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

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 (K-d) discerned for the dye and HRP-II to Ni2+ were 1.4 x 10(-6) and 6.8 x 10(-9) M-1, respectively. The progress of the reaction could be monitored from the change of color from orange (similar to lambda(482 nm)) to pink (similar to lambda(515 nm)) with the concomitant increase in HRP-II concentration in the mixture. A linear response (R-2 = 0.995) curve was generated by plotting the ratio of absorbance (similar to lambda(515 nm)/similar to lambda(482 nm)) 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 (mu PAD), 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 mu L 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.