The albumin-to-alkaline phosphatase ratio (AAPR) has been a cancer prognostic indicator. This paper presents the concept of a dual-color change distance-based paper device (dPAD) for albumin (Alb) and alkaline phosphatase (ALP) detection to evaluate this cancer prognostic index. Whereas Alb interacts with the bromocresol green (BCG) indicator to form a bluish-green complex, ALP hydrolyzes l-ascorbic acid-2-phosphate (AAP) to produce ascorbic acid (AA), which reacts with KIO3 to generate I2 and I-. I2/I- reacts with silver hexagonal nanoprisms (purple color) in the presence of Cu2+, resulting in a color change from purple to colorless. The distance of the color change from yellow to the bluish-green and purple to colorless correlates to Alb and ALP concentration, respectively. The angle index for the AAPR is then defined by drawing a straight line that connects the tops of the two changed band lengths in the detection area. The highest bluish-green color band length on the Alb region is the midpoint, which is the position set of the protractor at 0°, and the angle is measured using a simple protractor. The results indicate that an AAPR below 0.57 will have an angle greater than 40° and correlates with a risk factor for lung cancer. The naked-eye detection limits for Alb and ALP were found to be 0.8 g/L and 5 U/L (n = 10), respectively. The practical application of the developed dPAD was successfully demonstrated by Alb and ALP analysis in human serum and validated against standard methods. The proposed method does not require incubation conditions for the ALP assay, which strongly reduces the overall analysis steps and time. Moreover, our device provides a low-cost, simple, sensitive, selective, accurate, and precise determination of the AAPR.
Keywords: albumin; albumin-to-alkaline phosphatase ratio; alkaline phosphatase; distance-based paper device; microfluidic paper-based analytical devices.