Colorimetric detection of alkaline phosphatase on paper microfluidic test strip based on the in-situ formation of gold nanoparticles

Abstract

Affordable alkaline phosphatase enzyme detection methods are essential for diagnosing hepatic diseases. In this work, an in-situ formation of gold nanoparticles was utilized to develop a colorimetric method to detect alkaline phosphatase. Alkaline phosphatase dephosphorylates the substrate 2-phospho-L-ascorbic acid and produces ascorbic acid. Ascorbic acid, thus formed, is a good reducing agent that reduces Au3+ ions to gold nanoparticles. Alkaline phosphatase concentrations from 20 U/L to 400 U/L were spectrophotometrically detected using the surface plasmon resonance properties of the in-situ formed gold nanoparticles. The detection shows a sensitivity of 0.004 ± 0.0002 (a.u)/(U/L), a limit of detection of 2.8 mU/L, and a limit of quantification of 9.5 mU/L. A three-dimensional microfluidic paper test strip was fabricated to translate this detection method into a point-of-care testing device. The blue color formed on the paper was scanned, and the color intensities were analyzed using image processing. The three-dimensional microfluidic paper-based analytical devices could detect alkaline phosphatase concentration from 20 U/L to 400 U/L with an improved sensitivity of 0.0980 ± 0.0016 (a.u)/(U/L). The limit of detection and limit of quantification of the analysis on the paper strip was 0.748 U/L and 2.49 U/L, respectively.