Paradoxical Amplification of a AuNP-Enabled Enzyme-Coupled System for Ultrasensitive Bidirectional Catalytic ECL Immunoassay

Distinct from most electrochemiluminescence sensors that rely on elaborate materials, this study develops an immunosensor using simple materials for low-cost and highly sensitive biological analysis. We elucidated the mechanism of paradoxical amplification of gold nanoparticles (AuNPs), designed strategies of bidirectional catalysis, and expanded the application for multibiomarker detection. First, a new understanding of AuNPs’ catalytic role was established through investigating the inhibitory effects of five phenolic compounds on the luminol–H2O2 ECL system, and an AuNP-induced “paradoxical amplification” of phenolic electrochemiluminescence (ECL) inhibition was first identified. Contrary to the common view that AuNPs always enhance ECL, here, AuNPs amplified the inhibitory effect of phenolics, thereby reducing the ECL signal. Such a mechanism was validated via electrochemistry-mass spectrometry. Second, a methodological innovation is presented: a switch-type immunosensor with bidirectional catalytic amplification via dynamic conversion. Specifically, the same catalyst flexibly switches between enhancement and inhibition: horseradish peroxidase (HRP) and AuNPs first act synergistically to enhance the initial ECL signal of the luminol–H2O2 system; then, alkaline phosphatase serves as a “switch” and introduces phenolic compounds, triggering HRP and AuNPs to automatically convert into signal-inhibiting catalysts. This induces multiple signal attenuation via ECL energy resonance transfer, radical quenching, and mass transfer hindrance, leading to a rapid decrease in ECL response. Finally, a highly sensitive coupled enzyme-linked attenuated ECL immunoassay involving AuNPs was constructed and successfully applied to the specific detection of cardiac troponin I and erythropoietin with a wide linear range (from fg/mL to ng/mL), and a detection limit as low as fg/mL.