## 3 Results and Discussion
### Binding Isotherms  
Binding tests were implemented using PBS (pH 7.0, 10 mM) with or without 40 mM NaCl for comparison. Batch equilibrium binding tests were performed at various initial concentrations of BSA ranging from 0.3 to 6.0 \(\upmu\)M. As shown in Figure 7, within this concentration range, the PVC@TAA-MIP exhibited much higher binding capacities than the PVC@TAA-NIP control. This suggested that molecular binding sites were produced inside the imprinted film by the involvement of the template in the polymerization process. When PBS (pH 7.0, 10 mM) was employed, the saturation binding capacities of PVC@TAA-MIP and PVC@TAA-NIP were measured to be 1.054 and 0.2806 \(\upmu\)mol\(\cdot\)g\({}^{-1}\), respectively, achieving an imprinting factor of 3.76, which was higher than the previously reported value (2.66) using randomly oriented charged functional monomers [29]. These results confirmed the remarkable enhancement of the imprinting efficiency when using the anion exchange affinity-based immobilization imprinting strategy. When 10 mM PBS (pH 7.0) containing 40 mM NaCl was used, the saturation binding capacities of PVC@TAA-MIP and PVC@TAA-NIP were 0.7368 and 0.2518 \(\upmu\)mol\(\cdot\)g\({}^{-1}\), respectively, achieving an imprinting factor of 2.93. This result revealed that, even with a NaCl solution with a relatively high concentration, at which anion exchange affinity-based interactions were inactivated, the imprinted materials still exhibited a high imprinting factor. This outcome confirmed the presence of a low-affinity binding mode, mainly attributed to the hydrogen bonding interaction between the amide groups of the binding sites and the amino groups of BSA in PBS (pH 7.0, 10 mM). Figure 6: Influences of NaCl concentration on binding capacity and imprinting factor of PVC@TAA\(-\)MIP and PVC@TAA\(-\)NIP toward BSA. Figure 7: Binding isotherms of PVC@TAA\(-\)MIP and PVC@TAA\(-\)NIP toward BSA in absence or presence of NaCl. The Scatchard model was employed to study the binding properties of PVC@TAA-MIP [45]:  
\[\frac{Q}{C}=\frac{Q_{\text{max}}}{K_{d}}-\frac{Q}{K_{d}},\]  
where \(Q\) and \(Q_{\text{max}}\) (\(\mu\text{mol}\cdot\text{g}^{-1}\)) are the experimental binding capacity at the equilibrium concentration and the theoretical maximum adsorption, respectively; \(C\) (\(\mu\text{mol}\cdot\text{L}^{-1}\)) is the equilibrium concentration of BSA in the bulk solution; and \(K_{d}\) (\(\mu\text{mol}\cdot\text{L}^{-1}\)) is the dissociation constant. When PBS (pH 7.0, 10 mM) was employed, the Scatchard plots of the PVC@TAA-MIP deviated from a single straight line. In contrast, they consisted of two individual straight lines with different slopes. This suggests that their affinities could be approximated by two binding association constants (\(K_{\text{a}}\)) corresponding to the high- and low-affinity sites [46]. The values of \(K_{d}\) and \(Q_{\text{max}}\) were calculated to be 0.04590 \(\mu\text{mol}\cdot\text{L}^{-1}\) and 0.9265 \(\mu\text{mol}\cdot\text{g}^{-1}\) for high-affinity, and 0.1839 \(\mu\text{mol}\cdot\text{L}^{-1}\) and 1.079 \(\mu\text{mol}\cdot\text{g}^{-1}\) for low-affinity binding sites, respectively (Figure S3, ESI \(\dagger\)). When PBS (pH 7.0, 10 mM) containing 40 mM NaCl was utilized, the Scatchard plot of the PVC@TAA-MIP was linear. Fitting the binding data using a 1:1 Langmuir model yielded an acceptable correlation coefficient (R\({}^{2}\) = 0.98, Figure S3, ESI \(\dagger\)). The value of \(K_{d}\) was calculated to be 0.2607 \(\mu\text{mol}\cdot\text{L}^{-1}\), which was very close to the binding dissociation constant of the low-affinity binding sites of PVC@TAA-MIP in the absence of NaCl. This provided further support to the hypothesis by indicating that the two affinity binding models could originate from similar interaction patterns. Thus, high-affinity binding sites might be derived from anion exchange-based affinity interactions in the absence of NaCl. Furthermore, the binding strength could be tuned to a certain extent by changing the NaCl concentration.