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

Abstract

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, 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 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.

Enzyme Catalysis for Reversible Deactivation Radical Polymerization

“…ATRPase is particularly suited for sensing applications because ATRPase acts on alkyl bromides (ATRP initiator) to generate radicals, which can avoid possible interference from easily produced, unsourced radicals. This distinct feature of ATRPase was recently exploited by the Bruns group for sensing hemoglobin and hemozoin (a marker of the malaria parasite) [40d,f,g] . Both hemoglobin and hemozoin were able to catalyze the precipitation ATRP of NIPAM, and quantitative detection was made possible through the solution turbidity (Figure 10).…”

Radical polymerization reactions for amplified biodetection signals

“…Given that hemoglobin itself can catalyze ATRP, Bruns and coworkers also demonstrated the use of hemoglobin-catalyzed precipitation polymerization to quantify the amount of hemoglobin in biological fluids. 59 In this method, the polymerization conditions were the same as the hemozoin-catalyzed ATRP 56 except that sample preparation method and buffer pH were slightly different. The dose-response curve between logarithmic hemoglobin concentration and the rate of turbidity increase showed that the rate of turbidity increase was positively correlated with the concentration of hemoglobin although the rate reached a plateau at high concentration.…”

“…The dose-response curve between logarithmic hemoglobin concentration and the rate of turbidity increase showed that the rate of turbidity increase was positively correlated with the concentration of hemoglobin although the rate reached a plateau at high concentration. 59 According to authors, the plateau could appear likely because hemoglobin precipitated with PNIPAAm as polymerization proceeded. At low hemoglobin concentrations, the detection limit was 100 nM in buffer solution.…”

“…Biocatalytic ATRP is a new approach to detection of specific biomarkers with catalytic activity. As demonstrated by Bruns and coworkers, the biocatalyzed precipitation polymerization is a promising technology for point-of-care malaria 56 and hemoglobin 59 tests given that all reagents are stable during long-term storage at 50°C and the method induces extinction changes depending on the concentration of biocatalysts.…”

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

“…Hemoglobin can also be detected by the assay in a sensitive way. 29 Hemozoin is a product of hemoglobin digestion by Plasmodium sp. parasites.…”