Marina Resmini

Protein‐Size Molecularly Imprinted Polymer Nanogels as Synthetic Antibodies, by Localized Polymerization with Multi‐initiators

A new approach is proposed for the synthesis of molecularly imprinted polymers (MIPs) (synthetic antibodies) as soluble nanogels with sizes close to the size of real antibodies. To imprint a molecular memory in particles consisting of only a few polymer chains, an initiator carrying multiple iniferter moieties is used. This allows for the simultaneous initiation of several polymer chains, and yields molecularly imprinted nanogels (17 nm, molecular weight (MW) = 97 kDa) with good affinity and selectivity for the target.

Imprinted nanomaterials: a new class of synthetic receptors

The molecular imprinting approach provides a unique opportunity for the creation of three-dimensional cavities with tailored recognition properties. Over the last decade this field has expanded considerably, across a variety of disciplines, leading to novel approaches and many potential applications. Progress in the field of materials science has led to significant breakthroughs and the application of the imprinting approach to novel polymeric formats offers new insights and attractive methods for the preparation of synthetic receptors. In particular, nanomaterials have received considerable attention in the developing field of nanotechnology. With a large number of recent developments in the field of molecular imprinting available, this article is focused on a selection of new systems, in particular the different formats of nanomaterials, such as nanogels, nanofibres, nanowires and nanotubes.

Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine.

Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P‐loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P‐loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild‐type enzyme. The structure of the K15M mutant protein has been determined at 1.8 Å, and shows that the organization of the P‐loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P‐loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.

A Versatile Fiber‐Optic Fluorescence Sensor Based on Molecularly Imprinted Microstructures Polymerized in Situ

A method using laser-induced photopolymerization was developed to generate highly selective fiber optic sensors in a few seconds that are based on molecularly imprinted polymer (MIP) microtips. The fluorescence detection signal was enhanced using gold nanoparticles. The sensor also detects nonfluorescent analytes when a fluorescent signaling monomer is incorporated into the MIP.

Critical evaluation of the applicability of capillary zone electrophoresis for the study of hapten-antibody complex formation

The applicability of capillary zone electrophoresis (CZE) for the determination of association constants of an hapten-antibody complex with values as high as 10(7) (mol/I)-1 was investigated (dissociation constant in the nmol range). As a reference method the well known, enzyme-linked immunosorbent assay (ELISA) was selected. The study describes the optimisation of the experimental conditions of the CZE technique. The CZE measurements were optimised according to an experimental design. The results of the CZE and ELISA methods are compared giving consideration to the reproducibility (repeatability) of the two methods.

Pentablock star shaped polymers in less than 90 minutes via aqueous SET-LRP

The synthesis of multi-block star-shaped copolymers via SET-LRP in aqueous solution has been reported for the first time. This aqueous polymerization technique allows rapid and direct access to acrylamide based star-shaped polymers. It is possible to synthesize an A-B-A-B-C penta-block copolymer in <90 minutes in total. To achieve this, we have investigated a water-soluble 3-arm initiator based on a glycerol structure for the first time. Using N-isopropylacrylamide 3-arm star-shaped polymers were prepared with DP = 60 to 240 with full conversions in <30 minutes with polydispersities <1.11. The scope of the reaction was demonstrated by synthesizing diblock copolymers using a combination of NIPAM, DMA and HEAm in different ratios. In addition a sequence controlled 3-arm pentablock copolymer has been obtained with excellent control over molecular weight distribution (PDI < 1.14) as evidenced by GPC, 1H NMR, and MALDI-TOF MS.

Molecularly Imprinted Polymer Coated Quantum Dots for Multiplexed Cell Targeting and Imaging.

Advanced tools for cell imaging are of great interest for the detection, localization, and quantification of molecular biomarkers of cancer or infection. We describe a novel photopolymerization method to coat quantum dots (QDs) with polymer shells, in particular, molecularly imprinted polymers (MIPs), by using the visible light emitted from QDs excited by UV light. Fluorescent core-shell particles specifically recognizing glucuronic acid (GlcA) or N-acetylneuraminic acid (NANA) were prepared. Simultaneous multiplexed labeling of human keratinocytes with green QDs conjugated with MIP-GlcA and red QDs conjugated with MIP-NANA was demonstrated by fluorescence imaging. The specificity of binding was verified with a non-imprinted control polymer and by enzymatic cleavage of the terminal GlcA and NANA moieties. The coating strategy is potentially a generic method for the functionalization of QDs to address a much wider range of biocompatibility and biorecognition issues.

Modulation of imprinting efficiency in nanogels with catalytic activity in the Kemp elimination

The interactions between the template and the functional monomer are a key to the formation of cavities in the imprinted nanogels with high molecular recognition properties. Nanogels with enzyme‐like activity for the Kemp elimination have been synthesized using 4‐vinylpyridine as the functional monomer and indole as the template. The weak hydrogen bond interaction in the complex is shown to be able to induce very distinctive features in the cavities of the imprinted nanogels. The percentage of initiator used in the polymerisation, ranging from 1% to 3%, although it does not have a substantial effect on the catalytic rate, reduces considerably the imprinting efficiency. The alteration of the template/monomer ratio is also investigated, and the data show that there is considerable loss of imprinting efficiency. In terms of substrate selectivity, a number of experiments have been performed using 5‐Cl‐benzisoxazole as substrate analogue, as well as 5‐nitro‐indole as template analogue for the preparation of a different set of nanogels. All the kinetic data demonstrate that the chemical structure of the template is key to the molecular recognition properties of the imprinted nanogels that are closely tailored and able to differentiate among small structural changes. Copyright

Tuning Molecular Recognition in Water‐Soluble Nanogels with Enzyme‐Like Activity for the Kemp Elimination

The synthesis and characterization of water-soluble imprinted nanogels with enzyme-like activity in the Kemp elimination is reported together with studies that demonstrate how the recognition properties, morphology, and catalytic activity of the nanoparticles can be tuned by the use of surfactants, such as Tween 20. A detailed kinetic investigation is carried out, which shows clear evidence of saturation kinetics and rule out the effects of mass transfer. This is supported by characterization of the polymeric materials that confirms the morphological changes resulting from the use of surfactants. These results provide an important tool for the development of nanoparticle-based, new catalyst-mimicking enzymes.

Microgels and nanogels with catalytic activity.

Molecular imprinting has grown considerably over the last decade with more and more applications being developed. The use of this approach for the generation of enzyme-mimics is here reviewed with a particular focus on the most recent achievements using different polymer formats such as microgels and nanogels, beads, membranes and also silica nanoparticles.