## 3 Results and Discussion
### Binding Specificity  
Selectivity tests were carried out using structurally analog proteins such as OVA, Hb, and Lys as control compounds. OVA and Hb were selected because they have isoelectric points (pIs) and molecular weight similar to those of BSA. Compared with BSA, Lys has a lower molecular weight; however, it was selected because it has the opposite charge at pH 7.0. Table 1 lists the binding capacities of PVC@TAA-MIP and PVC@TAA-NIP for these proteins at a feed concentration of 3 \(\mu\)M, in the absence of NaCl. As shown in Table S3, PVC@TAA-MIP exhibited significant binding selectivity toward BSA, with an imprinting factor of 3.77 and specific binding of 1.068 \(\mu\)mol\(\cdot\)g\({}^{-1}\), which were much higher than those toward the control proteins, indicating the specific recognition of BSA.  
When PBS (pH 7.0, 10 mM) containing NaCl was employed, the PVC@TAA-MIP exhibited completely different binding capacities and imprinting factors (Table S4, ESI \(\dagger\)), indicating that the presence of NaCl had a significant effect on the binding mechanism of PVC@TAA-MIP toward all the analytes. PVC@TAA-MIP exhibited a lower binding capacity toward OVA than that in the absence of NaCl, mainly because of the suppression of anion exchange-based high-affinity interactions in the presence of NaCl. The binding capacity of Hb, which was almost neutral at pH 7.0, showed relatively small variation despite its similar molecular weight to BSA, which could have been caused by the weak anion exchange-based affinity interaction between the Hb and imprinted polymer. Compared to OVA and Hb, Lys had a higher binding capacity, mainly because of the weak electrostatic repulsion between the positive charges of Lys and the imprinted materials in the presence of NaCl. These results indicated that PVC@TAA-MIP exhibited excellent specificity even when the anion exchange-based high-affinity interactions were suppressed, thereby expanding the application scope of this imprinted material.  
Competitive binding tests were performed using a binary protein mixture of BSA and Hb. As shown in Figure 9, PVC@TAA-MIP exhibited acceptable specific selectivity toward the template, despite the fact that a small portion of the template was not adsorbed and a portion of Hb was captured by the sorbent material. In addition, the competition parameter, which was defined as the \(\beta_{\text{tem}}/\beta_{\text{ana}}\) ratio, was calculated as an index of the specific selectivity. In the absence of NaCl, the competition parameter was calculated to be 3.09, which was significantly higher than that in the presence of NaCl (2.03). Therefore, the high competition parameter resulted from the combination of anion exchange affinity-based interactions and amino group affinity-based hydrogen bondings. Furthermore, PVC@TAA-MIP provided an outstanding ability to resist interference, which is considered beneficial for BSA separation and enrichment in multi-component complicated systems.  
\begin{table}
\begin{tabular}{c c c c c} \hline \multirow{2}{*}{**Compound**} & \multicolumn{3}{c}{**Q (umol\(\cdot\)g\({}^{-1}\))**} & \multirow{2}{*}{\(\mathbf{\alpha}\)} & \multirow{2}{*}{\(\mathbf{\beta}\)} \\ \cline{2-2} \cline{4-5}  & **PVC@TAA-MIP** & & & \\ \hline BSA & 1.068 \(\pm\) 0.0235 & 0.2830 \(\pm\) 0.0184 & 3.77 & — \\ OVA & 0.3636 \(\pm\) 0.0166 & 0.2010 \(\pm\) 0.0143 & 1.81 & 2.08 \\ Hb & 0.2039 \(\pm\) 0.0189 & 0.2295 \(\pm\) 0.0125 & 0.89 & 4.24 \\ Lys & 0.0776 \(\pm\) 0.0138 & 0.0919 \(\pm\) 0.0116 & 0.84 & 4.49 \\ \hline \end{tabular}
\end{table}
Table 1: Imprinting efficiencies and selectivity coefficients of PVC@TAA-MIP and PVC@TAA-NIP toward BSA in absence of NaCl.