Fast multiplex analysis of antibodies in complex sample matrix using the microfluidic Evalution™ platform

•The Evalution™ platform was evaluated for high-throughput immunoassay development.•Fast and multiplex analysis of antibodies' performance was shown using Evalution™.•The obtained results were in accordance with ELISA and SPR measurements.•Influenza A nucleoprotein was detected on Evalution™ in nasopharyngeal swabs.•The detection limits for nucleoprotein were comparable to standard ELISA

Biosensor development requires comprehensive research for establishing the optimal bioassay conditions that determine the final kinetics, sensitivity and specificity. Different systems have been developed to study bioreceptor-target interactions but they often have drawbacks, such as long hands-on time, low throughput, high sample consumption and high cost. In this work, the potential of the novel microfluidic Evalution™ platform has been evaluated for developing sandwich-based assays in a fast and high-throughput fashion. An immunoassay for the detection of influenza A nucleoprotein was used as a model system. Exploiting the platform's unique features, various typical bioassay parameters (e.g. aspecific binding between assay components, different reagent concentrations and incubation times) were tested for three capture antibodies in a simple and fast manner (2 runs of 80 min). The selected conditions, giving the highest signal-to-noise ratio, were directly employed on the same platform to detect nucleoprotein in buffer and nasopharyngeal swabs. Two antibodies with a higher dissociation constant (Ab11 and Ab12) required longer incubation times (60 min) for sensitive detection (limit of detection (LOD) of 0.48 and 0.26 ng mL−1, respectively) compared to an antibody with lower dissociation constant (LOD of 0.04 ng mL−1 for Ab66 within 30 min). Moreover, one antibody (Ab12) showed limited capacity to capture nucleoprotein directly in sample matrix. The obtained results were in accordance with previous studies performed on an ELISA and SPR platform with the same antibodies. This positions the Evalution™ platform as a reliable platform for fast and multiplex analysis of antibodies' performance both in buffer and complex sample matrices.

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