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 nano- to microliter and can be coupled with numerous techniques, including colorimetry. However, although 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 (an important biomarker for kidney disease) in artificial urine. Optimized and selective Jaffé reaction was performed on the device, and reaction products were delivered to the PAD, which was subsequently analyzed with a bench scanner. The optimal operational parameters on DMF were 45 s reaction time with circular mixing and capture of the image 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 3.1 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. Creatinine-spiked synthetic urine samples were subjected to analysis via 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-PAD and spectrophotometry, respectively. Furthermore, the recovery values were within the acceptable range, with DMF-PAD yielding 96-108% and spectrophotometry producing 95-102%. Finally, the greenness levels of DMF-PAD and spectrophotometric methods were evaluated using the Analytical Greenness 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 characteristic 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.