Development of an Indicator Displacement Based Detection of Malaria Targeting HRP-II as Biomarker for Application in Point-of-Care Settings.

Abstract

A novel label free spectrophotometric detection of malarial biomarker HRP-II following an indicator displacement assay has been developed. The assay is based on competitive displacement of murexide dye from its complex with Ni2+ by HRP-II present in serum samples. The binding constant (Kd) discerned for the dye and HRP-II to Ni2+ were 1.4 × 10-6 M-1 and 6.8 × 10-9 M-1, respectively. The progress of the reaction could be monitored from the change of color from orange (∼λ482nm) to pink (∼λ515nm) with the concomitant increase in HRP-II concentration in the mixture. A linear response (R2 = 0.995) curve was generated by plotting the ratio of absorbance (λ515nm/λ482nm) against the HRP-II concentrations. The method offers to detect HRP-II as low as 1 pM without any interference from some common salts and the major protein, HSA, present in the blood serum. The detection method was reproduced in a microfluidic paper based analytical device (μPAD), fabricated by printing hydrophobic alkyl ketene dimer on a chromatographic paper to create hydrophilic microchannels, test zone, and sample application zone. The device offers to use a maximum sample volume of 20 ± 0.06 μL and detects HRP-II within 5 min with LOD of 30 ± 9.6 nM in a dynamic range of 10 to 100 nM. The method has thus immense potential to develop as rapid, selective, simple, portable, and inexpensive malarial diagnostic device for point-of-care and low resource setting applications.