一种温敏型超大孔生物分离介质的制备及表征

Preparation and Characterization of a Thermoresponsive Gigaporous Bioseparation Medium

采用改性琼脂糖对超大孔聚苯乙烯微球进行亲水化修饰(Agap-PS),通过酰基化反应在微球表面引入溴乙酰基(Agap-PS-Br),然后利用原子转移自由基聚合(ATRP)反应在Agap-PS-Br表面接枝温敏聚合物刷,得到一种温敏型超大孔生物分离介质(Agap-PS-PNIPAM).考察了配体、催化剂、溶剂和温度对N-异丙基丙烯酰胺ATRP反应的影响,在优化条件下PNIPAM的接枝量达到了15.07 mg/m2.采用红外光谱(FTIR)、扫描电镜(SEM)、压汞分析、激光共聚焦和蛋白吸附等手段对温敏型超大孔生物分离介质进行一系列表征,结果表明接枝温敏聚合物刷后Agap-PS-PNIPAM具有良好的温敏性,没有堵塞微球的超大孔,微球对蛋白的非特异性吸附大大降低.由于温敏聚合物刷发生了从亲水到疏水构象的转变,40℃时Agap-PS-PNIPAM对蛋白的吸附量是25℃时的2.69倍.压力流速实验表明Agap-PS-PNIPAM柱具有背压低、渗透性和机械稳定性好的优点,同样地由于PNIPAM链在40℃时收缩,此时Agap-PS-PNIPAM柱的床层渗透系数比25℃时提高了15.7%.

Gigaporous PS microspheres, prepared in a previous study, were hydrophilized with modified agarose （Agap-PS）, and then a thermoresponsive gigaporous bioseparation medium （Agap-PS-PNIPAM）was prepared through a two-step reaction. Bromoacetyl groups were coupled on Agap-PS through acylation to obtain a solid atom transfer radical polymerization （ATRP） initiator （Agap-PS-Br）, and poly（N-isopro- pylacrylamide） （PNIPAM） brushes were grafted onto Agap-PS-Br via ATRP afterwards. The effects of ligands, catalysts,solvent and temperature on NIPAM solution ATRP were investigated in detail, and the amount of PNIPAM grafted on Agap-PS-Br reached to 15.07 mg/m^2 under optimal conditions. Agap-PS-PNIPAM was characterized by Fourier transform infrared （FTIR） spectra, scanning electron microscope （SEM） , mercury porosimetry measurements,laser eonfoeal scanning microscope （LCSM） , and protein adsorption experiments. Results showed that thermoresponsive PNIPAM brushes were successfully grafted on the PS microspheres and that the Agap-PS-PNIPAM had good thermo-responsibility. Meanwhile, the gigaporous structure of PS mierospheres was well maintained. After modification, nonspecific adsorption of proteins on PS microspheres was greatly reduced. The adsorption amount of bovine serum albumin （BSA） on Agap-PS-PNIPAM at 40℃ was 2.69 times to that at 25℃ due to the phase transition of PNIPAM brushes from hydrophilicity to hydrophobicity. Flow experiments showed that the Agap-PS-PNIPAM column had low backpressure, good permeability and mechanical stability. Similarily,the bed permeability （K） of Agap-PS-PNIPAM column at 40℃ increased by 15.7% than that at 25℃ owing to the shrinking of PNIPAM brushes. All results indicate that Agap-PS-PNIPAM has great potentials in green high-speed protein chromatography.