A single thiolated-phage displayed nanobody-based biosensor for label-free detection of foodborne pathogen.

Rapid and sensitive detection of bacterial pathogens present in food and environmental samples is of crucial importance to ensure human health and safety. Here, we present a one-step label-free colorimetric strategy based on M13 bacteriophage-displayed nanobody (phage-Nb) derived from camelid heavy-chain antibodies specific to Vibrio parahaemolyticus (V. parahaemolyticus). The thiolation of phage-Nb (Phage-Nb-SH) on pVIII shell proteins induces the aggregation of gold nanoparticles (AuNPs), whereas the specific interaction between nanobody and bacteria prevents the aggregation of AuNPs, resulting in visible color change due to alteration of surface plasmon resonance properties. Based on this phenomenon, a simple and sensitive colorimetric immunosensor for V. parahaemolyticus was developed. The assay can be accomplished within 100 min, and exhibits a visual detection limit of 104 cfu/mL and a quantitative detection limit of 103 cfu/mL, with no cross-reactivity towards other bacterial species. This strategy takes full advantages of both the high specificity of phage-Nbs and the optical properties of AuNPs, enabling simple and rapid detection of bacterial pathogens.

Development of a streptavidin-bridged enhanced sandwich ELISA based on self-paired nanobodies for monitoring multiplex Salmonella serogroups.

Salmonella are major pathogens that cause foodborne diseases. In this work, a broad-spectrum Salmonella nanobody-01 (Nb-01) was isolated and applied in the development of a streptavidin-bridged sandwich ELISA (SAB-ELISA) for simultaneously identifying five Salmonella serovars, including Salmonella Enteritidis (S. Enteritidis), Salmonella Typhimurium (S. Typhimurium), Salmonella London (S. London), Salmonella Paratyphi B (S. Paratyphi B) and Salmonella Hadar (S. Hadar). In this work, streptavidin (SA) was utilized as a scaffold to directionally immobilize biotinylated nanobody (BiNb) and Salmonella was detected by phage-displayed nanobodies. The SAB-ELISA can be accomplished within 180 min with a limit of detection (LOD) of 6.31 × 103 colony forming units (CFU) mL-1, 9.15 × 103 CFU mL-1, 4.23 × 103 CFU mL-1, 7.31 × 103 CFU mL-1 and 7.25 × 103 CFU mL-1 towards S. Typhimurium, S. Enteritidis, S. London, S. Paratyphi B and S. Hadar, respectively. In comparison of sandwich ELISA by passive immobilization of Nb-01 on polystyrene plate, the sensitivity was increased by around 6-fold, which confirmed the enhanced immobilization efficacy of SAB-ELISA. Furthermore, the feasibility of the assay for S. Typhimurium determination in actual samples was evaluated, showing excellent recovery, inter-day and intra-day precision.

Ultrasensitive label-free immunochromatographic strip sensor for Salmonella determination based on salt-induced aggregated gold nanoparticles.

Here we present an innovative label-free immunochromatographic strip (ICTS) sensor, in which salt-induced aggregated gold nanoparticles (SIA-AuNPs) act as the signal probe, allowing in 14 min the identification and sensitive quantification of Salmonella as model targets. It has been evidenced that SIA-AuNPs could be absorbed on the surface of bacteria based on van der Waals forces. The SIA-AuNPs@Salmonella complex was captured by anti-Salmonella polyclonal antibody deposited on the test zone. With the label-free ICTS sensor, we successfully detected Salmonella in a concentration range of 103-108 CFU/mL and a visual detection limit of 1 × 103 CFU/mL. The band of test zone could be distinguished at a concentration of 103 CFU/mL by naked eye, which is 100-fold lower than the cationic AuNPs based method. The strip sensor was further validated with real samples including cabbage and drinking water with excellent precision and showed to provide excellent recovery.

Development of a specific nanobody and its application in rapid and selective determination of Salmonella enteritidis in milk.

The variable domain of heavy chain only antibody (VHH), also named as nanobody obtained from camelid antibody libraries, has shown great potential in immunochemistry, with many advantages over conventional antibodies. Here, nanobodies towards Salmonella enteritidis (S. enteritidis) were isolated for the first time after biopanning of an immune camelid nanobody library. Those nanobodies showed high thermostability and good specificity. A sandwich-ELISA was developed with nanobody Nb 13, which exhibited a detection limit of 1.4 × 105 CFU/mL. The proposed assay was also applied to detect S. enteritidis in milk samples, which could detect 6 CFU/mL of S. enteritidis in milk after 10 h of enrichment. This study demonstrated an alternative strategy for S. enteritidis determination, and further proved the utility and practicality of nanobody-based reagents in bioanalytical chemistry.