Sensitive lateral flow immunoassay enabled by signal amplification strategy with high active oxidase-like nanozymes

Abstract

The emergence of nanozymes has significantly propelled the advancement of lateral flow immunoassays (LFIAs). Among these, atomically dispersed single-atom nanozymes are considered particularly advantageous for enhancing sensor performance due to their high density of active sites, superior catalytic activity, and favorable surface properties. In this study, we designed a colorimetric-catalytic signal amplification LFIA mediated by the Fe-Fe single atom nanozyme (Fe₂NC) for the detection of capsaicin (CAP), a critical indicator of gutter oil that persists even after rough and fine processing. The Fe₂NC exhibited high oxidase-like activity and was capable of directly catalyzing the conversion of O₂ to reactive oxygen species (•O²⁻ and ¹O₂) without the need of H₂O₂. The V_max of Fe₂NC was 3.73 × 10⁻⁷Ms⁻¹ and the K_m value was 0.199 mM according to the enzymatic reaction kinetics calculation. The colorless 3,3′,5,5′-tetramethylbenzidine (TMB) was oxidized to the blue ox-TMB by Fe₂NC, which was then superimposed on the strip for signal amplification. It was evident that the color intensity of the T-line of Fe₂NC-TMB-LFIA underwent noticeable attenuation when the CAP concentration was 0.05 ng/mL, and the color completely disappeared when the CAP concentration reached 1 ng/mL. Concurrently, the quantitative detection range of Fe₂NC-TMB-LFIA was calculated to be 0.01–1 ng/mL. Compared to AuNPs-LFIA, the Fe₂NC-LFIA and Fe₂NC-TMB-LFIA demonstrated 2.5-fold and 10-fold improvements in sensitivity, respectively, and achieved a 2.5-fold and 5-fold reduction in antigen concentration. As a consequence, antigen cost was effectively decreased. This research offers a valuable reference for enhancing the sensitivity of immunoassay-based detection methods.