Here, we report gold nanoparticle-coated starch magnetic beads (AuNP@SMBs) that were prepared by in situ synthesis of AuNPs on the surface of SMBs. Upon functionalization of the surface with a specific antibody, the immuno-AuNP@SMBs were found to be effective in separating and concentrating the target pathogenic bacteria, Escherichia coli O157:H7, from an aqueous sample as well as providing a hotspot for surface-enhanced Raman scattering (SERS)-based detection. We employed a bifunctional linker protein, 4× gold-binding peptide-tagged Streptococcal protein G (4GS), to immobilize antibodies on AuNP@SMBs and AuNPs in an oriented form. The linker protein also served as a Raman reporter, exhibiting a strong and unique fingerprint signal during the SERS measurement. The amplitude of the SERS signal was shown to have a good correlation with the concentration of target bacteria ranging from 100 to 105 CFU/mL. The detection limit was determined to be as low as a single cell, and the background signals derived from nontarget bacteria were negligible due to the excellent specificity and colloidal stability of the immuno-AuNP@SMBs and SERS tags. The highly sensitive nature of the SERS-based detection system will provide a promising means to detect the pathogenic microorganisms in food or clinical specimen.

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Polymeric magnetic particles (PMPs) have become a powerful tool for the separation and concentration of microorganisms from a heterogeneous liquid matrix. The functionalization of PMPs with polycationic polymers, such as chitosan, provides an effective means of capturing a broad spectrum of pathogenic bacteria through the intrinsic nature of chitosan interacting with the surface components of bacteria. Here, we report a fairly simple approach for the preparation of starch magnetic microparticles (SMMPs) through molecular rearrangement of short-chain glucans (SCGs) produced by enzymatic debranching of waxy maize starch. The surfaces of SMMPs were readily functionalized with chitosan through electrostatic interaction and hydrogen bonding. The chitosan-functionalized SMMPs (CS@SMMPs) showed high capture efficiency (>90%) for both Gram-positive and Gram-negative bacteria. To further investigate the mechanisms of chitosan–bacteria interaction, we employed model bacteria with different surface compositions. The outer-core lipopolysaccharides as well as the surface charge of bacteria were found to be important for the specific interactions of chitosan to bacteria. The biocompatible paramagnetic materials developed in this study would be promising in removing or separating bacteria from contaminated water for hygienic purposes or subsequent biochemical analysis of certain pathogenic bacteria present in the sample.

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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.