Insight into the Covalently Oriented Immobilization of Antibodies on Gold Nanoparticle Probes to Improve Sensitivity in the Colorimetric Detection of <i>Listeria monocytogenes</i>

Abstract

In this work, the covalently oriented conjugation of monoclonal <i>Listeria monocytogenes</i> antibody (mAb-<i>Lis</i>) to amino-terminated oligo(ethylene glycol)-capped gold nanoparticles (NH₂-TEG-AuNPs) was studied. After NH₂-TEG-AuNPs were synthesized, the amino-terminated ligands on the particles were then linked to the carboxyl groups in the mAb-<i>Lis</i> through EDC/NHS chemistry. By maintaining the pH of the solution at ∼5, the Fc region of the antibody could preferably attach to the particle surface, providing a specific Fab region that was available for binding with the target pathogen. The resulting mAb-NH-TEG-AuNPs could act as a colorimetric probe for <i>L. monocytogenes</i> based on a particular antigen-antibody interaction, which resulted in a drastic aggregation of particles. This caused the color of the colloidal solution to transition from red-pink to purple or even gray depending on the pathogen concentration. To perform quantitative analysis, the absorbance ratio of <i>A</i>₆₅₀/<i>A</i>₅₃₄ was monitored as a function of <i>L. monocytogenes</i> concentration using a spectrophotometer. The detection limit was very low at 11 CFU/mL. Furthermore, a lateral flow strip (LFS) was fabricated as a portable device for onsite utilization. LFS detection could be completed by the naked eye and by a smartphone. The detection limit of LFS was estimated to be 10³ CFU/mL. Our methods exhibited a substantial improvement in sensitivity compared to that of previous studies on immuno-based nanoparticles. The assay could be completed in 15 min, and no cross reactivity by any pathogen was found. Hence, the designed AuNPs exhibit great promise for use in monitoring <i>L. monocytogenes</i> for food safety and in other applications.