Sensitive fluorescence ELISA for the detection of zearalenone based on self-assembly DNA nanocomposites and copper nanoclusters

Abstract

Zearalenone (ZEN), produced by Fusarium species, is a potential risk to human health. Traditional enzyme-linked immunosorbent assay (ELISA) is restricted due to low sensitivity for the detection of ZEN. Herein, enzyme nanocomposites (ALP-SA-Bio-ssDNA, ASBD) were prepared with the self-assembly strategy based on streptavidin-labeled alkaline phosphatase (SA-ALP) and dual-biotinylated ssDNA (B²-ssDNA). The enzyme nanocomposites improved the loading amount of ALP and catalyzed more ascorbic acid 2-phosphate to generate ascorbic acid (AA). Subsequently, Cu²⁺ could be reduced to copper nanoclusters (CuNCs) having strong fluorescence signal by AA with poly T. Benefiting from the high enzyme load of nanocomposites and the strong signal of CuNCs, the fluorescence ELISA was successfully established for the detection of ZEN. The proposed method exhibited lower limit of detection (0.26 ng mL^-1) than traditional ELISA (1.55 ng mL^-1). The recovery rates ranged from 92.00% to 108.38% (coefficient of variation < 9.50%) for the detection of zearalenone in corn and wheat samples. In addition, the proposed method exhibited no cross reaction with four other mycotoxins. This proposed method could be used in trace detection for food safety.

Keywords: Copper nanoclusters; Fluorescence enzyme-linked immunosorbent assay; Self-assembly strategy; Zearalenone.