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

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.