The Amount of Cross-Linker Influences Affinity and Selectivity of NanoMIPs Prepared by Solid-Phase Polymerization Synthesis

Abstract

The cross-linker methylene-bis-acrylamide is usually present in nanoMIPs obtained by solid-phase polymerization synthesis at 2 mol% concentration, with very few exceptions. Here, we studied the influence of variable amounts of methylene-bis-acrylamide in the range between 0 (no cross-linker) and 50 mol% concentration on the binding properties of rabbit IgG nanoMIPs. The binding parameters were determined by equilibrium binding experiments and the results show that the degree of cross-linking defines three distinct types of nanoMIPs: (i) those with a low degree of cross-linking, including nanoMIPs without cross-linker (0-05 mol%), showing a low binding affinity, high density of binding sites, and low selectivity; (ii) nanoMIPs with a medium degree of cross-linking (1-18 mol%), showing higher binding affinity, low density of binding sites, and high selectivity; (iii) nanoMIPs with a high degree of cross-linking (32-50 mol%), characterized by non-specific nanopolymer-ligand interactions, with low binding affinity, high density of binding sites, and no selectivity. In conclusion, the results are particularly relevant in the synthesis of high-affinity, high-selectivity nanoMIPs as they demonstrate that a significant gain in affinity and selectivity could be achieved with pre-polymerization mixtures containing quantities of cross-linker up to 10-20 mol%, well higher than those normally used in this technique.

Keywords: N,N′-methylene-bis-acrylamide; binding affinity; binding isotherm; binding selectivity; cross-linking; molecular imprinting; nanoMIP; rabbit IgG.

Figure 1

Binding isotherm curves for rabbit immunoglobulin G (rIgG). (a) NanoMIPs containing from 0% to 2% BIS (yellow: P0; red: P02; green: P05; blue: P1; violet: P2); (b) nanoMIPs containing from 4% to 50% BIS (yellow: P4; red: P8; green: P18; blue: P32; violet: P50). Error bars: ±1 s.d.

Figure 2

Binding isotherm curves for bovine immunoglobulin G (bIgG). (a) NanoMIPs containing from 0% to 2% BIS (yellow: P0; red: P02; green: P05; blue: P1; violet: P2); (b) nanoMIPs containing from 4% to 50% BIS (yellow: P4; red: P8; green: P18; blue: P32; violet: P50). Error bars: ±1 s.d.

Figure 3

Apparent equilibrium binding constants (K_eq) measured for rIgG (red circles) and bIgG (blue circles). Error bars: ±1 s.d.

Figure 4

Binding site density (B_max) measured for rIgG (red circles) and bIgG (blue circles). Error bars: ±1 s.d.

Figure 5

Green circles: binding selectivity (α) measured as the ratio between binding capacities (β) of nanoMIPs for bIgG and rIgG. Error bars: ±1 s.d. The pale red area indicates no (α ≥ 1) or marginal (0.8 ≤ α < 1) binding selectivity.