Cost-effective distance-based paper microfluidic sensor for hemoglobin quantification in human serum samples for rapid diagnosis and follow-up of blood disorders

Blood disorders such as anemia are among the most prevalent and debilitating disease conditions worldwide that require routine monitoring. Hemoglobin is clinically used as a key indicator for the rapid diagnosis of these blood-based diseases. However, a low-cost diagnostic tool to quantify hemoglobin remains a significant challenge. In this study, we present the first demonstration of a distance-based paper analytical device (dPAD) for the sensitive quantification of hemoglobin in human serum samples. The device is designed to incorporate a paper-based microfluidic delay zone to enhance its detection ability. The detection method relies on the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hemoglobin to form oxidized TMB (oxTMB) in the presence of H₂O₂, resulting in a blue-green color. The change in the distance of this color is proportional to hemoglobin levels, which can be measured using a printed ruler on the device via naked-eye readout. The sensor exhibits a working linear range of 2.0-10.0 mg dL^-1 (R² = 0.9983) with a limit of detection (LOD) of 2.0 mg dL^-1, demonstrating high detection sensitivity while using only 2.0 μL of sample solution and requiring 10 min for analysis. It is also selective and shows no interferent effects for detecting hemoglobin. Furthermore, the method demonstrates excellent accuracy and precision for the determination of hemoglobin, with recoveries ranging from 99.16% to 102.50% and the highest RSD of 1.46%. With this characteristic, the method is superior to previous methods that employ expensive instruments and require skilled individuals for operation. Therefore, the developed dPAD sensor offers a simple, affordable, and accessible alternative tool for the rapid monitoring of hemoglobin levels for blood analysis, enabling rapid clinical diagnostics and prognosis, especially in resource-limited settings.