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.
Imprinting of an ion-pair in presence of mutually compatible anion and cation host monomers leads to polymers showing enhanced ion uptake in competitive high ionic strength buffers.
Aberrant glycosylation has been proven to correlate with various
diseases including cancer. An important alteration in cancer progression
is an increased level of sialylation, making sialic acid one of the
key constituents in tumor-specific glycans and an interesting biomarker
for a diversity of cancer types. Developing molecularly imprinted
polymers (MIPs) with high affinity toward sialic acids is an important
task that can help in early cancer diagnosis. In this work, the glycospecific
MIPs are produced using cooperative covalent/noncovalent imprinting.
We report here on the fundamental investigation of this termolecular
imprinting approach. This comprises studies of the relative contribution
of orthogonally interacting functional monomers and their synergetic
behavior and the choice of different counterions on the molecular
recognition properties for the sialylated targets. Combining three
functional monomers targeting different functionalities on the template
led to enhanced imprinting factors (IFs) and selectivities. This apparent
cooperative effect was supported by
1
H NMR and fluorescence
titrations of monomers with templates or template analogs. Moreover,
highlighting the role of the template counterion use of tetrabutylammonium
(TBA) salt of sialic acid resulted in better imprinting than that
of sodium salts supported by both in solution interaction studies
and in MIP rebinding experiments. The glycospecific MIPs display high
affinity for sialylated targets, with an overall low binding of other
nontarget saccharides.
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 Kd<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.
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