response and highest apparent reporter-signal rising rate. PEC and FL calibrations were linear over 10–15–10–7 and 10–14–10–7 M, respectively, with equations I = 12.3328 + 0.7207 log10C (R2 = 0.9929) and F = 13631.32 + 845.64 log10C (R2 = 0.9926). Estimated blank-based limits of detection were 1.27 × 10–16 M (PEC) and 8.34 × 10–16 M (FL); these estimates are below the lowest tested standards and do not establish quantitation below the calibration ranges. Paired results were compared within the common calibration range using log-concentration differences and relative percent differences. Consensus recoveries were 94.3–103.6% in milk and 95.7–103.0% in river water. SCDA therefore combines sensitive dual-readout with an explicit preliminary screen for discordant measurements.