We investigated physicochemical properties of two types of poly(N-isopropylacrylamide) (PIPAAm)-grafted tissue culture polystyrene (TCPS) surfaces, to elucidate the influential factors for thermally regulated cell adhesion and detachment to PIPAAm-grafted surfaces. The two types of PIPAAm-grafted surfaces were prepared by the electron beam polymerization method. Attenuated total reflection Fourier transform infrared spectroscopy revealed that amounts of the grafted polymers were 1.4 +/- 0.1 microg/cm2 for PIPAAm-1.4 and 2.9 +/- 0.1 microg/cm2 for PIPAAm-2.9. Both PIPAAm-grafted surfaces showed hydrophobic/hydrophilic property alterations in response to temperature. However, PIPAAm-1.4 surfaces were more hydrophobic (cos theta = 0.21 at 37 degrees C and cos theta = 0.35 at 20 degrees C) than PIPAAm-2.9 (cos theta = 0.42 at 37 degrees C and cos theta = 0.50 at 20 degrees C) both above and below the PIPAAm's transition temperature. Thicknesses of the grafted PIPAAm layers were estimated to be 15.5 +/- 7.2 nm for PIPAAm-1.4 and 29.5 +/- 8.4 nm for PIPAAm-2.9, by the use of UV excimer laser and atomic force microscope. Bovine carotid artery endothelial cells (ECs) adhere to the surfaces of PIPAAm-1.4 and proliferate to form confluent cell monolayers. The cell monolayers were harvested as single cell sheets by temperature decrease from 37 to 20 degrees C. On the contrary, ECs did not adhere to the surfaces of PIPAAm-2.9. This phenomenon was correlated with an adsorption of cell adhesion protein, fibronectin, onto surfaces ofPIPAAm-1.4 and -2.9. In the case of nano-ordered thin grafted surfaces, the surface chain mobility is strongly influenced by the thickness of PIPAAm grafted layers because dehydration of PIPAAm chains should be enhanced by the hydrophobic TCPS surfaces. PIPAAm graft amounts, that is, thickness of the PIPAAm grafted layers, play a crucial role in temperature-induced hydrophilic/hydrophobic property alterations and cell adhesion/detachment behavior.

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Dense poly(N-isopropylacrylamide) (PIPAAm) brushes were created on silica bead surfaces by surface-initiated atom transfer radical polymerization (ATRP). Interfacial properties of PIPAAm brushes were characterized by thermoresponisve interaction with biomolecules. The grafted amounts of PIPAAm on silica bead surfaces exceeded that from previously reported polymer-hydrogel-modified silica beads prepared by conventional radical polymerization by nearly 1 order of magnitude. Temperature-dependent chromatographic interactions with soluble analytes were modulated by changing the grafted PIPAAm chain lengths. Short PIPAAm-grafted silica beads produce insufficient dehydration and chain aggregation to separate steroids using weak hydrophobic interactions. In contrast, broad unresolved peaks were observed on silica beads column grafted with long PIPAAm chains due to steroid partitioning into thick, densely grafted PIPAAm brush layers. Thus, silica beads column grafted with PIPAAm chains of proper length can demonstrate baseline separation of steroids with relatively high resolution among the tested columns. Relatively longer retention times for steroid analytes were observed on all columns compared to those previously reported for other PIPAAm-grafted silica beads. This indicates that densely PIPAAm-grafted chains enable control of strong hydrophobic interactions with steroids by changing the column temperature. Densely grafted PIPAAm columns were also successful in separating two peptides into two peaks as the column temperature was increased to 40 degrees C. This provides an effective separation alternative for peptides using substantial hydrophobicity without modification of hydrophobic surfaces and/or low mobile phase pH. In conclusion, densely PIPAAm-grafted surfaces exhibit strong, reversible temperature-modulated hydrophobic interactions, facilitating baseline separations of steroids and peptides in aqueous milieu without changes in the mobile phase pH and high ionic strength.

We have prepared various poly(N-isopropylacrylamide) (PIPAAm)-grafted silica bead surfaces through surface-initiated atom transfer radical polymerization (ATRP) by changing graft densities and brush chain lengths. The prepared surfaces were characterized by chromatographic analysis using the modified silica beads as chromatographic stationary phases. ATRP initiator (2-(m,p-chloromethylphenyl)ethyltrichlorosilane) density on silica bead surfaces was modulated by changing the feed composition of the self-assembled monolayers (SAMs) of mixed silane coupling agents consisting of ATRP initiator and phenethyltrichlorosilane on the surfaces. IPAAm was then polymerized on SAM-modified silica bead surfaces by ATRP in 2-propanol at 25 degrees C. The chain length of the grafted PIPAAm was controlled by simply changing the ATRP reaction time at constant catalyst concentration. The thermoresponsive surface properties of the PIPAAm-grafted silica beads were investigated by temperature-dependent elution behavior of hydrophobic steroids from the surfaces using Milli-Q water as a mobile phase. On the surfaces grafted with shorter PIPAAm chains, longer retention times for steroids were observed on sparsely grafted PIPAAm surfaces compared to dense PIPAAm brushes at low temperature, because of hydrophobic interactions between the exposed phenethyl groups of SAMs on silica surfaces and steroid molecules. Retention times for steroids on dilute PIPAAm chain columns decreased with temperature similarly to conventional reverse-phase chromatographic modes on octadecyl columns. This effect was due to limited interaction of solutes with the PIPAAm-grafted surfaces. Retention times for steroids on dilute PIPAAm brush surfaces with longer PIPAAm chains became greater above the PIPAAm transition temperature. At low-temperature regions, hydrated and expanded PIPAAm at low temperatures prevented hydrophobic interactions between the phenethyl group of SAMs on the silica bead surfaces and steroid molecules. Retention times for steroids on a dense PIPAAm brush column increased with temperature since solvated polymer segments within the dense brush layer undergo dehydration over a broad range of temperatures. In conclusion, PIPAAm graft density has a crucial influence on the elution behavior of steroids because of the interaction of analytes with silica bead interfaces, and because of the characteristic dehydration of PIPAAm in dense-pack brush surfaces.