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.Report abuse »