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In order to assist the refolding of recombinant nitrilase inclusion bodies, a series of thermoresponsive media were prepared by grafting poly(N-isopropylacrylamide-co-butyl-methacrylate) [P(NIPAM-co-BMA)] brushes onto PS microspheres with various particles and pore sizes via an atom transfer radical polymerization (ATRP) method. The effects of particle sizes, pore sizes, and brush grafting amounts of thermoresponsive microspheres on nitrilase refolding were investigated preliminarily. The results showed that the PS-P(NIPAM-co-BMA) microspheres with the medium particle size (74 μm), gigapore size (320 nm), and high grafting amount (35.6 mg/m2) were the most effective candidates. The final nitrilase activity yield could be up to 84.5% with a high initial protein concentration (1 mg/mL) at 30 °C, which was 52.5% higher than that of a simple dilution refolding method at the initial protein concentration (0.1 mg/mL). After the refolding process, the PS-P(NIPAM-co-BMA) microspheres can be easily separated by self-precipitation, and the activity yield of nitrilase still reached 74.5% after being reused for five batches. These results indicated that the thermoresponsive gigaporous medium was an ideal alternative as an artificial chaperone.

In order to assist the refolding of recombinant nitrilase inclusion bodies, a series of thermoresponsive media were prepared by grafting poly(N-isopropylacrylamide-co-butyl-methacrylate) [P(NIPAM-co-BMA)] brushes onto PS microspheres with various particles and pore sizes via an atom transfer radical polymerization (ATRP) method. The effects of particle sizes, pore sizes, and brush grafting amounts of thermoresponsive microspheres on nitrilase refolding were investigated preliminarily. The results showed that the PS-P(NIPAM-co-BMA) microspheres with the medium particle size (74 μm), gigapore size (320 nm), and high grafting amount (35.6 mg/m2) were the most effective candidates. The final nitrilase activity yield could be up to 84.5% with a high initial protein concentration (1 mg/mL) at 30 °C, which was 52.5% higher than that of a simple dilution refolding method at the initial protein concentration (0.1 mg/mL). After the refolding process, the PS-P(NIPAM-co-BMA) microspheres can be easily separated by self-precipitation, and the activity yield of nitrilase still reached 74.5% after being reused for five batches. These results indicated that the thermoresponsive gigaporous medium was an ideal alternative as an artificial chaperone.

The hierarchically bicontinuous polystyrene monoliths (HBPMs) with homogeneous skeletons and glycopolymer surfaces are fabricated for the first time based on the medium internal phase emulsion (MIPE) templating method via activator generated by electron transfer for atom transfer radical polymerization (AGET ATRP). The synergistic self-assembly of amphiphilic diblock glycopolymer (ADG) and Pluronic F127 (PF127) at the oil/water interface via hydrogen bonding interaction contributes to the formation of bicontinuous MIPE with deformed neighboring water droplets, resulting in the highly interconnected HBPM after polymerization. There is a bimodal pore size distribution in the HBPM, that is, through pores (150-5000 nm) and mesopores (10-150 nm). The HBPMs as prepared show excellent biocompatibility, homogeneous skeletons, strong mechanical strength, and high bed permeability, overcoming the practical limitations of the second generation of polystyrene (PS) monoliths. Glycoprotein concanavalin A (Con A) can be easily and quickly separated by the HBPM in hydrophilic interaction chromatography (HILIC) mode. These results suggest the HBPMs have great potentials in catalysis, separations, and biomedical applications.

A temperature-responsive solid-phase microextraction (SPME) coating was prepared via in-situ atom transfer radical polymerization (ATRP) method. By controlling the temperature of solution below and above the lower critical solution temperature (LCST) of the coating, it can switch between hydrophilic and hydrophobic, thus providing a convenient approach for the selective extraction of analytes with different polarities. The average extraction amount of temperature-responsive coating for polar analytes is about 1.5-fold to that of non-polar ones below LCST, and vice versa. Effective extraction of three biomacromolecules was also obtained by controlling the temperature below or above LCST. The adsorption capacity of the coating for the hydrophilic biomacromolecules at 15 °C is 1.5-2 folds that of 50 °C, whereas the adsorption capacity of the coating to BSA at 50 °C is about 3 folds that of 15 °C. This approach holds great promise for SPME because it provides a simple strategy to prepare bifunctional coatings for various applications.