Paper-based colorimetric assay has become a promising diagnostic tool in the field of food industry due to their unique properties that can rapidly identify pathogenic bacteria on site with naked eyes. A novel radial flow immunoassay (RFIA) employing gold nanoparticles (AuNPs) as a chromatic agent with well-oriented antibody on the surface was developed for the detection of Escherichia coli O157:H7. 4-repeated Gold binding polypeptide-tagged protein G (4X-GBP-SPG) fusion protein was employed as a bifunctional linker that render well-oriented immobilization of antibodies on the surface of AuNPs based on the specific affinity of GBP and SPG to the gold and Fc portion of antibody, respectively. The resulting immuno-AuNPs were shown to have an excellent ability to capture the target bacteria, E. coli O157:H7. Colorimetric signals were generated on nitrocellulose membrane by the radial flow of test solution containing the target bacteria, E. coli O157:H7, labeled with the immuno-AuNPs. In the absence of target bacteria the immuno-AuNP spread radially by capillary action. When target bacteria were present at solution, the immuno-AuNPs were capture by the bacteria and their radial diffusion was limited, forming a small inner ring. The intensity of colorimetric signals in small inner ring were dependent on the concentration of target bacteria. The developed RFIA successfully detected E. coli O157:H7 with a detection limit of 102 CFU and103 CFU in PBS and milk sample, respectively. The quantification of target bacteria could be achieved by analyzing the intensity of colorimetric signal using Image J program. This paper-based detection system would provide an effective means of monitoring the presence of food-borne pathogens on site with naked eyes.

Paper-based radial flow immunoassay for the detection of Escherichia coli O157:H7 using surface-engineered gold nanoparticles

2019 Fall National Meeting

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