A direct, sensitive, near-real-time, handheld optical immunoassay device was developed to detect Salmonella typhimurium in the naturally occurring liquid from fresh poultry packages (hereafter "chicken matrix"), with just single pipetting of sample (i.e., no filtration, culturing and/or isolation, thus reducing the assay time and the error associated with them). Carboxylated, polystyrene microparticles were covalently conjugated with anti-Salmonella, and the immunoagglutination due to the presence of Salmonella was detected by reading the Mie scatter signals from the microfluidic channels using a handheld device. The presence of chicken matrix did not affect the light scatter signal, since the optical parameters (particle size d, wavelength of incident light λ and scatter angle θ) were optimized to minimize the effect of sample matrix (animal tissues and blood proteins, etc.). The sample was loaded into a microfluidic chip that was split into two channels, one pre-loaded with vacuum-dried, antibody-conjugated particles and the other with vacuum-dried, bovine serum albumin-conjugated particles. This eliminated the need for a separate negative control, effectively minimizing chip-to-chip and sample-to-sample variations. Particles and the sample were diffused in-channel through chemical agitation by Tween 80, also vacuum-dried within the microchannels. Sequential mixing of the sample to the reagents under a strict laminar flow condition synergistically improved the reproducibility and linearity of the assay. In addition, dried particles were shown to successfully detect lower Salmonella concentrations for up to 8 weeks. The handheld device contains simplified circuitry eliminating unnecessary adjustment stages, providing a stable signal, thus maximizing sensitivity. Total assay time was 10 min, and the detection limit 10 CFU mL(-1) was observed in all matrices, demonstrating the suitability of this device for field assays.
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