Rotary manifold for automating a paper-based Salmonella immunoassay

Abstract

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.

Fig. 1. CAD rendering (A) and images (B) of the rotational manifold. In the CAD renderings all gray portions are 3D printed, dark blue are lamination or transparency sheets, and white are exposed Fusion 5 paper, and light blue are Fusion 5 paper covered by lamination or transparency sheets.

Fig. 2. Schematic demonstrating the concept of sequential reagent delivery using a rotating reagent storage card and a stationary sample layer (A) add sample containing magnetic beads conjugated to your target analyte to the sample layer. (B) Add the sample layer to the device. Biotinylated antibodies will be introduced. (C) Rotate the device to a washing step used to remove excess biotinylated antibodies. (D) Continue rotating until streptavidin β-galactosidase has been introduced and washed. (E) Remove sample layer, add substrate CPRG, and observe color change.

Fig. 3. Flow through the device begins in the buffer reservoir where PBS is wicked through a paper wick into the reagent channel. Buffer flows through the reagent channel and delivers reagents to the conjugated system on the sample layer before washing away excess reagents to the waste pad.

Fig. 4. Effect of sample layer shape and blocking with BSA. The blank sample was an image of the sample layer with CPRG without any β-galactosidase ever introduced. All other samples had β-galactosidase washed through the sample layer into the waste pads.

Fig. 5. Dose response curve (n = 3) for Salmonella in media detected using the rotational manifold. The curve was fit to a 4-parameter logistic model and a LOD of 2.9 × 10³ was achieved.

Fig. 6. (A) Specificity study using E. coli at 10⁷ CFU mL⁻¹ compared to blank samples in milk, growth media, and a positive Salmonella sample. (B) Dose–response curve (n = 3) of Salmonella in milk detected using the rotational manifold. The curve was fit to a 4-parameter logistic model and a LOD of 6.4 × 10² CFU mL⁻¹ was achieved.