Highly Efficient Synthesis and Assay of Protein‐Imprinted Nanogels by Using Magnetic Templates

Abstract

We report an approach integrating the synthesis of protein‐imprinted nanogels (“plastic antibodies”) with a highly sensitive assay employing templates attached to magnetic carriers. The enzymes trypsin and pepsin were immobilized on amino‐functionalized solgel‐coated magnetic nanoparticles (magNPs). Lightly crosslinked fluorescently doped polyacrylamide nanogels were subsequently produced by high‐dilution polymerization of monomers in the presence of the magNPs. The nanogels were characterised by a novel competitive fluorescence assay employing identical protein‐conjugated nanoparticles as ligands to reversibly immobilize the corresponding nanogels. Both nanogels exhibited K d <10 pM for their respective target protein and low cross‐reactivity with five reference proteins. This agrees with affinities reported for solid‐phase‐synthesized nanogels prepared using low‐surface‐area glass‐bead supports. This approach simplifies the development and production of plastic antibodies and offers direct access to a practical bioassay.

Molecular Imprints Frozen by Strong Intermolecular Interactions in Place of Cross‐Linking

“…The structure of the grafted layer is a dense polymer brush [62, 63] with strong lateral stacking of PANI strands and packing defects where the molecular imprints are frozen. Such defects are similar to grain boundaries of polycrystalline materials, they allow for molecular imprints but would not be suitable for keeping the shape of large templates such a nanoparticles or proteins [64, 65] . A method that may be considered as being similar is building a self‐assembled monolayer (SAM) of fatty thiol molecules on a gold surface where the imprints are the “shadows” of the template molecules interacting with the gold surface, which leave holes inside the layer [66–68] .…”

Flexible Ferrofluids: Design and Applications

“…Benefitting from their dynamic, flexible, and superparamagnetic properties, as well as the resulting fast, reversible response to magnetic fields, ferrofluids are employed not only as tunable templates for fabrication of orderly lined microarrays, but also as carrier solutions for multiple kinds of nanoparticle self‐assembly . Besides, they could serve as indispensable components of smart materials such as field responsive soft robots, magnetically assembled photonic structures, and bioinspired arrays with the combination of other curable materials .…”

Strategies for Molecular Imprinting and the Evolution of MIP Nanoparticles as Plastic Antibodies—Synthesis and Applications

“…The new format of the test system was based on magnetically-induced decolorization, caused by removal of erythrocytes from the solution by magnetic nanoMIPs (Figure 8). A similar principle is used in the magnetic imprinted nanoparticle-based assay (MINA) for detection of methyl parathion [151], biotin [152], and proteins [153]. This assay was based on competition of fluorescent nanoMIPs for binding to free analyte and analyte immobilized on magnetic inserts (Figure 9).…”

“…Reproduced from [152] with permission. Recently, an integrating approach for the synthesis and direct assay for protein-imprinted nanoMIPs has been reported using magnetic nanoparticles [153]. The enzymes trypsin and pepsin were immobilized on solid support, i.e., functionalized magnetic nanoparticles (magNPs).…”

“…Fluorescence intensity of nanoMIPs displaced from the corresponding magNPs upon addition of incremental amounts of protein and incubation for 2 h. The excitation/emission filters used were 485 and 520 nm, adapted from data presented by Mahajan et al[153].…”