From Leuco to Blue: Photochemical Redox Amplification for Small-Molecule Immunodetection

Sensitive enough detection of small-molecule toxins and many other target analytes remains challenging for immunoassays that rely on stoichiometric reporters or enzymatic cascades. Here we introduce an enzyme-free, light-gated amplification strategy that teams up stabilized leucomethylene blue (LMB) derivatives with antibody recognition and photoredox cycling. As a proof-of-concept, we designed a stable LMB precursor (δ-BLMB) and a BLMB-zearalenone (ZON) conjugate that undergo rapid, selective photo-oxidation to methylene blue (MB) only under the assay illumination. Electrochemical analysis has established δ-BLMB as a redox precursor to MB, while time-resolved luminescence of [Ru(phen)₃]²⁺ (RP3) photocatalyst revealed diffusion-limited quenching (k_q = 2.68 × 10⁹ M^-1 s^-1) consistent with dynamic electron transfer to the precursor. Mechanistic studies show that blue light activates RP3 to generate acyl-radical and MB^- intermediates via O₂-independent pathways; red light is then used to excite nascent MB to drive a secondary photocatalytic reductive cycle that regenerates MB through radical/anionic intermediates, yielding exponential signal growth. We have validated immunoassay integration using ZON-specific monoclonal antibodies in bead-based and plate formats, demonstrating specific, light-triggered fluorescence that reports competitive binding. Its implementation with sequential two-color illumination demonstrates selective system activation and precise control of the signal amplification (300-fold). This photochemical approach provides stable, long-term reagents that can be tailored to the target species and offers a potentially general strategy for developing enzyme-free, high sensitivity immunoassays for small analytes.