Dual-spot ratiometric microfluidic paper-based analytical device for accuracy and precision improvement.

Instrumental and environmental fluctuations are common sources of error in smartphone-based optical detection, significantly affecting the accuracy of analytical measurements. In this regard, spotting the sample and reference simultaneously and in close proximity compensates for the fluctuations. This "dual-spot" design is similar to the double-beam technique used in spectrophotometry, which reduces fluctuations in the results. The underlying hypothesis is that any instrumental and/or environmental factors influencing the color intensity in the detection zones will similarly impact the color intensity in the control zone under the same conditions. To test our design, a ratiometric microfluidic paper-based analytical device (μPAD), functionalized with a mixture of green-emissive carbon dots (CDs) and red-emissive ethidium bromide, was developed for the selective detection of ascorbic acid (AA). The green emission of the CDs is quenched by both AA and Fe; NaF was thus loaded onto the 3D connector as a masking agent to remove the interference effect of the Fe ions. The color variations were monitored under a UV lamp, using a smartphone to capture the images, and the RGB intensities were processed using the Color Grab application. The proposed double-spot method greatly enhanced the analytical precision and accuracy of the device. A linear working range from 0 to 125 μM was obtained, and the limit of detection was 2.71 μM. The μPAD was successfully used for the detection of AA in human serum, with recoveries from 87.27 to 98.52 %.