## 2 Thermo-Responsive Molecularly Imprinted Polymers
Moreover, unlike those described above, the approach only requires the facile PNIPAm-grafting on the preformed MIP microspheres and no PNIPAm blocks in the imprinted network, thus avoiding many complicated and time-consuming optimizations for MIPs formulations and making the preparation more flexible. This represents a novel and promising way to develop advanced MIP materials not only with thermo-responsive binding property, but also with other stimuli-responsive properties such as when replacing the thermally responsive polymer with a pH or light sensitive polymer, _etc_. Furthermore, by the combined use of PNIPAm brushes on the surface and PNIPAm blocks into the imprinted network, [PERSON] _et al_. prepared a molecularly imprinted bead with double thermo-sensitive gates for selective recognition and release of proteins [33]. The MIP beads were prepared by two-step surface-initiated inferter polymerization on mesoporous chloromethylated polystyrene (MCP) beads, which formed double layers, an internal thermo-sensitive lysozyme imprinted layer, and an external linear PNIPAm brush layer. The two layers showed different volume phase transition temperature. At 43 \({}^{\circ}\)C, the outer linear PNIPAm layer and the inner imprinting layer were all collapsed, resulting in a hydrophobic shell and distorted recognition sites in the core, respectively. This led to nonspecific  
Figure 3: Protocol for the preparation of water-compatible and stimuli-responsive MIP microspheres with surface-grafted functional polymer brushes. Reprinted with permission from Reference 36. Copyright 2010, Elsevier. protein binding on the MIP bead surface. When the temperature changed to 38 \({}^{\circ}\)C, the MIP bead expanded to form a hydrophilic PNIPAm shell and a spatial integrity of the internal recognition site in the core, so a suppression of nonspecific protein binding was observed accompanied by improved specific protein binding. At even lower temperatures around 23 \({}^{\circ}\)C, the internal recognition site showed a more swollen state and a weak binding capacity to the template protein. The results further indicate that fabricating the thermo-sensitive surface layer or the interior imprinted sites for the MIPs can both create thermo-responsive binding capacity in spite of their different manner and different effect. Besides, if we regulate the performance (e.g., an identical volume phase transition temperature) of the surface and interior of MIPs to an optimal level, fantastic thermo-responsive MIPs could be obtained. To enhance cell adhesion during culture and facilitate the rapid harvest of cell sheets, [PERSON] _et al._ reported a novel system for harvesting cell sheets which relies on a PNIPAm-based MIP hydrogel layer with thermo-responsive affinity toward specific biomolecules by redox-initiated polymerization (Figure 4) [37]. Molecular imprinting methodology was employed to introduce the cell-adhesive peptide RGDS onto a thermo-responsive cell culture substrate, which was innovatively used as a highly efficient novel system for harvesting cell sheets. With the reversible thermo-responsive \"specific recognition sites\", the imprinted hydrogel could recognize and bind RGDS molecules at a temperature common for cell culture (37 \({}^{\circ}\)C), and then rapidly release RGDS when the temperature was lowered. Compared with simple physical absorption and covalent immobilization approaches, the thermo-responsive \"specific  
Figure 4: Strategy to introduce the RGDS peptide to a thermo-responsive cell culture substrate by means of molecular imprinting and the schematic illustration for the cell adhesion and the harvesting of a cell sheet. Reprinted with permission from Reference 37. Copyright 2013, Wiley. binding\" in the imprinted hydrogel promotes cell adhesion during cell culture and facilitates cell detachment during cell sheet harvest.