Coffee Rings as Low-Resource Diagnostics: Detection of the Malaria Biomarker Plasmodium falciparum Histidine-Rich Protein-II Using a Surface-Coupled Ring of Ni(II)NTA Gold-Plated Polystyrene Particles

We report a novel, low-resource malaria diagnostic platform inspired by the coffee ring phenomenon, selective for Plasmodium falciparum histidine-rich protein-II (PfHRP-II), a biomarker indicative of the P. falciparum parasite strain. In this diagnostic design, a recombinant HRP-II (rcHRP-II) biomarker is sandwiched between 1 mu m Ni(II)nitrilotriacetic acid (NTA) gold-plated polystyrene microspheres (AuPS) and Ni(II)NTA-functionalized glass. After rcHRP-II malaria biomarkers had reacted with Ni(II)NTA-functionalized particles, a 1 mu L volume of the particle-protein conjugate solution is deposited onto a functionalized glass slide. Drop evaporation produces the radial flow characteristic of coffee ring formation, and particle-protein conjugates are transported toward the drop edge, where, in the presence of rcHRP-II, particles bind to the Ni(II)NTA-functionalized glass surface. After evaporation, a wash with deionized water removes nonspecifically bound materials while maintaining the integrity of the surface-coupled ring produced by the presence of the protein biomarker. The dynamic range of this design was found to span 3 orders of magnitude, and rings are visible with the naked eye at protein concentrations as low as 10 pM, 1 order of magnitude below the 100 pM PfHRP-II threshold recommended by the World Health Organization. Key enabling features of this design are the inert and robust gold nanoshell to reduce nonspecific interactions on the particle surface, inclusion of a water wash step after drop evaporation to reduce nonspecific binding to the glass, a large diameter particle to project a large two-dimensional viewable area after ring formation, and a low particle density to favor radial flow toward the drop edge and reduce vertical settling to the glass surface in the center of the drop. This robust, antibody-free assay offers a simple user interface and clinically relevant limits of biomarker detection, two critical features required for low-resource malaria detection.