Development of a Semiquantitative Barcode Readout Approach for Paper-Based Analytical Devices (PADs) for Enzymatic H₂O₂ and Glucose Detection

The integration of barcode technology with smartphones on paper-based analytical devices (PADs) presents a promising approach to bridging manual detection with digital interpretation and data storage. However, previous studies of 1D barcode approaches have been limited to providing only a "yes/no" response for analyte detection. Herein, a method of using barcode readout for semiquantitative signal detection on PADs has been achieved through the integration of barcode technology with a distance-based measurement concept on PADs. To demonstrate the feasibility of this concept, a PAD fabrication strategy incorporating barcodes was explored, using the enzymatic reaction between horseradish peroxidase (HRP), 3,3'-diaminobenzidine (DAB), and H₂O₂ as a model system. The enzyme-catalyzed polymerization of DAB to polyDAB in the presence of hydrogen peroxide results in the appearance of color observable by the naked eye inside a paperfluidic channel, with the color-changed length depending on the H₂O₂ concentration. At the same time, the barcode pattern displayed as a result of this distance-based color evolution overlaid with a paper-based barcode layer can be read using a smartphone application. Parameters affecting the signal readout performance were studied. The developed device can be used to detect H₂O₂ concentrations in the range of 0.25 to 10 mM within 90 min with 79.6% of barcode signals correctly readable. Additionally, results from different smartphone models showed a consistent reading performance (78.4-79.6%). Finally, the quantification of glucose levels in artificial urine samples was demonstrated. This developed PAD signaling strategy offers end-users more simplicity and can be used as a standalone device or in conjunction with other digital devices.