A Critical Comparison between Flow-through and Lateral Flow Immunoassay Formats for Visual and Smartphone-Based Multiplex Allergen Detection

(1) Background: The lack of globally standardized allergen labeling legislation necessitates consumer-focused multiplexed testing devices. These should be easy to operate, fast, sensitive and robust. (2) Methods: Herein, we describe the development of three different formats for multiplexed food allergen detection, namely active and passive flow-through assays, and lateral flow immunoassays with different test line configurations. (3) Results: The fastest assay time was 1 min, whereas even the slowest assay was within 10 min. With the passive flow approach, the limits of detection (LOD) of 0.1 and 0.5 ppm for total hazelnut protein (THP) and total peanut protein (TPP) in spiked buffer were reached, or 1 and 5 ppm of THP and TPP spiked into matrix. In comparison, the active flow approach reached LODs of 0.05 ppm for both analytes in buffer and 0.5 and 1 ppm of THP and TPP spiked into matrix. The optimized LFIA configuration reached LODs of 0.1 and 0.5 ppm of THP and TPP spiked into buffer or 0.5 ppm for both analytes spiked into matrix. The optimized LFIA was validated by testing in 20 different blank and spiked matrices. Using device-independent color space for smartphone analysis, two different smartphone models were used for the analysis of optimized assays.