Rotary manifold for automating a paper-based Salmonella immunoassay

Foodborne pathogens are responsible for hundreds of thousands of deaths around the world each year. Rapid screening of agricultural products for these pathogens is essential to reduce and/or prevent outbreaks and pinpoint contamination sources. Unfortunately, current detection methods are laborious, expensive, time-consuming and require a central laboratory. Therefore, a rapid, sensitive, and field-deployable pathogen-detection assay is needed. We previously developed a colorimetric sandwich immunoassay utilizing immuno-magnetic separation (IMS) and chlorophenol red-β-d-galactopyranoside for Salmonella detection on a paper-based analytical device (μPAD); however, the assay required many sample preparation steps prior to the μPAD as well as laboratory equipment, which decreased user-friendliness for future end-users. As a step towards overcoming these limitations in resource-limited settings, we demonstrate a reusable 3D-printed rotational manifold that couples with disposable μPAD layers for semi-automated reagent delivery, washing, and detection in 65 minutes. After IMS to clean the sample, the manifold performs pipette-free reagent delivery and washing steps in a sequential order with controlled volumes, followed by enzymatic amplification and colorimetric detection using automated image processing to quantify color change. Salmonella was used as the target pathogen in this project and was detected with the manifold in growth media and milk with detection limits of 4.4 × 10² and 6.4 × 10² CFU mL^-1 respectively. The manifold increases user friendliness and simplifies immunoassays resulting in a practical product for in-field use and commercialization.