Tongchang Zhou

Molecular imprinting of protein in Pickering emulsion

A new strategy of molecular imprinting to prepare spherical hydrogels via water-in-oil Pickering emulsion polymerization was developed. The imprinted hydrogels exhibited fast adsorption kinetics and significant selectivity for the target protein.

Molecularly imprinted polymer beads for nicotine recognition prepared by RAFT precipitation polymerization: a step forward towards multi- functionalities†

A nicotine imprinted polymer was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization using methacrylic acid (MAA) as a functional monomer. The resulting molecularly imprinted polymers were monodispersed beads with an average diameter of 1.55 μm. The molecular selectivity of the imprinted polymer beads was evaluated by studying the uptake of nicotine and its structural analogs by the polymer beads. Equilibrium binding results indicate that the amount of nicotine bound to the imprinted polymer beads is significantly higher than that bound to the non-imprinted polymer in both acetonitrile and in a mixture of acetonitrile and water. The RAFT reagent present on the surface of the polymer beads allowed straightforward grafting of hydrophilic polymer brushes on the particle surface. In addition to the demonstrated molecular selectivity and the straightforward surface modification of the imprinted polymer beads, we also show that the dithioester end groups on the surface of the polymer beads can be converted into new thiol groups without sacrificing the specific molecular recognition. Through the new terminal thiol groups, a fluorescent dye was conveniently conjugated to the imprinted polymer beads via Michael addition reaction. The living characteristic of RAFT and the versatile thiol groups that can be derived from the RAFT reagent provide many new possibilities for realizing multi-functionalities for molecularly imprinted polymers.

Preparation of protein imprinted polymer beads by Pickering emulsion polymerization

We present a new method for preparation of protein-specific polymer beads based on surface molecular imprinting in Pickering emulsion. In the first step, adult human hemoglobin (Hb) was adsorbed on silica nanoparticles. The protein-coated silica particles were then used to stabilize an oil-in-water emulsion (Pickering emulsion) composed of cross-linking monomer in the oil phase. After free radical polymerization of the oil phase, the protein-silica particles were removed to leave Hb-imprinted sites on the polymer surface. The protein-imprinted polymer microspheres were characterized by scanning electron microscopy and their selectivity was investigated by protein binding analysis. The new synthetic method based on Pickering emulsion polymerization produced easily accessible Hb binding sites on the surface of spherical polymer particles, which are useful for protein separation, purification and analysis.

Implementation of Molecularly Imprinted Polymer Beads for Surface Enhanced Raman Detection

Molecularly imprinted polymers (MIPs) have a predesigned molecular recognition capability that can be used to build robust chemical sensors. MIP-based chemical sensors allow label-free detection and are particularly interesting due to their simple operation. In this work we report the use of thiol-terminated MIP microspheres to construct surfaces for detection of a model organic analyte, nicotine, by surface enhanced Raman scattering (SERS). The nicotine-imprinted microspheres are synthesized by RAFT precipitation polymerization and converted into thiol-terminated microspheres through aminolysis. The thiol groups on the MIP surface allow the microspheres to be immobilized on a gold-coated substrate. Three different strategies are investigated to achieve surface enhanced Raman scattering in the vicinity of the imprinted sites: (1) direct sputtering of gold nanoparticles, (2) immobilization of gold colloids through the MIPs thiol groups, and (3) trapping of the MIP microspheres in a patterned SERS substrate. For the first time we show that large MIP microspheres can be turned into selective SERS surfaces through the three different approaches of assembly. The MIP-based sensing surfaces are used to detect nicotine to demonstrate the proof of concept. As synthesis and surface functionalization of MIP microspheres and nanoparticles are well established, the methods reported in this work are handy and efficient for constructing label-free chemical sensors, in particular for those based on SERS detection.

Nanohybrid polymer brushes on silica for bioseparation

Boronic acid based affinity materials are of great importance for effective enrichment of biomolecules containing a cis-diol structure, for example glycoproteins. In this work, we developed a new pH- and temperature-responsive boronate affinity material for effective separation of glycoproteins. A nanohybrid material composed of silica cores and flexible polymer brushes, denoted as Si@poly(NIPAm-co-GMA)@APBA, was prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) in combination with Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. The size, morphology and composition of the obtained nanohybrid were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), elemental analysis and thermogravimetric analysis (TGA). The density of polymer brushes on the surface of silica nanoparticles was determined to be 0.7 molecules per nm2. The maximum binding capacities of the nanohybrid Si@poly(NIPAm-co-GMA)@APBA for ovalbumin (OVA) and horseradish peroxidase (HRP) were determined to be 87.6 mg g-1 and 22.8 mg g-1, respectively. Glycoprotein binding on the nanohybrid could be controlled by varying the pH of the binding buffer. By increasing the temperature from 20 °C to 35 °C, glycoprotein binding onto the nanohybrid was decreased. This new pH- and temperature-responsive nanohybrid will be useful for a number of biotechnological and biomedical applications, for example, for protein separation and drug delivery.

Synthesis of naproxen-imprinted polymer using Pickering emulsion polymerization

For the last decades, molecular imprinting is developing intensively, especially in the case of the application of new imprinting techniques. In this work, for the first time, a Pickering emulsion polymerization was used to synthesize the S‐naproxen–imprinted polymer spheres following a noncovalent protocol. To enhance the knowledge about imprinting process using mentioned technique, thorough analysis of the synthesis process was performed. Optimization of polymerization conditions included the selection of functional monomer, cross‐linking agent, type of porogen, surfactant, and the choice of appropriate amount of the template and porogen. Prepared materials were characterized using scanning electron microscopy and nitrogen adsorption. To study the binding properties, the sorption studies, including adsorption isotherms and competitive binding, were performed. Investigation of the effect of the functional monomer on the selective recognition of S‐naproxen showed that the interactions between the template molecule and 4‐vinylpyridine resulted in the best recognizing ability. Moreover, the synthesis with application of ethylene glycol dimethacrylae as a cross‐linker, toluene as a porogen, and Tween 20 as an additional emulsion stabilizer gave the most desired result. The optimal ratio of the porogen to monomers mixture was 0.1, due to the fact that the increase of the porogen volume resulted in the significant increase of nonspecific uptake. In addition, the tenfold molar excess of functional monomer relative to the template turned out to be optimal. Subsequent binding studies demonstrated that the material synthesized using optimized polymerization conditions consists of imprinted sites that are sensitive for the S‐naproxen.

Synthesis and characterization of epitope-imprinted polymers for purification of human hemoglobin

One promising method to prepare protein-selective polymers is the epitope-imprinting approach, where surface-accessible peptides from a target protein are used as templates to create surface-exposed binding sites on molecularly imprinted polymers (MIPs). However, selection of a suitable peptide target is not always straightforward, and synthesis of peptide on a large scale can be costly. In this work, we developed a new approach that can be used to select peptide epitopes on protein surface to be used as templates to prepare protein-selective MIPs. In this case study, human hemoglobin (Hb) was immobilized on silica nanoparticles and then fragmented by tryptic digestion. The particle-supported peptides were then used as templates to synthesize the Hb-selective MIPs, which were obtained after removal of the silica support and the peptides. The MIPs were tested in equilibrium binding experiments to evaluate their protein separation performance. The new surface imprinted MIPs displayed high selectivity for Hb, and was able to separate different variants of Hb from protein mixtures and crude cell extracts.

Preparation of diclofenac-imprinted polymer beads for selective molecular separation in water.

Molecular imprinting technique is an attractive strategy to prepare materials for target recognition and rapid separation. In this work, a new type of diclofenac (DFC)–imprinted polymer beads was synthesized by Pickering emulsion polymerization using 2‐(dimethylamino)ethyl methacrylate as the functional monomer. The selectivity and capacity of the molecularly imprinted polymers (MIPs) were investigated in aqueous solution. Equilibrium binding results show that the MIPs have a high selectivity to bind DFC in a wide range of pH values. Moreover, in liquid chromatography experiment, the imprinted polymer beads were packed into column to investigate the binding selectivity under nonequilibrium conditions. The retention time of DFC on the MIP column is significantly longer than its structural analogues. Also, retention of DFC on the MIP column was significantly longer than on the nonimprinted polymer column under aqueous condition. As the new MIP beads can be used to achieve direct separation of DFC from water, the synthetic method and the affinity beads developed in this work opened new possibilities for removing toxic chemicals from environmental and drinking water.

Characterization of Protein-Protein Interactions in Recombinant Hemoglobin Producing Escherichia coli Cells Using Molecularly Imprinted Polymers

The worldwide blood shortage has generated demands for alternatives to transfusible human blood. One such important option is based on recombinant hemoglobin-based oxygen carriers (rHBOCs). Most efforts have been focused on various E. coli based production systems. One of the key challenges in these systems is to devise an efficient and economical protein production strategy involving selection of suitable host cell and Hb variant, growth conditions and media engineering. Hb also influences the heterologous host cell metabolism and therefore the identification of modified protein-protein interactions is critical for optimizing Hb production. In this study, molecularly imprinted polymers (MIPs) directed against Hb were used to identify the human Hb protein interaction network in E. coli. One E. coli host protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), interacted strongly with Hb, especially fetal Hb (HbF).

Molecular imprinting in particle-stabilizedemulsions: enlarging template size from smallmolecules to proteins and cells

Abstract Molecular imprinting of small organic compounds is now a standard procedure for preparation of tailor-designed affinity materials. Molecularly imprinted polymers (MIPs) have outstanding stability and can be prepared in a large quantity, therefore are useful replacements for biological receptors for a number of applications including product purification, analytical separation, chemical sensing and controlled delivery and biomineralization. Although preparation of MIPs, in particular using the non-covalent imprinting strategy, has become a routine practice in many research laboratories, new synthetic methods continued to be invented, which contribute to new MIPs with unprecedented functional performances. As the size of the template increases from small organic compounds to biomacromolecules to large virus particles and cells, the traditional methods of imprinting often fail to give useful MIP products. Another important aspect is the shift from organic solvents to water for MIPs designed for treatment or analysis of biological samples. The demand on water-compatibility and recognition of larger entities for MIPs call for new and efficient synthetic methods. This mini review will summarize the recent progress of molecular imprinting using particle-stabilized emulsion as a general synthetic platform to furnish the new MIPs with the desired functions.