Study of paper-based assaying system for diagnosis of total serum bilirubin by colorimetric diazotization method

Accumulation of excessive total serum bilirubin (TSB) induces hyperbilirubinemia and jaundice, especially in neonates. Diagnostics of TSB from bench to bedside is a clinical need requiring development of low-cost point-of-care analytical devices. This work describes a bilirubin paper-based analytical device (BiliPAD), for detection of TSB following diazotization by a colorimetric method. The chemistry of diazotization of TSB by using different accelerating reagents (dimethyl sulfoxide (DMSO) and caffeine-benzoate) on paper substrate is investigated with focus. It is found that paper assay is sensitive to the DMSO solvent system, which has interaction with the sample matrix; such sensitivity cannot be observed in solution reactions. Further study shows that the use of DMSO results in the precipitation of albumin on BiliPAD, interfering with the colorimetric evaluation of TSB; however, the caffeine-benzoate accelerating system produces a homogeneous colorimetric result. This work has also shown that the interferent tolerance of hemoglobin by the BiliPAD method for TSB assay is up to 100 mg/dL. The accuracy of the BiliPAD is verified by comparison with the accredited hospital laboratory method, obtaining a good linearity with the slope of 0.99 and the correlation coefficient R² of 0.96. The 3D-BiliPAD device reported in this study also includes the design of 3D-printed wells, which is essential for realizing pre-treatment of serum samples using accelerating reagent and quantitative measurement of TSB without requirement of lab-based micropipettes.