Shear Stress-Responsive Polymersome Nanoreactors Inspired by the Marine Bioluminescence of Dinoflagellates.

Some marine plankton called dinoflagellates emit light in response to the movement of surrounding water, resulting in a phenomenon called milky seas or sea sparkle. The underlying concept, a shear-stress induced permeabilisation of biocatalytic reaction compartments, is transferred to polymer-based nanoreactors. Amphiphilic block copolymers that carry nucleobases in their hydrophobic block are self-assembled into polymersomes. The membrane of the vesicles can be transiently switched between an impermeable and a semipermeable state by shear forces occurring in flow or during turbulent mixing of polymersome dispersions. Nucleobase pairs in the hydrophobic leaflet separate when mechanical force is applied, exposing their hydrogen bonding motifs and therefore making the membrane less hydrophobic and more permeable for water soluble compounds. This polarity switch is used to release payload of the polymersomes on demand, and to activate biocatalytic reactions in the interior of the polymersomes.

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.

Repurposing Biocatalysts to Control Radical Polymerizations.

Reversible-deactivation radical polymerizations (controlled radical polymerizations) have revolutionized and revitalized the field of polymer synthesis. While enzymes and other biologically derived catalysts have long been known to initiate free radical polymerizations, the ability of peroxidases, hemoglobin, laccases, enzyme-mimetics, chlorophylls, heme, red blood cells, bacteria, and other biocatalysts to control or initiate reversible-deactivation radical polymerizations has only been described recently. Here, the scope of biocatalytic atom transfer radical polymerizations (bioATRP), enzyme-initiated reversible addition-fragmentation chain transfer radical polymerizations (bioRAFT), biocatalytic organometallic-mediated radical polymerizations (bioOMRP), and biocatalytic reversible complexation mediated polymerizations (bioRCMP) is critically reviewed, and the potential of these reactions for the environmentally friendly synthesis of precision polymers, for the preparation of functional nanostructures, for the modification of surfaces, and for biosensing is discussed.

Controlling Enzymatic Polymerization from Surfaces with Switchable Bioaffinity.

The affinity of surfaces toward proteins is found to be a key parameter to govern the synthesis of polymer brushes by surface-initiated biocatalytic atom transfer radical polymerization (SI-bioATRP). While the "ATRPase" hemoglobin (Hb) stimulates only a relatively slow growth of protein repellent brushes, the synthesis of thermoresponsive grafts can be regulated by switching the polymer's attraction toward proteins across its lower critical solution temperature (LCST). Poly(N-isopropylacrylamide) (PNIPAM) brushes are synthesized in discrete steps of thickness at temperatures above LCST, while the biocatalyst layer is refreshed at T < LCST. Multistep surface-initiated biocatalytic ATRP demonstrates a high degree of control, results in high chain end group fidelity and enables the synthesis of multiblock copolymer brushes under fully aqueous conditions. The activity of Hb can be further modulated by tuning the accessibility of the heme pocket within the protein. Hence, the multistep polymerization is accelerated at acid pH, where the enzyme undergoes a transition from its native to a molten globule conformation. The controlled synthesis of polymer brushes by multistep SI-bioATRP highlights how a biocatalytic synthesis of grafted polymer films can be precisely controlled through the modulation of the polymer's interfacial physicochemical properties, in particular of the affinity of the surface toward proteins. This is not only of importance to gain a predictive understanding of surface-confined enzymatic polymerizations, but also represents a new way to translate bioadhesion into a controlled functionalization of materials.