Macroporous polyacrylamide monolithic gels with immobilized metal affinity ligands: the effect of porous structure and ligand coupling chemistry on protein binding

Abstract

Macroporous polyacrylamide gels (MPAAG) with iminodiacetic acid (IDA) functionality were prepared by (i) chemical modification of polyacrylamide gel, (ii) co-polymerization of acrylamide with allyl glycidyl ether (AGE) and N,N'metylene-bis(acrylamide) (MBAAm) followed by coupling IDA ligand or (iii) by copolymerization of acrylamide and MBAAm with functional monomer carrying IDA-functionality (1-(N,N-bis(carboxymethyl)amino-3-allylglycerol). Screening for optimized conditions for the production of the MPAAG with required porous properties was performed in a 96-well chromatographic format that allowed parallel production and analysis of the MPAAG prepared from reaction mixtures with different compositions. Scanning electron microscopy of the fabricated MPAAG revealed two different types of the porous structures: monomodal macroporous structure with large interconnected pores separated by dense non-porous pore walls in case of plain gels or gels produced via copolymerization with AGE. The other type of the MPAAG (gel produced via co-polymerization with functional monomer carrying IDA-functionality) had bimodal pore structure with large interconnected pores separated by the pore walls pierced through with micropores. The effect of different modifications of MPAAG monoliths and of porous structure of the MPAAG (monomodal and bimodal porous structure) on protein binding has been evaluated.

Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate‐containing fluids and cell culture applications

“…A high monomer conversion (gelation yield) of MGs of 70 -90% is typically achieved during the formation of MGs at moderately low temperature, down to -208C [10,42]. The formation of pAAm MGs with bimodal pore size distribution resulted from a combination of two processes, namely the formation of macropores in the non-frozen liquid microphase (when solvent crystals act as porogen) and the phase separation of the polymer synthesized within the non-frozen liquid phase [10,21].…”

“…The ligand could be coupled to a reactive group, e. g. an epoxy group included in pAAm MGs, either during the production directly [18,32] or via a spacer [19,21] or by grafting of polymeric chains onto cryogel surface followed by derivatization of the grafted polymer with required functionality [24,25] or through incorporation of the functional group via copolymerization of AAm with the monomer bearing the required functionality [21] with no post-polymerization derivatization needed. An alternative way to modify the surface and porous properties of MGs is to form the additional cryogel network with required functionality and surface properties inside interconnected pores of the already prepared MGs [14].…”

“…The b-PVA MG perform efficiently when used in expanded bed chromatography mode [36] or as carriers for immobilization of cells to be used in well stirred tanks [27,54] or as carriers for immobilized enzymes [55 -58]. Typically, the back pressure depends on the porous structure of MG monoliths and is higher for MGs with bimodal pore size distribution (when large interconnected pores are surrounded by microporous pore walls) compared to the MGs with monomodal pore size distribution (when large interconnected pores are surrounded by dense and non-porous pore walls) [21].…”

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

“…Acrylamide (AAm) and N,N -methylenebisacrylamide (MBA), which have been extensively used in polyacrylamide gel electrophoresis [24,25], are regarded as beneficial monomers to form monoliths with excellent hydrophilicity [17,19,20]. However, the traditional poly (AAm-co-MBA) monolith, which was formed by free radical polymerization reaction in an aqueous medium [26][27][28][29][30], was cryogels with an elastic feature. With a different approach, heat polymerization, Frechet et al [31] prepared a poly (AAm-co-MBA) monolith which exhibited much better mechanical stability than cryogels.…”

“…doi:10.1016/j.talanta.2010.12.011 1,4-butanediol, dodecanol and dimethyl sulfoxide (DMSO) as porogens. After sequentially activated by ethylenediamine [28] and glutaraldehyde, the monolith was ready for trypsin immobilization. The mechanical stability and permeability of prepared poly (AAm-co-MBA) monolith were evaluated systematically.…”