Cellular micropatterning has become an important tool to precisely design cell-to-substrate attachment for cell biology studies, tissue engineering, cell-based biosensors, biological assays, and drug screens. This paper describes a new technique for micropatterning of cells that is based on the use of oxygen plasma as a patterning tool. The technique consists of (1) homogeneously grafting a glass substrate with a protein-repellent interpenetrating polymeric network (IPN) of poly(acrylamide) and poly(ethyleneglycol) [P(AAm-co-EG)] prepared with commercially available reagents and (2) selectively removing this coating using oxygen plasma. We use elastomeric stencils (i.e. self-sealing membranes with through-holes) and microchannels as removable masks for the selective oxygen plasma etch of the IPN areas that are not protected by the mask. The stencil or microchannels are peeled off to reveal cell-adhesive regions separated by the nonadhesive coating. Our method offers a convenient way of patterning a robust protein-repelling material, allows for independently controlling the chemistry of the regions reserved for cell attachment, and can be used to create coculture systems.
Block copolymers containing stimuli-responsive segments provide important new opportunities for controlling the activity and aggregation properties of protein-polymer conjugates. We have prepared a RAFT block copolymer of a biotin-terminated poly(N-isopropylacrylamide) (PNIPAAm)-b-poly(acrylic acid) (PAA). The number-average molecular weight (M(n)) of the (PNIPAAm)-b-(PAA) copolymer was determined to be 17.4 kDa (M(w)/M(n) = 1.09). The PNIPAAm block had an M(n) of 9.5 kDa and the poly(acrylic acid) (PAA) block had an M(n) of 7.9 kDa. We conjugated this block copolymer to streptavidin (SA) via the terminal biotin on the PNIPAAm block. We found that the usual aggregation and phase separation of PNIPAAm-SA conjugates that follow the thermally induced collapse and dehydration of PNIPAAm (the lower critical solution temperature (LCST) of PNIPAAm is 32 degrees C in water) is prevented through the shielding action of the PAA block. In addition, we show that the cloud point and aggregation properties (as measured by loss in light transmission) of the [(PNIPAAm)-b-(PAA)]-SA conjugate also depended on pH. At pH 7.0 and at temperatures above the LCST, the block copolymer alone was found to form particles of ca. 60 nm in diameter, while the bioconjugate exhibited very little aggregation. At pH 5.5 and 20 degrees C, the copolymer alone was found to form large aggregates (ca. 218 nm), presumably driven by hydrogen bonding between the -COOH groups of PAA with other -COOH groups and also with the -CONH- groups of PNIPAAm. In comparison, the conjugate formed much smaller particles (ca. 27 nm) at these conditions. At pH 4.0, however, large particles were formed from the conjugate both above and below the LCST (ca. 700 and 540 nm, respectively). These results demonstrate that the aggregation properties of the block copolymer-SA conjugate are very different from those of the free block copolymer, and that the outer-oriented hydrophilic block of PAA shields the intermolecular aggregation of the block copolymer-SA bioconjugate at pH values where the -COOH groups of PAA are significantly ionized.
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