Rapid, High Affinity Binding by a Fluorescein Templated Copolymer Combining Covalent, Hydrophobic, and Acid⁻Base Noncovalent Crosslinks

Abstract

A new type of biomimetic templated copolymer has been prepared by reverse addition fragmentation chain transfer polymerization (RAFT) in dioxane. The initial formulation includes the template fluorescein, N-isopropylacrylamide (NIPAM, 84 mol %), methacrylic acid (MAA, 5-mol %), 4-vinylpyridine (4-VP, 9 mmol %), and N,N′-methylenebis(acrylamide) (MBA, 2 mol %). PolyNIPAM is a thermosensitive polymer that comes out of aqueous solution above its lower critical solution temperature forming hydrophobic ‘crosslinks’. MAA and 4-VP interact in dioxane forming acid⁻base crosslinks. The excess 4-VP serves as a recognition monomer organizing around the template fluorescein to form a binding site that is held in place by the noncovalent and covalent crosslinks. The MBA is a covalent crosslinker. The RAFT agent in the resulting copolylmer was reduced to a thiol and attached to gold nanoparticles. The gold nanoparticle bound copolymer binds fluorescein completely in less than two seconds with an affinity constant greater than 10⁸ M^−1. A reference copolymer prepared with the same monomers by the same procedure binds fluorescein much more weakly.

Keywords: binding affinity; binding kinetics; fluorescein; moleculary imprinted polymers; noncovalent crosslinks; poly(N-isopropylacrylamide); templates.

Figure 1

DLS of the TMP and REF at 1 g/L in DI Water (filtered twice with a 0.45 PET syringe filter) changing polymer size) versus temperature. x-x-x is for TMP, •-•-• is REF.

Figure 2

Transmission electron microscopy images of AuNP stabilized with TEM polymer. The scale bare is equal to 20 nm.

Figure 3

TMP and REF (0.0355 g/L) stabilized on to multiple 20 nm AuNP samples then added to varying concentrations of fluorescein (10–1000 nM) aliquots. The various solutions were all heated to the corresponding LCST and then spun down, the supernatant was removed and analyzed for remaining unbound fluorescein.

Figure 4

Bound fluorescein to the TMP/REF stabilized AuNP versus initial fluorescein present before it was introduced and heated to the LCST of TMP or REF. Concentration of the bound fluorescein is calculated from the data from Figure 3.

Figure 5

TMP and REF (0.035 g/L) stabilized on to multiple 20 nm AuNP samples then added to varying concentrations of rhodamine-B (10–1000 nM) aliquots. The various solutions were all heated to the corresponding LCST then spun down and the supernatant was removed and analyzed for remaining unbound rhodamine-B.

Figure 6

Initial amount of 100 nM fluorescein in DI water was taken for a fluorescence baseline. Equal parts of fluorescein (100 nM) and TMP polymer solution (0.0355 g/L) were mixed together and normalized fluorescence intensity was measured over time (milliseconds). The same experiment was repeated with equal parts fluorescein (100 nM) and REF polymer solution (0.0355 g/L).