Aqueous two-phase systems evolved double-layer film for enzymatic activity preservation: A universal protein storage strategy for paper based microdevice

Integration and storage of bioactive reagents is an important and challenging task in microfluidic paper-based analytical devices (μPADs). Here, we developed a convenient and universal method to store proteins and preserve their activities in μPADs by using aqueous two-phase systems (ATPs) evolved film. A polyethylene glycol (PEG)–dextran (DEX) double-layer film was formed through dehydration of ATPs. Functional biomolecules were stored in the bottom DEX layer on the basis of the biased partitioning and rehydrated conveniently by simple addition of buffer solution at usage. As a demonstration, enzyme immunoassay (EIA) of carcinoembryonic antigen was performed successfully on μPAD integrated with antibodies. Even after 104 days of storage at 4 °C and ambient conditions, the EIA signal just lost less than 10% and 30%, which meet the storage requirements of invitro diagnosis reagents. The ATPs evolved double-layer film has double functions of stabilization and insulation, and provide a high efficiency of biomolecule preservation, thereby promoting the applications of μPADs in POC diagnostic assay.