## 2 Results
### The Self-Assembly Approach
#### 2.1.6 Binding Isotherms  
The minimum time required to reach optimal template binding for MIPs E71-MIPCHC13, E72-MIPCHC13, T71-MIPCHC13 and T72-MIPCHC13 was determined prior to undertaking any other binding assays. No marked difference in the rebinding of **1** was observed for incubation times between 30 min and 24 h. Thus, we opted to use 60 min for subsequent binding tests. As only one MIP from the CH\({}_{3}\)CN series (E71-CH3 CN) exhibited noteworthy imprinting, no further analysis of this series was undertaken. Figure 3: SEM images for (**A**) E72-MIP\({}_{\text{CH3 CN}}\) and (**B**) E72-MIP\({}_{\text{CHCl3}}\) obtained at 20,000\(\times\) magnification, at 15.0 kV. The binding isotherms were generated by varying the rebinding solution concentration of **1** between 0.5 and 10 mM, using a constant polymer loading of 20 mg and 60 min incubation (Supplementary Figure S19A-D). Maximum binding (B\({}_{\text{max}}\)) and affinity constants (K\({}_{\text{d}}\)) were calculated from non-linear regression of the Langmuir binding isotherms, using GraphPad Prism 9.4.1; the best-fit values obtained from the one-site-total-binding equation are presented in Table 1. These data show MIPs bound twice the amount of **1** than their corresponding NIPs, and this is attributable to the imprinting effect. E7\({}_{2}\) and T7\({}_{2}\) polymers (MIPs and NIPs) bound twice the amount of **1** than E7\({}_{1}\) and T7\({}_{1}\) polymers, a result that is unsurprising given that the E7\({}_{2}\) and T7\({}_{2}\) feed formulations contain twice the amount of functional monomer **7**. The K\({}_{\text{d}}\) values for the MIPs (0.22\(-\)0.62 uM) were consistently lower than the corresponding NIPs (0.71-1.52 uM) because of the higher affinity template binding arising from the imprinting process. Furthermore, the K\({}_{\text{d}}\) values were higher for the 1:1 (E7\({}_{1}\) and T7\({}_{1}\) \begin{table}
\begin{tabular}{c c c c c} \hline \multicolumn{1}{c}{**Polymers**} & \multicolumn{1}{c}{**K\({}_{\text{d}}\) (uM)**} & \begin{tabular}{c} **B\({}_{\text{max}}\)** \\ **(umol/g)** \\ \end{tabular} & \
\begin{tabular}{c} **K\({}_{\text{d}}\) Ratio** \\ **(NIP/MIP)** \\ \end{tabular} &
\begin{tabular}{c} **R\({}^{2}\)** \\ **(NIP/MIP)** \\ \end{tabular} \\ \hline \multirow{3}{*}{**E7\({}_{\text{1-MIPCHCI3}}\)**} & MIP & 0.27 \(\pm\) 0.07 & 56.2 \(\pm\) 5.6 & \multirow{3}{*}{5.0} & 0.966 \\  & NIP & 1.36 \(\pm\) 0.83 & 30.0 \(\pm\) 10.4 & & 0.884 \\  & MIP & 0.22 \(\pm\) 0.04 & 111.7 \(\pm\) 9.3 & \multirow{3}{*}{4.5} & 0.988 \\  & NIP & 0.98 \(\pm\) 0.50 & 66.2 \(\pm\) 19.3 & & 0.909 \\  & MIP & 0.60 \(\pm\) 0.16 & 45.9 \(\pm\) 6.0 & \multirow{3}{*}{2.5} & 0.933 \\  & NIP & 1.52 \(\pm\) 0.73 & 22.7 \(\pm\) 6.3 & & 0.934 \\  & MIP & 0.31 \(\pm\) 0.07 & 90.1 \(\pm\) 9.7 & \multirow{3}{*}{2.3} & 0.984 \\  & NIP & 0.71 \(\pm\) 0.26 & 35.7 \(\pm\) 7.2 & & 0.937 \\  & MIP & 0.09 \(\pm\) 0.02 & 16.8 \(\pm\) 1.2 & - & 0.949 \\  & MIP & 0.02 \(\pm\) 0.01 & 21.5 \(\pm\) 1.7 & - & 0.869 \\ \hline \end{tabular} \(\text{K}_{\text{d}}\) and B\({}_{\text{max}}\) are the best-fit values at 95% confidence interval from non-linear regression of the binding isotherms (Supplementary Figure S19), using the one-site–total-binding equation generated using GraphPad Prism 9.4.1.  
\end{table} 
Table 1: Binding and thermodynamic parameters for E7\({}_{\text{1-MIPCHCI3}}\), E7\({}_{\text{2-MIPCHCI3}}\), T7\({}_{\text{1-MIPCHCI3}}\) and T7\({}_{\text{2-MIPCHCI3}}\) towards **1**. Figure 4: Imprinting factors (IFs) for the better-performing **1**-MIPs (\(\geq\)2) from the set of formulations utilising **6**, **7** and **12** as FMs, EGDMA and TRIM as XLs and CH\({}_{\text{3}}\)CN and CHCl\({}_{\text{3}}\) as porogens. Rebinding studies were conducted using 1 mL of 0.8 mM solution of benzylpiperazine in the original porogen, with the uptake measured after 30 min. All binding tests were performed in triplicate. than for the 1:2 (E7\({}_{2}\) and T7\({}_{2}\)) formulations, suggesting that the affinity for **1** was stronger at a higher functional monomer content.