Exploiting pH stability of nanozymes for low-background signal amplification in lateral flow immunoassays

Nanozymes are known for their exceptional stability under extreme pH conditions, yet this property has never been exploited to overcome practical analytical limitations. Here, we report the first utilization of the extreme acid tolerance to address a long-standing challenge in wash-free assays - background signal caused by endogenous peroxidases. By performing in situ signal amplification at pH 1.0, we selectively inactivate natural peroxidases while preserving sufficient catalytic activity of Au@Pt nanozyme, thereby eliminating false-positive signals without additional washing steps. We applied this strategy to lateral flow immunoassay (LFIA) using monoclonal antibodies and nanobodies for the detection of paraquat in spinach characterized by high endogenous peroxidase activity. Under standard pH conditions, strong background interference prevented reliable visual readout. In contrast, low-pH amplification completely suppressed matrix-induced background, significantly improved the signal-to-noise ratio, and enabled detection limits (60 pg/mL) unachievable with conventional protocols and yielded satisfactory paraquat recoveries of 90-130% This work establishes a new design paradigm in biosensing and chemical engineering: instead of avoiding harsh conditions, they can be served on purpose to gain selectivity against biological background.