Double-layered microfluidic paper-based device with multiple colorimetric indicators for multiplexed detection of biomolecules

In this work, a double-layered microfluidic paper-based device (mu PAD) with multiple colorimetric indicators was presented for the sensitive simultaneous detection of four kinds of small biomolecules, including glucose, uric acid, lactate, and choline in mixture. The double-layered mu PAD were composed of the top detection layer and the bottom auxiliary layer to construct 3D microfluidic channels. The detection zones on the top detection layer were modified with various colorimetric reagents, different kinds of oxidase (glucose oxidase, uricase, lactate oxidase or choline oxidase) and horseradish peroxidase (HRP). The immobilized chromogenic reagents could be oxidized by the generated H2O2 from the specific enzymatic reactions between the oxidases and their corresponding substrates to form different colored products with co-immobilized HRP as catalyst. As a result, four linear calibration curves were obtained for the determination of glucose (0.01-10.0 mmol/L), uric acid (0.01-5.0 mmol/L), lactate (0.04-10.0 mmol/L), and choline (0.04-24.0 mmol/L), respectively. The proposed double-layered mu PAD with multiple colorimetric indicators exhibited good colorimetric performance, effectively improved sensitivity, and extended linear range, which was further applied to the simultaneously determination of glucose, uric acid, lactate, and choline in serum samples.