Development of a sensitive immunoassay for Helicobacter pylori based on scFv coupled with porous PtCu nanozyme

A nanozyme-amplified immunoassay was developed for Helicobacter pylori detection. • Avian-derived IgY and scFv enabled a dual-recognition sandwich format. • Porous PtCu nanozymes provided stable colorimetric signal amplification. • The assay achieved an LOD of 1.91 × 10 3  CFU/mL with good selectivity. • The method supports sensitive screening in matrix-rich samples. Helicobacter pylori ( H. pylori ) is a major gastric pathogen associated with chronic gastrointestinal diseases, creating a need for simple and reliable analytical methods for its detection, particularly in complex sample matrices. Herein, we developed a nanozyme-amplified colorimetric sandwich immunoassay by integrating a unified avian-derived dual-recognition format with porous PtCu nanozyme-based catalytic signal amplification. Egg yolk-derived immunoglobulin yolk (IgY) was used as the capture antibody, while a phage display-derived single-chain variable fragment (scFv) selected after chicken immunization and biopanning was assembled onto porous PtCu nanomicrobeads (PtCu NPMs) as the catalytic detection probe. This design combines complementary IgY/scFv recognition with the catalytic robustness of porous PtCu nanozyme for sensitive H. pylori detection. Under optimized conditions, the assay achieved a limit of detection of 1.91 × 10 3  CFU/mL and showed good selectivity toward H. pylori . The method was further evaluated in saliva, milk, water, and vegetable juice samples, where satisfactory recoveries were obtained at moderate-to-high spiking levels. These results indicate that the proposed assay is suitable for sensitive screening and semi-quantitative analysis of H. pylori in complex matrices, and provides a practical strategy for combining avian-derived recognition reagents with nanozyme-based signal amplification in pathogen detection. Scheme 1 . Schematic illustration of the PtCu NPMs@scFv-based nanozyme-amplified sandwich immunoassay for H. pylori detection. (A) Selection of avian-derived scFv against H. pylori by hen immunization and phage display. (B) Construction of the PtCu NPMs@scFv probe through electrostatic assembly on PtCu NPMs. (C) Proposed detection mechanism: egg-yolk-derived IgY captures H. pylori on the solid phase, scFv recognizes cell-surface antigenic determinants of H. pylori , and the resulting IgY H. pylori -PtCu NPMs@scFv sandwich complex enriches porous PtCu nanozyme at the interface, thereby catalyzing the TMB/H 2 O 2 reaction to generate an amplified colorimetric signal. Download: Download high-res image (153KB) Download: Download full-size image