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

Abstract

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 goldbinding 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 welloriented 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 10 3 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.

Recent Advances in Microfluidic Devices for Contamination Detection and Quality Inspection of Milk

“…Thus, the problem of developing microfluidic platforms for the on-site detection of common foodborne pathogens in milk samples has attracted great interest in the recent literature. Typically, these platforms have been designed for the detection of E. coli [ 86 , 90 , 91 , 92 , 93 ], S. aureus [ 94 , 95 , 96 ], Listeria [ 97 , 98 , 99 ], and Salmonella [ 100 , 101 , 102 , 103 , 104 ].…”

“…However, in severe cases, it can cause more serious problems such as sepsis and hemolytic uremic syndrome [ 105 ]. Various microfluidic platforms have been developed for the detection of E. coli in milk samples, including lab-on-a-chip devices combined with immunoassay [ 78 ], aptamer and fluorescence detection [ 76 ], rolling circle amplification (RCA), and fluorescence detection [ 91 ], and lab-on-paper devices based on RCA and colorimetric detection [ 105 ], colorimetric detection [ 86 , 106 ], and immunoassay [ 90 , 92 , 93 ]. Sun et al [ 105 ] developed a folding microfluidic lab-on-paper RCA device with DNAzyme-containing DNA superstructures for E. coli detection in juice and milk samples.…”

The Components of Functional Nanosystems and Nanostructures

“…Luo et al [34] detected E. coli bacteria in milk by using radial flow chromatographic immunoassay (RFCI) method using gold nanoparticles as a chromatic agent. Hunter et al [35] synthesized nanobiosensors to quickly detect pathogenic bacteria in serum with the optofluidic (surface-enhanced Raman scattering) SERS method.…”

Gold Nanoparticle-Coated Starch Magnetic Beads for the Separation, Concentration, and SERS-Based Detection of E. coli O157:H7

“…coli O157:H7, was immobilized on the surface of gold nanoparticles via a bifunctional linker protein, gold-binding peptide-tagged Staphylococcal protein G (GBP-SPG). The linker protein comprises two domains, GBP and SPG, that have a specific affinity toward the gold substrate and the Fc region of the antibody, respectively, enabling a facile immobilization of the antibody on the gold nanoparticles in the oriented form. , To increase the binding affinity of GBP to the gold substrate, a recombinant protein having four repeated GBP domains was constructed and fused to the SPG domain. The gold nanoparticle-coated magnetic bead functionalized with the antibody via the recombinant linker protein was used to separate and concentrate target bacteria by utilizing its strong superparamagnetic properties.…”