Guoqi Fu

Enhanced lysozyme imprinting over nanoparticles functionalized with carboxyl groups for noncovalent template sorption.

Surface molecular imprinting, in particular over nanosized support materials, is very suitable for a template of bulky structure like protein. Inspired by the surface template immobilization method reported previously, we herein demonstrate an alternative strategy for enhancing specific recognition of core-shell protein-imprinted nanoparticles through prefunctionalizing the cores with noncovalent template sorption groups. For proof of this concept, silica nanoparticles chosen as the core materials were modified consecutively with 3-aminopropyltrimethoxysilane and maleic anhydride to introduce polymerizable double bonds and terminal carboxyl groups, hence capable of physically adsorbing the print protein. With lysozyme as a template, thin protein-imprinted shells were fabricated according to our newly developed approach for surface protein imprinting over nanoparticles. The rebinding experiments confirmed that the introduction of the carboxyl groups could remarkably improve the imprinting effect in relation to a significantly increased imprinting factor and specific rebinding capacity. Moreover, in contrast to the harsh template removal conditions required for the covalent template coupling approach, the template removal during the imprinted particle synthesis as well as desorption after rebinding could be mildly achieved via washing with salt solution.

Imprinting of protein over silica nanoparticles via surface graft copolymerization using low monomer concentration.

Surface imprinting and adopting a nano-sized physical form are two effective approaches to overcome the template transfer difficulty within molecularly imprinted polymers and in particular advantageous to the imprinting of macromolecular structures like proteins. The surface protein-imprinted nanoparticles based on these two strategies are attractive for biosensor development. We here demonstrate a facile way for imprinting protein over nanoparticle supports. It was achieved simply via radical induced graft copolymerization of low concentration monomers on the surface of vinyl modified silica nanoparticles dispersed in aqueous media with lysozyme as a model protein. With a total monomer concentration of 0.4 wt%, less than tenth that employed conventionally, the possible gelation of the dispersion after polymerization was avoided and hence the unagglomerated imprinted particles could be readily collected. It was proved that thin polymer shells with imprinted sites had been formed over the support particles. In batch rebinding tests, the imprinted particles reached saturated adsorption within 5 min and exhibited significant specific recognition toward the template protein. The presented approach may be a versatile way for the fabrication of surface protein-imprinted nanoparticles via rational design of the surface chemistry of the support particles and choice of functional monomers according to the properties of the print protein.

Synthesis of surface molecularly imprinted nanoparticles for recognition of lysozyme using a metal coordination monomer.

Molecularly imprinted polymers against proteins are regarded as promising substitutes for natural antibodies, but have been frustrated with the problems including reduced interaction between functional monomers and protein template in the aqueous media required during their synthesis and restricted mass transfer across the resulting crosslinked polymer matrixes. For addressing these issues, herein we proposed a strategy for imprinting of a protein on the surface of nanoparticles using a metal chelating monomer. With lysozyme as a model protein template and Cu(2+) chelating N-(4-vinyl)-benzyl iminodiacetic acid as the coordination monomer along with other monomers, protein imprinted polymer nanoshells were formed over vinyl-modified silica nanoparticles via surface polymerization in high-dilution monomer solution. The feed concentration of the crosslinking monomer was optimized toward achieving the best imprinting effect. Compared with the related imprinted materials reported previously, the resultant core-shell imprinted particles showed greatly faster binding kinetics, elevated rebinding capacity and selectivity. More importantly, noticeably high binding affinity was achieved with an estimated dissociation constant of 4.1 × 10(-8)M which is comparable to that of conventional antibodies.

Imprinting effect of protein-imprinted polymers composed of chitosan and polyacrylamide: a re-examination.

In this paper the imprinting effects recently reported with protein-imprinted polymers based on chitosan and polyacrylamide are re-assessed. Molecularly imprinted polymers (MIPs) from which the embedded template molecules were removed by washing with a solution of sodium dodecyl sulfate and acetic acid were prepared. In batch template rebinding experiments, the MIPs displayed quite high template binding capacity as reported previously. However, the non-imprinted polymers (NIPs), after washing with the same solution, also showed large binding capacity nearly equal to that of the MIPs. X-ray diffraction and scanning electronic microscope investigations confirmed remarkable property changes of the NIPs after the washing process. These findings indicate that the non-specific adsorption resulting from the template removing process rather than the imprinted sites generated on the MIPs themselves may account for the high template rebinding capacity of the reported protein-imprinted polymers.

Silica nanoparticle supported molecularly imprinted polymer layers with varied degrees of crosslinking for lysozyme recognition

Surface imprinting over nanosized support materials is particularly suitable for protein templates, considering the problems with mass transfer limitation and low binding capacity. Previously we have demonstrated a strategy for surface protein imprinting over vinyl-modified silica nanopartiles with lysozyme as a model template by polymerization in high-dilution monomer solution to prevent macrogelation. Herein, the synthesis process was further studied toward enhancement of the imprinting performance by examining the effect of several synthesis conditions. Interestingly, the feed crosslinking degree was found to have a great impact on the thickness of the formed imprinting polymer layers and the recognition properties of the resulting imprinted materials. The imprinted particles with a crosslinking degree up to 50% showed the best imprinting effect. The imprinting factor achieved 2.89 and the specific binding reached 23.3 mg g(-1), which are greatly increased compared to those of the lowly crosslinked imprinted materials reported previously. Moreover, the relatively high crosslinking degree led to no significant retarding of the binding kinetics to the imprinted particles, and the saturated adsorption was reached within 10 min. Therefore, this may be a promising method for protein imprinting.

Fabrication of Surface Protein-Imprinted Nanoparticles Using a Metal Chelating Monomer via Aqueous Precipitation Polymerization

Molecular imprinting is a promising way for constructing artificial protein recognition materials, but it has been challenged by difficulties such as restricted biomacromolecule transfer in the cross-linked polymer networks, and reduced template-monomer interactions that are due to the required aqueous media. Herein, we propose a strategy for imprinting of histidine (His)-exposed proteins by combining previous approaches such as surface imprinting over nanostructures, utilization of metal coordination interactions, and adoption of aqueous precipitation polymerization capable of forming reversible physical crosslinks. With lysozyme as a model template bearing His residues, imprinted polymer nanoshells were grafted over vinyl-modified nanoparticles by aqueous precipitation copolymerization of a Cu(2+) chelating monomer with a temperature-responsive monomer carried out at 37 °C, above the volume phase-transition temperature (VPTT) of the final copolymer. The imprinted nanoshells showed significant temperature sensitivity and the template removal could be facilitated by swelling of the imprinted layers at 4 °C, below the VPTT. The resultant core-shell imprinted nanoparticles exhibited strikingly high rebinding selectivity against a variety of nontemplate proteins. An imprinting factor up to 22.7 was achieved, which is among the best values reported for protein imprinting, and a rather high specific binding capacity of 67.3 mg/g was obtained. Moreover, this approach was successfully extended to preliminary imprinting of hemoglobin, another protein with accessible His. Therefore, it may be a versatile method for fabrication of high-performance surface-imprinted nanoparticles toward His-exposed proteins.

Enhanced surface imprinting of lysozyme over a new kind of magnetic chitosan submicrospheres

Surface protein imprinting over nano- or micron-sized substrates is an effective approach for improving the biomacromolecule mass transfer and rebinding capacity. For achieving high recognition performance, it is necessary to introduce certain functional groups onto the surface of the support materials which can interact with the template protein. Herein, we report a surface protein imprinting approach using a new kind of core-shell magnetic chitosan submicrospheres as the supports. The surface of these magnetic chitosan particles is tethered with uncross-linked chitosan chains, hence bearing plenty of amino and hydroxyl groups, where a large amount of functional ligands can be readily coupled for capturing of the protein template. With lysozyme as a model print protein, the magnetic supports were functionalized with maleic acid and then coated with imprinted polymer layers. The resulting imprinted microspheres show significantly selective rebinding for lysozyme. In particular, they exhibit a specific rebinding capacity about three times higher than achieved with our previous lysozyme-imprinted particles synthesized in similar way but with maleic acid modified silica nanoparticles as the supports. This can be attributed to the much higher template binding capacity to the modified magnetic chitosan submicrospheres. Also, the resultant imprinted particles can be easily collected by a magnet. Therefore, such kind of chitosan submicrospheres may be a versatile carrier for constructing high-capacity and magnetically recyclable surface protein-imprinted particles.

Facile synthesis of hairy core–shell structured magnetic polymer submicrospheres and their adsorption of bovine serum albumin

Highly magnetic polymer submicrospheres with a hairy core-shell structure were facilely synthesized by combining distillation-precipitation polymerization (DPP) with subsequent surface-initiated atom transfer radical polymerization (SI-ATRP), and then investigated for protein adsorption. A robust polymer shell consisting of poly(divinylbenzene-co-chloromethylstyrene) (P(DVB-co-CMS)) was coated on superparamagnetic submicrometer-sized magnetite colloid nanocrystal clusters (MCNCs) via DPP. With the benzyl chloride groups on the shell as initiator, poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) hairs were grafted by SI-ATRP approach. The resulting hairy core-shell structured Fe3O4@ P(DVB-co-CMS)-PDMAEMA microspheres showed pH- and temperature-sensitivity, and high-magnetization. The composite microspheres were further investigated for adsorption of a typical acidic protein, i.e. bovine serum albumin (BSA). They exhibited a high binding capacity up to over 660 mg/g (corresponding to 158 DMAEMA monomer units cooperating for binding one BSA molecule) and could rapidly reach binding equilibrium within 5 min. Moreover, the adsorption of BSA was found to be remarkably dependent on the pH and salt concentration of the protein solutions, and the bound protein could be quantitatively desorbed by washing with a medium with lowered pH or raised salt concentration.