[Show abstract][Hide abstract] ABSTRACT: Porous nylon membranes modified with poly(acid) brushes and their derivatives can rapidly purify proteins via ion-exchange and metal-ion affinity interactions. Membranes containing poly(2-(methacryloyloxy)ethyl succinate) (poly(MES)) brushes bind 118 +/- 8 mg of lysozyme per cm(3) of membrane and facilitate purification of lysozyme from chicken egg white. Moreover, functionalization of the poly(MES) brushes with nitrilotriacetate (NTA)-Ni(2+) complexes yields membranes that bind poly(histidine)-tagged (His-tagged) ubiquitin with a capacity of 85 +/- 2 mg of protein per cm(3) of membrane. Most importantly, the membranes modified with poly(MES)-NTA-Ni(2+) allow isolation of His-tagged cellular retinaldehyde-binding protein directly from a cell extract in <10 min, and the protein purity is comparable to that achieved with commercial affinity columns. Therefore, porous nylon membranes containing functionalized poly(MES) brushes are attractive candidates for rapid, high-capacity purification of His-tagged proteins from cell extracts.
Full-text · Article · Mar 2010 · Biomacromolecules
[Show abstract][Hide abstract] ABSTRACT: This review examines the application of polymer brush-modified flat surfaces, membranes, and beads for protein immobilization and isolation. Modification of porous substrates with brushes yields membranes that selectively bind tagged proteins to give 99% pure protein at capacities as high as 100 mg of protein per cubic centimeter of membrane. Moreover, enrichment of phosphopeptides on brush-modified matrix-assisted laser desorption/ionization (MALDI) plates allows detection and characterization of femtomole levels of phosphopeptides by MALDI mass spectrometry. Because swollen hydrophilic brushes can resist nonspecific protein adsorption while immobilizing a high density of proteins, they are attractive as substrates for protein microarrays. This review highlights the advantages of polymer brush-modified surfaces over self-assembled monolayers and identifies some research needs in this area.
No preview · Article · Jul 2009 · Annual Review of Analytical Chemistry (2008)
[Show abstract][Hide abstract] ABSTRACT: The combination of a highly reactive monomer, 2-(methacryloyloxy)ethyl succinate (MES), and active catalyst systems, e.g., Cu(I) complexed with 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), allows rapid formation of poly(carboxylic acid) brushes by atom transfer radical polymerization (ATRP) from a surface. ATRP from initiators immobilized on Au-coated Si wafers yields films with an ellipsometric thickness of 120 nm in less than 15 min. To the best of our knowledge, this is the first example of direct ATRP of protonated acidic monomers that is capable of yielding such thick films, and MES polymerizes much more rapidly than methacrylic acid both in solution and on a surface. The MES films are attractive for a number of applications, including immobilization of proteins. Assuming that a lysozyme monolayer has a thickness of 2 nm, a 55 nm poly(MES) brush binds 70 monolayers of lysozyme via ion-exchange interactions. Additionally, when derivatized with nitrilotriacetate−Cu2+ complexes, poly(MES) films bind 34 monolayers of BSA through metal-ion affinity interactions (assuming a BSA monolayer thickness of 4 nm). These binding capacities are similar to those of poly(acrylic acid) brushes prepared by polymerization of tert-butyl acrylate and subsequent hydrolysis, but the poly(MES) synthesis is a one-step, aqueous process.
[Show abstract][Hide abstract] ABSTRACT: Over the last 15 years, the layer-by-layer deposition of polyelectrolytes and the growth of polymer brushes from surfaces have become established techniques for the formation of a wide range of thin films. This article discusses the use of these techniques in creating the skin layer of nanofiltration or gas-separation membranes and in functionalizing the interior of membranes for protein adsorption or catalysis. In the case of separation membranes for nanofiltration, the minimal thickness of layer-by-layer films allows for high flux, and the wide range of available polyelectrolytes that can form these films permits the tailoring of membranes for separations such as water softening, the reduction of F (-) concentrations, and the removal of dyes from wastewater. For gas separation, polymers grown from surfaces are more attractive than layer-by-layer coatings because most polyelectrolyte films are not highly gas-selective. Cross-linked poly(ethylene glycol dimethacrylate) films grown from porous alumina exhibit CO(2)/CH(4) selectivities of around 20, and the careful selection of monomers should further improve the selectivity of similar membranes. Both layer-by-layer methods and polymer brushes can also be employed to modify the interior of membranes, and we have utilized these techniques to create catalysts, antibody arrays in membranes, and membrane absorbers for protein purification. Polymer brushes are particularly attractive because they allow the absorption of multilayers of protein to yield membranes with binding capacities as high as 150 mg protein/cm(3). Some challenges in the practical implementation of these systems, such as the economical formation of membranes using highly permeable polymeric supports, and future directions in research on membrane modification with multilayer films and polymer brushes are also discussed herein.
[Show abstract][Hide abstract] ABSTRACT: The growth of polymer brushes on polymer substrates is often challenging because of substrate incompatibility with the organic solvents used for initiator attachment. This letter reports the use of layer-by-layer adsorption of macroinitiators and subsequent aqueous ATRP from these immobilized initiators to prepare polymer brushes on polymeric substrates. Polyethersulfone (PES) films and porous membranes were modified with polyelectrolyte multilayer films, and a previously developed polycationic initiator, poly(2-(trimethylammonium iodide)ethyl methacrylate-co-2-(2-bromoisobutyryloxy)ethyl acrylate), was then electrostatically adsorbed onto these polyelectrolyte films. The immobilized macroinitiator is very efficient in initiating the growth of polymer brushes on PES, as demonstrated by aqueous syntheses of poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) films. PHEMA (250 nm thick) and PDMAEMA (40 nm thick) brushes were grown in 2 h from surfaces modified with polycationic initiators. Moreover, this procedure is effective for growing brushes in the pores of PES membranes.
[Show abstract][Hide abstract] ABSTRACT: Porous membrane absorbers are attractive for increasing the rate of protein purification, but their binding capacity is low relative to porous beads. Modification of membranes with functionalized polymer brushes, however, can greatly enhance capacity. This work demonstrates that membrane modification with poly(2-hydroxyethyl methacrylate) (PHEMA) brushes derivatized with nitrilotriacetate-Ni2+ (NTA-Ni2+) complexes allows purification of polyhistidine-tagged ubiquitin (HisU) in less than 30 min with a binding capacity of 120 mg of HisU/cm3 of porous alumina membrane. Adsorption isotherms show that saturation of the brushes occurs at HisU concentrations as low as 0.04 mg/mL and that these brushes can bind up to 23 monolayers of HisU. Gel electrophoresis reveals that the purity of eluted HisU is more than 99%, even when the initial feed solution contains 10% bovine serum or a 20-fold excess of BSA. Thus, reusable porous membranes modified by PHEMA-NTA-Ni2+ brushes are attractive candidates for rapid purification of polyhistidine-tagged proteins.
No preview · Article · Nov 2007 · Biomacromolecules