Highly efficient synthesis and assay of protein imprinted nanogels using magnetic templates

We here report an approach integrating synthesis and a highly sensitive as-say of protein imprinted nanogels ("plastic antibodies") employing templates attached to magnetic carriers. The proteolytic enzymes trypsin and pepsin were immobilized on aminofunctionalized solgel coated magnetic nanoparticles (magNPs). Lightly crosslinked fluorescently doped polyacrylamide nanogels were subsequently produced by high dilution polymerization of monomers in presence of 50 mg of the magNPs. This was followed by multiple washes of the magnetically trapped particles and collection of each fraction. Finally, a wash of the particles at elevated temperature led to elution of ca 10 mg of fluorescent nanogel taken on to further characterisation. This corresponded to a gravimetric yield of nanogel of ca 12% based on added monomer. The nanogels were characterised by a novel competitive fluorescence assay employing the identical protein conjugated nanoparticles as ligands to reversibly immobilize the corresponding nanogels. Both nanogels exhibited Kd < 10 pM for their respective target protein and low crossreactivity for five reference proteins. This agrees with affinities reported for solid phase synthesized nanogels prepared using low surface area glass bead supports. The integrated approach simplifies in a major way the development and produc-tion of plastic antibodies and offers direct access to a practical bioassay