An ultrasensitive label-free impedimetric immunosensor for the rapid detection of foodborne bacteria

Publication Date: 2020-04-02

Execution Methods	Purification of Salmonella-Specific Monoclonal Antibodies. Hybridoma cells 7F1A and 5E6A were cultured in IMDM/fetal calf serum (FBS). The optimal condition for the highest antibody production was first determined by Western blotting of the culture supernatants collected at different time points with anti-mouse antibodies. Antibodies were purified by Protein-A-sepharose affinity chromatography and the purity was monitored by SDS-PAGE. The purified antibodies were stored at 4℃. Validation of Antibody Specificity. The specificity of purified 7F1A and 5E6A was verified by Western blotting, ELISA, and immunomicroscopy. The capability of antibody to capture Salmonella in solution was performed using biotinylated 7F1A and magnetic bead conjugated with streptavidin followed by plating the pull-down sample on an agar plate (for live bacteria) or examined by electron microscopy. Besides, colloidal gold-labeled 5E6A and 7F1A were prepared to test whether these two antibodies can be used to develop a rapid screening immunochromatography strip. The temperature effect on antibody stability was monitored by SDS-PAGE, ELISA, and dot immunoblotting. Preparation of immune-magnetic bead (IMBs): the 7F1A antibody was immobilized on the COOH-activated MB as IMBs. Collection efficiency of bacteria with IMBs: to discuss the adequate ratio between the 10⁷ CFU/mL Salmonella and IMBs Fabrication of microfluidic chip integrating electrochemical impedimetric detectors: holes of 10 mm wide diameter were formed on the gold working electrode (WE) and a channel with a 1 mm width and 100 mm height was combined with the WE and counter electrode (CE) and reference electrode (RE). WE and CE are placed in opposite position. Detection of real bacterial sample: after collecting the IMB-Salmonella complex, the complex was placed in the microchannel, and then collected in the hole structures by using dielectrophoretic force. Subsequently, the collected IMB-Salmonella complex was quantified by electrochemical impedance spectrum.
Performance Evaluation
Conclusion & Suggestion	Salmonella, facultative anaerobic Gram-negative bacteria, is a genus of the family Enterobacteriaceae. Based on the DNA similarity, this genus comprises two species, S. enterica and S. bongori, the former is divided into six subspecies. However, according to the antigenicity of the flagella FimH protein, surface polysaccharide antigen (Vi), and the O antigen of LPS, Samonella includes more than 2600 serotypes. More than 20 antibodies will be needed in the detection process since the identification range of individual antibodies is limited. Thus, antibodies such as 7F1A and 5E6A monoclonal antibodies that recognize the common antigens on the surface of Salmonella are key reagents in the development of methods that allow identifying a wide range of Salmonella serotypes. The low-serum-demand hybridoma clones obtained in this study can reduce the production cost of antibodies. Also, these two antibodies can be used in other immunoassays, such as rapid screening immunochromatography strips for detection of a wide range of Salmonella or specific seroptypes in combination with other serotype-specific antibodies. In this project, 7F1A anti-Salmonella antibody was successfully immobilized on MB to form IMBs, which is feasible for Salmonella collection. The collected IMB- Salmonella complex can be concentrated in the hole structure by dielectrophoresis and quantified by electrochemical impedimetry. Moreover, the chip can be regernerated by using gravity and flow shear force to remove the collected complex. The microfluidic chip has no request of antibody immobilization on the electrochemical detectors. The IMB can perform specific collection for different bacteria. The dielectrphoretic concentration can promote the collection efficiency on the hole structure for the promotion of the sensing properties . The method can reduce the immunoreaction time and lower the detection limit. The microfluidic chip integrating electrochemical impedimetric detector can be used for the fast detection of food-borne bacteria. In the future this technique can be used as an efficient platform for the source traces of food safety in meat and egg products, which has promising potential for the monitoring of bacterial pollution of poultry and meat.
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