Paper-Based Radial Chromatographic Immunoassay for the Detection of Pathogenic Bacteria in Milk

Here, a paper-based radial flow chromatographic immunoassay (RFCI) employing gold nanoparticles (AuNPs) as chromatic agents was developed for the detection of Escherichia coli O157:H7 in whole milk. A 4-repeated gold-binding peptide-tagged (4GBP) streptococcal protein G (SPG) fusion protein was constructed as a bifunctional linker to immobilize antibodies on the surface of AuNPs with a well-oriented form based on the specific affinity of GBP and SPG to the gold and Fc portion of the antibody, respectively. 4GS@AuNPs prepared with the bifunctional linker protein exhibited excellent colloidal stability even at high salt concentrations of up to 500 mM, which is a critical requirement for its application to a broad range of biological and food samples. The enhanced colloidal stability and excellent binding capability of the immuno-4GS@AuNPs toward target bacteria lowered the detection limit of RFCI for target pathogenic bacteria in whole milk as low as 10³ CFU/mL, which is by an order of magnitude lower than that of conventional immuno-AuNPs prepared with physical adsorption of antibodies. The RFCI pattern could also be converted into a grayscale value by simple image processing for quantitative determination of target pathogenic bacteria. This paper-based detection system would provide an effective means of monitoring the presence of food-borne pathogens in real food samples with naked eyes.