This article presents a novel, simple and low-cost method for the fabrication of microfluidic paper using sticker templates with specific patterns and a highly accessible waterproof eye pencil. The entire fabrication process could be finished in 10 min by heating the patterned paper at 150° without using complicated instruments. Important parameters in the fabrication of microfluidic paper such as type of paper, heating time, melting temperature, reagent and sample volume were optimized. To verify the applicability of the fabricated microfluidic paper, colorimetric assays were performed for simultaneous analysis of calcium and magnesium by single and multiple indicators and water hardness was determined. The limit of detection is 8.3 mg L−1 and 1.0 mg L−1, and the relative standard deviation is 8.3% and 5.9% for calcium and magnesium, respectively. A linear range from 10 to 100 mg L−1 for calcium and two linear ranges of 4–20 mg L−1 and 20–100 mg L−1 for magnesium were obtained in the paper based microfluidic device. The concentration of calcium and magnesium were successfully determined in tap, river, mineral and household purifier water samples.

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