iridium-iii-luminescent-probe-for-detection-malarial-protein

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Vanderbilt University

We have demonstrated the utility of a self-contained extraction device for the selective isolation, purification, and concentration of the malaria diagnostic protein biomarker Plasmodium falciparum histidine-rich protein II (pfHRPII) from human plasma and whole blood. The extraction cassette consists of a small-diameter tube containing a series of preloaded processing solutions separated by mineral oil valves. Nickel(II) nitrilotriacetic acid-functionalized magnetic particles are added to a parasite-spiked sample contained within the loading chamber of the device for capture of pfHRPII. The biomarker-bound magnetic particles are then entrained by an external magnetic field and transported through three wash solutions. Processing removes sample interfering agents, and the biomarker target is concentrated in the final chamber for subsequent analysis. At parasitemias of 200 parasites/μL, purification and concentration of pfHRPII with extraction efficiencies in excess of 70% total protein target are achieved. The concentration of nonspecific protein interfering agents was reduced by more than 2 orders of magnitude in the final extracted sample without the need for hours of processing time and specialized laboratory equipment. We have demonstrated an application of this low-resource technology by coupling extraction and concentration of pfHRPII within the cassette to a commonly employed rapid diagnostic test. Sample preprocessing improved the visual limit of detection of this test by over 8-fold, suggesting that the combination of both low-resource technologies could prove to be useful in malaria eradication efforts.

Low-resource method for extracting the malarial biomarker histidine-rich protein II to enhance diagnostic test performance.

The need for robust reagents for biomarker detection has become an increasing necessity in designing point-of-care diagnostics. We report a non-emissive, cyclometalated iridium(III) complex, Ir(ppy)2(H2O)2(+) (Ir1), which, on coordination to a histidine-containing protein bound to the surface of a magnetic particle, elicits a rapid, long-lived phosphorescent signal. The interactions between Ir1 and numerous other amino acids were examined for activity, but only the addition of histidine resulted in a four orders of magnitude enhancement in signal intensity. Buffer conditions (pH and temperature) and composition (coordinating vs. non-coordinating and ionic strength) were optimized to achieve maximum signal and stability of Ir1. The activity of the probe under optimized conditions was validated with BNT-II, a histidine-containing branched peptide mimic of the malarial biomarker Plasmodium falciparum histidine-rich protein II (PfHRPII). By comparing Ir1 binding to BNT-II versus L-histidine, steric and quenching effects were noted in the peptide. Despite these deviations from ideal conditions, signal response reached saturation with both BNT-II and recombinant HRPII (rcHRPII). When immobilized on the surface of a 50 μM magnetic agarose particles, the limit of detection of rcHRPII was 14.5 nM. The robust signal response of this inorganic probe lends itself to future applications in on-particle enzyme-linked immunosorbent assay (ELISA)-based assays.

On-particle detection of Plasmodium falciparum histidine-rich protein II by a "switch-on" iridium(III) probe.

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 μm 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 μL 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.

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.

Article	Year
Low-resource method for extracting the malarial biomarker histidine-rich protein II to enhance diagnostic test performance. Analytical chemistry\| PubMed ID: 22734432	2012
On-particle detection of Plasmodium falciparum histidine-rich protein II by a "switch-on" iridium(III) probe. Analytical biochemistry\| PubMed ID: 24129120	2014
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... ACS applied materials & interfaces\| PubMed ID: 24758478	2014

Keersten M. Davis

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