Colorimetric absorbance mapping and quantitation on paper-based analytical devices

A wide range of microfluidic paper-based analytical devices (μPADs) have been developed in the last decade. Despite this, the quality of colorimetric analysis has not substantially improved as the data is vulnerable to heterogeneous color distribution (e.g., coffee ring effects), non-uniform shapes of colored detection area, and noise from the underlying paper structure. These limitations are here addressed by a colorimetric method to quantify freely discharged dye on paper substrate, without the need for a defined channel or hydrophobic barrier. For accurate quantification, colorimetric absorbance values are calculated for each pixel based on the recorded RGB values and noise from the paper structure eliminated, to extract accurate absorbance information at the pixel level. Total analyte quantity is then calculated through the conversion of absorbance values into quantity values for each pixel followed by integration across the entire image. The resulting quantity is shown to be independent of the shape of the applied colored dye spot, with a cross, circle or rod shape all giving the same quantity information. The approach is applied to a capillary-based potassium-selective sensor, where the sample solution is loaded with the dye thioflavin T (ThT) obtained by quantitative exchange with K⁺ in a sensing capillary, which is discharged onto a bare paper substrate without any channels. The resulting dye quantity is successfully obtained by flatbed scanner and smartphone. The successful automated computation of colorimetric data on μPADs will help realize simpler paper-based assay and reaction systems that should be more applicable to addressing real world analytical problems.