A microfluidic paper-based analytical device that uses gelatin film to assay protease activity via time readout

Food processing, detergents, and pharmaceuticals frequently employ proteases, which are enzymes that break the chemical bonds of both proteins and peptides. In this work, we developed a microfluidic paper-based analytical device (µPAD) for protease activity assays via time readout. To accomplish this, we folded the µPAD to form layers, then inserted a water-insoluble gelatin film between the layers of paper to form the device. Lamination helps to maintain the gelatin film between the introduction zone, which is the upper layer, and the detection channel, which is the lower layer. Proteases decompose the gelatin film when it enters the introduction zone, which then allows it to flow into the detection channel. The protease activity in the sample solution determines the time required to dissolve the gelatin film, which leads to a linear relationship between the logarithm of the protease concentration and the time required to flow the solution a specific distance on the detection channel. The µPAD was used to measure proteases in concentrations that ranged from 0.25 to 1 mg L−1 for bromelain, 2.5 to 10 mg L−1 for papain, and 1 to 8 mg L−1 for trypsin. The limits of quantification for bromelain, papain, and trypsin were 0.41, 2.7, and 9.2 mg mL−1, respectively. The relative standard deviations for bromelain were smaller than 2 % for concentrations ranging from 0.5 to 1.0 mg L−1. We compared the µPAD to a commercially available protease activity assay kit, which relies on quenching fluorescein isothiocyanate-labeled casein. Both methods demonstrated the same order of activity: bromelain > papain > trypsin. The proposed device allowed the assay of bromelain in both pineapple pulp and juice, which were stored at room temperature. When first using the proposed device, the bromelain in the pulp gradually lost its activity, while the activity of the bromelain in the juice showed no significant change for five days. The µPAD requires no analytical instruments for quality control and monitoring of the protease activity in food.

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