The channel was patterned on paper by employing the wax
printing method [18], and later incubated in a vacuum oven at
150 ◦C for 30 s to melt the printed wax permanently onto the paper
(Fig. 1(B)). The unprinted hydrophilic regions function as microfluidic
channels that spontaneously transport the sample solution
along the channel via capillary force [19]. For sample analysis,
glucose and lactate oxidases (GOx and LOx, respectively), incorporated
into HRP-mediated colorimetric assays using chromogenic
substrates, were employed in the PAD for glucose and lactate
measurements, respectively [20]. As shown in Fig. 1(C), when a
sample solution is introduced to the drop zone at the top layer,
the solution is transferred to both the glucose and lactate detection
zones along the fluidic channel on the bottom layer. Next,
the solution participates in the HRP-mediated color-developing
reaction. In the detection zone of the PAD, GOx and LOx digest
glucose and lactate, respectively, generating H2O2. In the presence
of H2O2, the HRP converts 4-aminoantipyrine (4-AAP) and
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium
salt monohydrate (MAOS) to a blue-colored product, which can be
visualized in the detection zone. Its intensity is directly proportional
to the concentrations of glucose and lactate in the applied
sample solution (Fig. 2(A) and (B)) [21–23