# A Two‐Stage Cascading Amplification Strategy Based on Zn2+‐Doped WOX Nanozymes for Ultrasensitive Lateral Flow Immunoassays of Clostridium Difficile Toxin B

## TL;DR

A new ultrasensitive lateral flow immunoassay method is developed for detecting Clostridium difficile toxin B using a two-stage amplification strategy with nanozymes.

## Contribution

A novel two-stage cascading amplification strategy using Zn2+-doped WOX nanozymes significantly enhances the sensitivity of lateral flow immunoassays.

## Key findings

- A 500-fold enhancement in the visual limit of detection (LOD) is achieved for Clostridium difficile toxin B.
- The fitted LOD based on grayscale analysis reaches 0.01 ng mL−1 for Tcd B detection.

## Abstract

Clostridium difficile is one of the primary causative agents of nosocomial antibiotic‐associated diseases. Early detection and prevention are effective strategies to curb disease transmission, which often requires highly sensitive point‐of‐care testing (POCT) methods. Although lateral flow immunoassay (LFIA) technology—known for its convenience, speed, and cost‐effectiveness—has gained prominence in POCT, its application in early screening remains limited due to relatively low sensitivity. Herein, a two‐stage cascading enhancement strategy based on reductase‐like and peroxidase‐like activities of nanozyme to construct a colorimetrically enhanced LFIA platform is proposed. Specifically, Zn2+‐doped WOX (Zn/WOX) loaded with Au nanoparticles (Zn/WOX@Au) first generates an initial colorimetric signal through immunochromatography. Subsequently, in situ light‐induced deposition of Pt creates Zn/WOX@Au@Pt, which exhibits enhanced peroxidase‐like activity based on optimal band structure between the components for colorimetric signal amplification. As a result, a 500‐fold enhancement in the visual limit of detection (LOD) is achieved. Moreover, the fitted LOD based on grayscale analysis for Clostridium difficile toxin B (Tcd B) reaches 0.01 ng mL−1. This strategy not only maintains the operational simplicity of LFIA but also considerably strengthens the colorimetric signal, offering a novel approach for highly sensitive colorimetric‐enhanced LFIA detection.

Based on the modulation of active sites and electronic structure of substrate materials, along with a colorimetric two‐stage cascading enhancement strategy, an LFIA platform with highly efficient POD‐mimetic enzymes is developed, achieving highly sensitive detection of Clostridium difficile toxin B.