## 3 Results and Discussion  
[PERSON] et al. [1] showed how the \"sacrificial spacer\" methodology [19] could be applied to build a MIP with specific recognition sites for TCDD (MIP1). In fact, the application of a close analogue of dioxin, \(N\)-(2-(3,7,8-trichlorodibenzodioxinyl))-2-methacryloyloxybenzamide (6), fused with a salicylic acid moiety ensures the introduction of methacrylic acid residue into polymer after hydrolysis for template removal (Fig.4). In this way, dioxin binding was allowed by the enhanced hydrogen bond with chlorine atom as methacrylic acid residue is situated in a fixed position in the active site; it can be noticed that salicylic acid provides an intramolecular hydrogen bonding between oxygen atom and -NH group [1], essential for creating right space arrangement in the binding site.  
In our strategy, the production of methacrylic functionality under suitable geometric constraints was achieved by a more simplified scheme (see experimental) with less extensive synthetic work (the chlorination of (4) and the production of (6) are avoided).  
As it was discussed, the proposed MIPs differ in templates but the monomers employed in the polymerisation step are nearly the same (figs.4-5). In fact, previous study [1] underlined the importance of choosing a suitable co-monomer to improve \(\pi\)-\(\pi\) interactions with the electro-deficient dioxin system. On the basis of original results, 1-methoxy-3,5-bis(4-vinylbenzyloxy)benzene (7) was selected as a useful second recognition element into the binding sites. Comparison between figures 4 and 5 gives indication on the different imprinting approaches. While template for MIP1 (compound (6)) is covalently bonded to the methacrylic moiety to be polymerised, MIP2 is based on ionic interactions and/or hydrogen bonding between the template (dioxin analogue (3)) and (4) (bearing the methacrylic polymerisable group). The common drawback of non-covalent approach represented by template bleeding in analytical applications of MIPs is overcome in our proposed scheme by the use of a \"dummy\" template.  
To assess imprinting effect and evaluate specific binding capacity of MIP2, re-binding experiments were performed on MIP2 and NIP (Table 2). Results were also compared with performance of polymer MIP1, as evaluated in this work. A concentration of TCDD nearly 3 orders of magnitude lower than the one employed in the original paper was selected for re-binding experiments. This choice was aimed to lower influence of aspecific binding and evidence specific differences among tested polymers.  
It should be noted that polymer grain size distribution was not characterised. Even so, results of re-binding experiments were quite reproducible (Table 2).  
Data suggest that an imprinting effect is exhibited by both MIPs, higher for MIP1 as it could be expected. However, it is noteworthy that the imprinting effect is remarkably high also for the new proposed MIP; this feature was advantageously coupled to a simplified preparation scheme for MIP precursors. Thus, MIP2 could represent an interesting compromise between an easy polymer preparation and a good selectivity for dioxins.  
Our calculated ratios are significantly higher than in the original paper: we believe that this finding simply indicates that at the higher concentration employed by [PERSON] et al., aspecific binding has a significant role.  
Finally, it should be observed that extracted TCDD is lower in our measurements than in the original paper [1]: this could be tentatively attributed to the lower TCDD concentration and the coarser grain size polymer employed.