Rapid and Portable Gold Nanoparticle-Based Nanosensors for <i>Vibrio</i> spp. Detection from Aquaculture Water and Fish Tissues

Multiple species of Vibrio bacteria (Vibrio spp.) are pathogenic and can cause mortality to aquaculture organisms. Current Vibrio detection relies on bacteria plate count or molecular methods, both requiring sophisticated laboratory facilities and prolonged processes. We have developed a gold nanoparticle (AuNP)-based “Filter-and-Stain” and “Swab-and-Stain” methods for rapid and on-site detection of Vibrio spp. from aquaculture water and solid/semisolid fish tissues, respectively. An anti-Vibrio DNA aptamer with a generic selectivity to four prominent Vibrio species was conjugated to 13 nm AuNPs (Apt-AuNPs) as plasmonic nanostains. In the “Filter-and-Stain” method, water samples were filtered through a polyvinylidene fluoride (PVDF) syringe filter (0.22 μm pore size), followed by addition of the Apt-AuNPs solution. The selective binding of the Apt-AuNPs to the Vibrio cells captured by the membrane can be detected by the stain color, which is further amplified by a gold deposition reaction. A home-built hand-held optical detector was used to quantify the stain intensity for quantification of Vibrio spp. with a LOD of 14 CFU/mL within 30 min assay time. For fish tissues, on the other hand, the “Swab-and-Stain” method was used, where anti-Vibrio antibody-modified cotton swabs capture Vibrio cells from homogenized tissue samples, followed by Apt-AuNPs staining of the captured Vibrio cells (forming a sandwiched complex) and gold deposition. The stain intensity on the swabs, quantified by mobile phone image analysis, enabled a binary assessment of the Vibrio presence (“yes” or “no”) at a cutoff concentration of 100 CFU/mL in 30 min. Twenty-five fish tank water samples and six fish kidney samples were used to validate the methods, relative to Vibrio selective Thiosulfate Citrate Bile salts Sucrose (TCBS) agar plate count.