Integration of paper-based analytical devices with digital microfluidics for colorimetric detection of creatinine

Digital microfluidics (DMF) is a platform that enables the automated manipulation of individual droplets of sizes ranging from nanoliter to microliter and can be coupled with numerous techniques, including colorimetry. However, although the DMF electrode architecture is highly versatile, its integration with different analytical methods often requires either changes in sample access, top plate design, or the integration of supplementary equipment into the system. As an alternative to overcome these challenges, this study proposes a simple integration between paper-based analytical devices (PADs) and DMF for automated and eco-friendly sample processing aiming at the colorimetric detection of creatinine (CR, an important biomarker for kidney disease) in artificial urine. An optimized and selective Jaffé reaction was performed on the device, and the reaction products were delivered to the PAD, which was subsequently analyzed with a bench scanner. The optimal operational parameters on the DMF platform were a reaction time of 45 s with circular mixing and image capture after 5 min. Under optimized conditions, a linear behavior was obtained for creatinine concentrations ranging from 2 to 32 mg dL^-1, with limits of detection and quantitation equal to 1.4 mg dL^-1 and 2.0 mg dL^-1, respectively. For the concentration range tested, the relative standard deviation varied from 2.5 to 11.0%, considering four measurements per concentration. CR-spiked synthetic urine samples were subjected to analysis <i>via</i> DMF-PAD and the spectrophotometric reference method. The concentrations of CR determined using both analytical techniques were close to the theoretical values, with the resultant standard deviations of 2-9% and 1-4% for DMF-PADs and spectrophotometry, respectively. Furthermore, the recovery values were within the acceptable range, with DMF-PADs yielding 96-108% and spectrophotometry producing 95-102%. Finally, the greenness of the DMF-PAD and spectrophotometry methods was evaluated using the Analytical Greenness (AGREE) metric software, in which 0.71 and 0.51 scores were obtained, respectively. This indicates that the proposed method presents a higher greenness level, mainly due to its miniaturized characteristics using a smaller volume of reagent and sample and the possibility of automation, thus reducing user exposure to potentially toxic substances. Therefore, the DMF-PADs demonstrated great potential for application in the clinical analysis of creatinine, aiding in routine tests by introducing an automated, simple, and environmentally friendly process.