A hybrid paper-based microfluidic platform toward veterinary P-ELISA

This study describes a novel fabrication method to produce a hybrid paper-based microfluidic platform, termed Multi-Pad Paper Plate (MP³) for transfer and optimisation of enzyme-linked immunoassays (ELISA) on paper (P-ELISA). The new fabrication technique is based on a combination of laser micromachining of paper and packaging through thermal lamination. This method is simple, rapid and of high-resolution and can produce robust, versatile and low-cost devices, compatible with a standard 96-well microplate format. The MP³ was used to transfer a sandwich ELISA for detection of bovine haptoglobin (Hp), a marker of inflammation in animals allowing optimisation of the assay in the new format. Using the MP³, a standard curve was generated and the limit of detection achieved was 0.73 μg/ml. The optimised protocol was also applied to the detection of Hp in bovine serum, demonstrating the possibility of using this platform with biological samples. The new platform allowed for a reduction of 93% in time and of 88% in cost for performing the assay and represents a valid alternative to other commonly used device fabrication methods, especially in the context of low-resource settings. It has the potential to translate similar assays into P-ELISA and offers a starting point for achieving animal-side testing.