Purpose :With the aim of developing advanced method, we have devised a new sensing system that simultaneously detects 12 foodborne pathogens by using optical signals generated in designated wells for each pathogen on microfluidic chip.
Methods :Each capture probe specific for the genomic regions of 12 pathogens was spotted on the wells (400 μm in diameter per well) of micropatterned-COC (cyclic olefin copolymer) chip and directly immobilized by UV irradiation. The biotin-labeled target DNAs for the pathogens were prepared by multiplex PCR using biotin-labeled primers, then the target DNAs flowed on the chip and were incubated for 5 min at 60°C. Streptavidin and gold nanoparticles conjugated with biotin then sequentially flowed and were respectively incubated for 20 min at room temperature. Next silver solution flowed and silver aggregation only on the surface of gold nanoparticles leads to significant reduction of light transmission into the chip and high contrast signal visible under optical scanner.
Results : By employing the method, all 12 pathogens were correctly identified through the optical signals at the correct positions of the wells without any interference caused by non-specific binding or noise, demonstrating its high selectivity. Furthermore, pathogen concentrations were conveniently determined using optical intensities induced by silver aggregation proportional to the amount of target pathogens.
Significance : The results demonstrated that the new method displays high sensitivity (limit of detection= 5 x102 cells), precision (<10%), and convenience by automated multiplex analysis within 90 min. By eliminating the requirement of labor-intensive and time-consuming procedures, the new approach should be widely applicable in pathogens sensing.