Composite thermally sensitive particles were elaborated by chemical grafting of reactive poly(NIPAM) chains on colloidal silica particles or by encapsulation of iron oxide nanoparticles. Based on the high interest in magnetic colloidal particles in the biomedical field, various approaches have been developed as evidenced from the reported papers. The first work in this domain was reported by Kawaguchi et al.  who produced iron oxide nanocrystals in the preformed cross-linked poly(NIPAM) microgel particles. The amount of iron oxide incorporated was reported to be low so as to induce fast magnetic separation of such composite materials. Then thermoflocculation was induced to enhance magnetic separation. To increase the iron oxide amount in the colloidal composite particles, a new method has recently been reported by Sauzedde et al. [21,22] derived from the early method developed by Furusawa et al. . This approach is divided into two steps: (1) adsorption of iron oxide nanoparticles negatively charged onto polystyrene core cationic poly(NIPAM) shell, and (2) encapsulation and functionalization of adsorbed iron oxide particles via precipitation polymerization using NIPAM monomer, MBA as cross-linker, potassium persulfate, and itaconic acid as functional monomer. The elaborated composite particles contain at least 20% iron oxide material, which favors their magnetic separation.
The colloidal properties of such thermally sensitive particles were found to be dramatically affected by the environmental temperature. The hydrodynamic particle size decreased with increasing the incubation temperature, and the volume phase transition temperature was found to be in the poly(NIPAM) lower critical solution temperature (LCST) region. Consequently, the surface charge density (i.e., electrophoretic mobility) increased dramatically as the temperature increased above the TVPT.
III. PROTEIN ADSORPTION ON THERMALLY SENSITIVE PARTICLES
As mentioned in the previous section, single thermosensitive particles show a variety of surface properties. Their features are shown in Fig 4. Thermosensitive particles change in their hydrophilicity, softness, electric potential by virtue of the transition temperature of thermosensitive components in the particle. In the case of poly(NIPAM) particles, they are hydrophilic below the transition tem-
perature (32°C) as shown with the absorption peak of 1-anilinonaphthalene-8-sulfonic acid (ANS) at 515 nm or with the contact angle less than 28° , but become hydrophobic (or less hydrophilic) at a temperature higher than the transition temperature of poly(NIPAM) as shown with the absorption of ANS at 493 nm or with the contact angle of 45°. The absorption peak at 493 nm at high temperature indicates that the hydrophilicity of poly(NIPAM) atmosphere at a temperature close to that of acetone. Based on electrophoresis, the apparent surface charge of particles is low or negligible below the transition temperature but becomes high above 32°C. This was attributed to the change of density of ionic groups, e.g., sulfate group originated from the initiator fragment binding to the polymer chain end. Ohshima et al. argued that the electrophoretic mobility of hydrogel particles is a function of the volume density of the ionic group but not the surface density . Ohshima's equation included a term for softness of the thermosensitive gel phase. The experimental results indicated that softness significantly decreased with increasing temperature. The effect of temperature on hydro-dynamic particle size and electrokinetic properties is illustrated in Fig. 5.
As hydrophilicity and surface charge are two most important factors influencing the proteic material immobilization, the adsorption of proteins onto poly-(NIPAM) particles is significantly affected by the incubation temperature.
One point to consider is the permeation of proteins into the particles leading
to protein absorption. If the particle has a hairy or loosely cross-linked structure, not only adsorption but also absorption can take place. In the case of thermally sensitive particles whose degree of swelling changes sharply at a certain temperature, swollen particles may have the same situation and invite proteins inside if the cross-link density is not so high; however, shrunken particles do not accept protein absorption even if they are cross-linked very loosely. Anionic group-containing poly(NIPAM) gel particles selectively absorbed cationic proteins at temperatures lower than 32°C, and this technology was applied to bioseparation .
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