## 2 Thermo-Responsive Molecularly Imprinted Polymers
reported on a temperature-sensitive protein-imprinted hydrogel based on PNIPAm by using _N_-(3-(Dimethylamino)propyl)-methacrylamide (DMPMA) as functional monomer and bovine serum albumin (BSA) as a template through electrostatic interaction [88]. After removal of the template proteins by treatment with 500 mM aqueous NaCl solution, the polymer affinity to recognize the target protein was greatly influenced by external stimuli, such as temperature and ionic strength. The authors found that the optimal binding conditions (maximal affinity toward the template) were very close to the original imprinting state, proving that the origin of the specificity relied on the spatial integrity of the recognition site formed in the imprinting process. The changes in gel volume result in a decreased affinity of the MIPs. Considering the similarity with NIPAm, TBAm [47] and MBAAm [57] were also used as thermo-responsive co-monomers in MIPs to achieve responsive molecular recognition. TBAm has proven to be a temperature sensitive monomer that allowed for swelling and shrinking of the hydrogels in response to temperature [96]. According to this, [PERSON] _et al_. prepared poly(_N-tert_-butylacrylamide-_co_-acrylamide/maleic acid) imprinted hydrogels for recognizing BSA with a thermo-responsive performance [47]. Adsorption studies also showed that other stimuli, such as pH and initial BSA concentration, also influenced the BSA adsorption capacity of the MIP hydrogels. In the same manner, temperature sensitive binding capacity of dopamine-imprinted MBA cross-linked polymers was observed in 80% aqueous methanol solution around 35 \({}^{\circ}\)C by [PERSON] _et al_. [57]. Subsequently, the resulting MIP was tested for its application as a sorbent material of solid-phase extraction (SPE), utilizable in the selective extraction of dopamine from urine samples mixing with other adrenergic compounds (epinephrine, isoproterenol, salbutamol and serotonin). The above literature showed that the introduction of thermo-responsive co-monomers into the networks of MIPs was a general and efficient approach to fabricate thermo-responsive MIPs. Nevertheless, it is accompanied by a number of disadvantages. One obvious problem is the greater complexity of the self-assembly system between template and functional monomers caused by the addition of NIPAm. This, together with the decreased proportion of functional monomers, should lead to a lower affinity compared to conventional imprinted polymers without PNIPAm blocks. The problem is ubiquitous and similar to the development of water-compatible MIPs, whose affinity would be largely affected by the addition of hydrophilic co-monomers. Another problem is the contradiction between the affinity and the responsiveness of this kind of MIPs. One of the most attractive features of MIPs is their robustness with respect to reusability and compatibility with a range of solvents and test conditions. These properties depend strongly on the degree of cross-linking in the MIP. On the other hand, the volume inversion of PNIPAm based MIPs will be greatly limited in a case of a robust polymeric network, subsequently leading to a poor responsiveness to temperature. Overall, using this method, complicated and time-consuming optimizations are necessary for preparing a well-performing thermo-responsive MIP. Recently, [PERSON] _et al_. reported a novel approach to prepare both thermo-responsive and water-compatible MIP microspheres by the facile surface-grafting of PNIPAm brushes via RAFT polymerization [36] (Figure 3). The reported method demonstrated an efficient approach to avoid the disadvantages of PNIPAm based MIPs mentioned above by grafting a high-density layer of linear PNIPAm on the surface of preformed MIP microspheres. Distinctively, the hydrophilic/hydrophobic conversion on the surface of MIPs microspheres caused by the thermo-responsive PNIPAm brushes leads to a thermo-responsive binding property.