Rapid quantification of the malaria biomarker hemozoin by improved biocatalytically initiated precipitation atom transfer radical polymerizations.

The fight against tropical diseases such as malaria requires the development of innovative biosensing techniques. Diagnostics must be rapid and robust to ensure prompt case management and to avoid further transmission. The malaria biomarker hemozoin can catalyze atom transfer radical polymerizations (ATRP), which we exploit in a polymerization-amplified biosensing assay for hemozoin based on the precipitation polymerization of N-isopropyl acrylamide (NIPAAm). The reaction conditions are systematically investigated using synthetic hemozoin to gain fundamental understanding of the involved reactions and to greatly reduce the amplification time, while maintaining the sensitivity of the assay. The use of excess ascorbate allows oxygen to be consumed in situ but leads to the formation of reactive oxygen species and to the decomposition of the initiator 2-hydroxyethyl 2-bromoisobutyrate (HEBIB). Addition of sodium dodecyl sulfate (SDS) and pyruvate results in better differentiation between the blank and hemozoin-containing samples. Optimized reaction conditions (including reagents, pH, and temperature) reduce the amplification time from 37 ± 5 min to 3 ± 0.5 min while maintaining a low limit of detection of 1.06 ng mL-1. The short amplification time brings the precipitation polymerization assay a step closer to a point-of-care diagnostic device for malaria. Future efforts will be dedicated to the isolation of hemozoin from clinical samples.

Biocatalytically Initiated Precipitation Atom Transfer Radical Polymerization (ATRP) as a Quantitative Method for Hemoglobin Detection in Biological Fluids.

The hemoglobin content of blood is an important health indicator, and the presence of microscopic amounts of hemoglobin in places where it normally does not occur, e.g. in blood plasma or in urine, is a sign of diseases such as hemolytic anemia or urinary tract infections. Thus, methods to detect and quantify hemoglobin are important for clinical laboratories, blood banks, and for point-of-care diagnostics. The precipitation polymerization of N-isopropylacrylamide by hemoglobin-catalyzed atom transfer radical polymerization (ATRP) is used as an assay for hemoglobin quantification relying on the formation of turbidity as a simple optical read-out. Dose-response curves for pure hemoglobin and for hemoglobin in blood plasma, in urine, in erythrocytes, and in full blood are obtained. Turbidity formation increases with the concentration of hemoglobin. Concentrations of hemoglobin as low as 6.45 × 10-3 mg mL-1 in solution, 4.88 × 10-1 mg mL-1 in plasma, and 1.65 × 10-1 mg mL-1 in urine could be detected, which is below the clinically relevant concentrations in the respective body fluids. Total hemoglobin in full blood is also accurately determined. The reaction can be regarded as a polymerization-based signal amplification for the sensing of hemoglobin, as the analyte catalyzes the formation of radicals which add many monomer units into detectable polymer chains. While most established hemoglobin tests involve the use of highly toxic reagents such as potassium cyanide, the polymerization-based test uses simple and stable organic reagents. Thus, it is an environmentally friendlier alternative to established chemical assays for hemoglobin.

Hemozoin-catalyzed precipitation polymerization as an assay for malaria diagnosis.

Methods to diagnose malaria are of paramount interest to eradicate the disease. Current methods have severe limitations, as they are either costly or not sensitive enough to detect low levels of parasitemia. Here we report an ultrasensitive, yet low-resource chemical assay for the detection and quantification of hemozoin, a biomarker of all Plasmodium species. Solubilized hemozoin catalyzes the atom transfer radical polymerization of N-isopropylacrylamide above the lower critical solution temperature of poly(N-isopropylacrylamide). The solution becomes turbid, which can be observed by naked eye and quantified by UV-visible spectroscopy. The rate of turbidity increase is proportional to the concentration of hemozoin, with a detection limit of 0.85 ng mL-1. Malaria parasites in human blood can be detected down to 10 infected red blood cells µL-1. The assay could potentially be applied as a point-of-care test. The signal-amplification of an analyte by biocatalytic precipitation polymerization represents a powerful approach in biosensing.