We developed a simple and low-cost cell culture monitoring system utilizing a paper-based analytical
device (PAD) and a smartphone. The PAD simultaneously analyses glucose and lactate concentrations in
the cell culture medium. Focusing on the factthat animal cells consume glucose and produce lactate under
anaerobic conditions, oxidase- and horseradish peroxidase (HRP) enzyme-mediated colorimetric assays
were integrated into the PAD. The PAD was designed to have three laminated layers. By using a doublesided
adhesive tape as the middle layer and wax coating, a bifurcated fluidic channel was prepared
to manipulate sample flow. At the inlet and the outlets of the channel, a sample drop zone and two
detection zones for glucose and lactate, respectively, were positioned. When sample solution is loaded
onto the drop zone, it flows to the detection zone through the hydrophilic fluidic channel via capillary
force. Upon reaching the detection zone, the sample reacts with glucose and lactate oxidases (GOx and
LOx) and HRP, immobilized on the detection zone along with colorless chromophores. By the Trinder’s
reaction, the colorless chromophore is converted to a blue-colored product, generating concentrationdependent
signal. With a gadget designed to aid the image acquisition, the PAD was positioned to the
smartphone-embedded camera. Images of the detection zones were acquired using a mobile application
and the color intensities were quantified as sensor signals. For the glucose assay using GOx/HRP format,
we obtained the limit of detection (LOD ∼0.3 mM) and the limit of quantification (LOQ ∼0.9 mM) values
in the dynamic detection range from 0.3 to 8.0 mM of glucose. For lactate assay using LOx/HRP, the LOD
(0.02 mM) and the LOQ (0.06 mM) values were registered in the dynamic detection range from 0.02 to
0.50 mM of lactate. With the device, simultaneous analyses of glucose and lactate in cell culture media
were conducted, exhibiting highly accurate and reproducible results. Based on the results, we propose
that the optical sensing system developed is feasible for practical monitoring of animal cell culture.