Development of crosslinker-free antibody-enzyme conjugates for immunoassays in pesticide residue detection.

Journal: Analytical methods : advancing methods and applications
Published Date: May 9, 2025

Abstract

Immunoassays are biochemical analytical techniques based on the specific binding between antigens and antibodies. Due to their high sensitivity, rapid analysis, high throughput, cost-effectiveness, and broad applicability, immunoassays have been widely utilized across various fields. Antibody-enzyme conjugates are essential components for signal generation in immunoassays, with crosslinking chemical (crosslinker)-based methods traditionally employed for their production. However, these methods often involve hazardous reagents and complex procedures, highlighting the need for more eco-friendly alternatives. In this study, we developed a universal, crosslinker-free approach for preparing antibody-enzyme conjugates. This method eliminates the use of chemical reagents such as glutaraldehyde or periodate, as well as the intricate conjugation and purification steps typically required. Instead, gold nanoparticles (AuNPs) were employed as a versatile nanomaterial to passively adsorb horseradish peroxidase (HRP) and antibodies through a simple procedure completed within 60 minutes. The resulting conjugates were characterized using UV-vis spectroscopy, transmission electron microscopy (TEM), and zeta potential analysis. Using acetamiprid (ACE), a widely-used pesticide, as the target model, an enzyme-linked immunosorbent assay (ELISA) was developed based on the AuNP-mediated conjugates. The assay demonstrated performance comparable to that of traditional crosslinker-based methods, with an IC of 0.49 ng mL and a limit of detection (LOD) of 0.11 ng mL. Recovery rates for ACE in spiked fruit samples were consistent with those obtained using liquid chromatography-mass spectrometry (LC-MS). The proposed method not only provides a reliable alternative for HRP and antibody conjugation but also advances green analytical chemistry by minimizing the use of hazardous chemicals, reducing derivative waste, and improving energy efficiency.

Authors

  • Jiayi Chen
    College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, Zhejiang 310058, China. dylee@zju.edu.cn.
  • Qiyi He
    Department of Pharmaceutical Engineering, Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, People's Republic of China. Electronic address: chesto36@163.com.
  • Junkang Pan
    College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, Zhejiang 310058, China. dylee@zju.edu.cn.
  • El-Sayed A El-Sheikh
    Plant Protection Department, Faculty of Agriculture, Zagazig University, Zagazig 44511, Sharkia, Egypt.
  • Rubing Zou
    Institute of Pesticide and Environmental Toxicology, Zhejiang Key Laboratory of Biology and Ecology Regulation of Crop Pathogens and Insects, Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Zhejiang University, Hangzhou 310058, China.
  • Yirong Guo
    Institute of Pesticide and Environmental Toxicology, Zhejiang Key Laboratory of Biology and Ecology Regulation of Crop Pathogens and Insects, Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Zhejiang University, Hangzhou 310058, China.
  • Bruce D Hammock
    Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California, Davis, CA 95616, USA.
  • Li Li
    Department of Gastric Surgery, Sichuan Clinical Research Center for Cancer, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, Affiliated Cancer Hospital of University of Electronic Science and Technology of China, Chengdu, China.
  • Dongyang Li
    CAS Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China.

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