## 3 Results and Discussion
### Molecularly Imprinted Stir-Bars (MMISB) for Patulin Extraction  
In this study, 2-oxindole, a compound structurally related to PAT, was selected as a dummy template for the design of a MIP selective towards PAT. This analogue of patulin has already been used successfully to synthesize a MIP capable of selectively binding PAT molecules [9]. The removal of the template leaves binding sites within the polymeric matrix that are complementary in shape and functionality to the template and to the target molecule of PAT (Figure 1). Dummy templates are usually selected to overcome bleeding problems. Additional advantages of these templates may include lower cost and toxicity. In this context, a dummy-template approach was preferred to avoid the manipulation of patulin during MIP synthesis, as it could be hazardous to the personnel working in the laboratory. Patulin has been classified as having acute toxicity (oral, dermal, inhalation) for humans, while 2-oxindole has not been classified as hazardous (Sigma-Aldrich website). As for the costs, relatively high amounts of template are normally required to synthesize imprinted polymers. In that respect, 2-oxindole is much cheaper than patulin. To provide just one example, 5 mL of PAT correspond to the price of 25 g of 2-oxindole in the Spanish market (Sigma-Aldrich website).  
The polymer coated on the surface of the magnetic stir-bars was prepared by a non-covalent approach, based on the formation of the non-covalent interactions of MAA and the dummy template. MAA was selected because of its high capability to act both as a hydrogen bond and a proton donor and as a hydrogen bond acceptor [15]. The selected polymerization technique was bulk polymerization, because it does not require sophisticated instrumentation and the reaction conditions can be easily controlled. Furthermore, it is the most widely used method for the preparation of imprinted polymers [16]. In the design of any pre-polymerization mixture, the selection of the cross-linker and porogen solvent are two key factors, as they would determine the aspect, strength and even the color and porosity of the final polymer. DVB-80 and EGDMA were therefore tested separately and in combination with various solvent mixtures, in order to achieve the best option for MIP synthesis. The tested combinations were methanol and/or acetonitrile with different percentages of toluene, as follows: 0%, 20%, 50%, 70% and 90% toluene in methanol and 0%, 20%, 50%, 70% and 90% toluene in acetonitrile. The different combinations of monomer-solvent were introduced in an injection vial and allowed to polymerize under bulk conditions for 24 h at 60 \({}^{\circ}\)C. After polymerization, the glass vials were broken and the resulting material was tested visually and by touching. With the use of DVB-80 as a cross-linker, it was impossible to obtain a strong and tough polymer. The resulting polymer would be a plastic, yellowish, cracked and fragile material, not suitable for resisting the required stirring conditions. Thus, adequate divinylbenzene polymers could not be achieved using the different tested solvents (acetonitrile or methanol), even changing the toluene percentage (from 0% to 90% toluene). As for EGDMA, in every case the polymers were stronger than those obtained with DVB, showing more resistance to compressive strength and even more when using methanol (combined with toluene) instead of acetonitrile. As for the color, EGDMA polymers were white and DVB yellowish. The mixture of methanol and toluene (90:10) with EGDMA proved to be useful, resulting in a hard and white polymer. Consequently, this combination was used to prepare the magnetic molecularly imprinted stir-bar (MMISB).