Abstract
Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world as well as in the United States. Current detection methods have limitation to implement for rapid field-deployable detection with high volume of samples that are needed for regulatory purposes. Surface plasmon resonance imaging (SPRi) has proved to achieve rapid and label-free screening of multiple pathogens simultaneously, so it was evaluated in this work for the detection of Shiga toxins (Stx1a and Stx2a toxoids were used as the less toxic alternatives to Stx1 and Stx2, respectively). Multiple antibodies (Stx1pAb, Stx1-1mAb, Stx1-2mAb, Stx1d-3mAb, Stx1e-4mAb, Stx2pAb, Stx2-1mAb, Stx2-2mAb, and Stx2-10mAb) were spotted one by one by programed microarrayer, on the same high-throughput biochip with 50-nm gold film through multiple crosslinking and blocking steps to improve the orientation of antibodies on the biochip surface. Shiga toxins were detected based on the SPRi signal difference (ΔR) between immobilized testing antibodies and immunoglobulin G (IgG) control. Among the antibodies tested, Stx1pAb showed the highest sensitivity for Stx1 toxoid, with the limit of detection (LOD) of 50 ng/mL and detection time of 20 min. Both Stx2-1mAb and Stx2-2mAb exhibited high sensitivity for Stx2 toxoid. Furthermore, gold nanoparticles (GNPs) were used to amplify the SPRi signals of monoclonal antibodies in a sandwich platform. The LOD reached the level of picogram (pg)/mL with the help of GNP-antibody conjugate. This result proved that SPRi biochip with selected antibodies has the potential for rapid, high-throughput and multiplex detection of Shiga toxins.
Highlights
Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world
Toxins 2020, 12, 280 in samples directly obtained from the food industry and market, where the rapid screening and identification of foodborne pathogens are frequently demanded by regulatory agencies
The waswas covered withwith mercaptoundecanoic acid (MUA), and subsequently activated
Summary
Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Each method has advantages over others in terms of specificity and sensitivity, it is difficult to use one technique platform for rapid detection of STEC or Shiga toxins (Stx) contaminating. We expanded the label-free SPRi detection to the immunosensing of Shiga toxins (Stx, Stx2) produced by STEC. This study exploited a label-free and high-throughput SPRi platform to develop the immunosensor with rapid detection time of less than 20 min while maintaining high specificity and sensitivity. Two commercial Stx and Stx ELISA kits (Abraxis Inc., Warminster, PA, USA) are available to detect all 10 subtypes of Stx and Stx, with the LOD of 25 pg/mL These ELISAs require bulky instruments and extensive sample preparation processes, which hinder ELISA applications in the field. We developed a label-free and easy-to-use SPRi immunosensor capable of high-throughput microarray detection of Stx and Stx. Depending on the future development, this SPRi sandwich immunoassay could become a powerful tool for screening of agricultural products contaminated with foodborne pathogens and toxins
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