Microfluidic paper-based analytical devices (PADs) have gained a lot of attention for their attractive features owing to the use of paper as a substrate, such as being low-cost, easily and safely disposable by incineration and allowing pump-free sample transport driven by capillary forces. However, it is still challenging to perform sub-microliter sample analyses by PADs, mainly because of large dimensions of microfluidic structures and the open system prone to evaporation of the sample liquid. In this work, we demonstrate the advantages of using a hot laminator instead of a hot plate in the wax printing-based microfluidic patterning method. The shortened heating time and the pressure applied to the paper substrate by the hot rollers contribute to the formation of high-resolution microfluidic structures. Consideration of the device geometry and the influence of cellulose fiber direction in the filter paper substrate have led to a model PAD design with four microfluidic channels that can be filled with as low as 0.5 µL of liquid. A colorimetric protein assay was performed targeting tear fluid protein analysis. This PAD allows to obtain quadruplicate colorimetric data by single pipetting of a sub-microliter sample. Finally, the strength of fully enclosed microfluidic structures in PADs, achieved by device lamination, is demonstrated for sample volume-independent quantitative assays. Prevention of evaporation by lamination leads to controlled sample liquid uptake, resulting in constant colorimetric signals regardless of the sample volume applied to the PAD, which is a significant advantage for practical applications.
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