Yukiya Kitayama

Preparation of micrometer-sized, onionlike multilayered block copolymer particles by two-step AGET ATRP in aqueous dispersed systems: effect of the second-step polymerization temperature.

The polymerization rate, control/livingness, and particle morphology in seeded activators generated by electron transfer for the atom-transfer radical polymerization of styrene with PiBMA-Br macroinitiator particles were investigated at 70, 90, and 110 degrees C. At 110 degrees C, the polymerization proceeded quickly until 60% conversion was reached, but control/livingness was not observed. This seems to be the reason for the high activation rate and spontaneous initiation of styrene, which significantly increased the radical concentration, resulting in a number of radical terminations. As a result, the block copolymer was not sufficiently formed, leading to a sea-island structure. However, at 70 and 90 degrees C, the polymerizations were almost complete in 14 and 7 h, respectively. Control/livingness was maintained, resulting in PiBMA-b-PS. As a result, onionlike multilayered particles were successfully synthesized. These polymerization behaviors were discussed from the viewpoint of the radical concentration and propagation rate coefficient at various temperatures.

Conjugated-Protein Mimics with Molecularly Imprinted Reconstructible and Transformable Regions that are Assembled Using Space-Filling Prosthetic Groups†

Conjugated-protein mimics were obtained using a new molecular imprinting strategy combined with post-imprinting modifications. An antibiotic was employed as a model template molecule, and a polymerizable template molecule was designed, which was composed of the antibiotic and two different prosthetic groups attached through a disulfide bond and Schiff base formation. After co-polymerization with a cross-linker, the template molecule was removed together with the prosthetic groups, yielding the apo-type scaffold. Through conjugation of the two different prosthetic groups at pre-determined positions within the apo-type scaffold, the apo cavity was transformed into a functionalized holo cavity, which enables the on/off switching of the molecular recognition ability, signal transduction activity for binding events, and photoresponsive activity.

Localized Surface Plasmon Resonance Nanosensing of C-Reactive Protein with Poly(2-methacryloyloxyethyl phosphorylcholine)-Grafted Gold Nanoparticles Prepared by Surface-Initiated Atom Transfer Radical Polymerization

Highly sensitive and selective protein nanosensing based on localized surface plasmon resonance (LSPR) of gold nanoparticles (AuNPs) on which polymerized specific ligands were grafted as an artificial protein recognition layer for the target protein were demonstrated. As a model, optical nanosensing for C-reactive protein (CRP), a known biomarker for chronic inflammation that predicts the risk of arteriosclerosis or heart attacks, was achieved by measuring the shift of LSPR spectra derived from the change of permittivity of poly(2-methacryloyloxyethyl phosphorylcholine)-grafted AuNPs (PMPC-g-AuNPs) upon interacting with CRP, in which the PMPC-g-AuNPs layer were grafted on AuNPs by surface-initiated atom transfer radical polymerization (ATRP). This nanosensing system was effective even for detecting CRP concentrations in a human serum solution diluted to 1% (w/w), at which point a limit of detection was ~50 ng/mL and nonspecific adsorption of other proteins was negligible. The nanosensing system using specific ligand-grafted AuNPs has several strengths, such as low preparation cost, avoiding the need for expensive instruments, no necessary complex pretreatments, and high stability, because it does not contain biobased molecules. We believe this novel synthetic route for protein nanosensors, composed of AuNPs and a polymerized specific ligand utilizing surface-initiated controlled/living radical polymerization, will provide a foundation for the design and synthesis of nanosensors targeting various other biomarker proteins, paving the way for future advances in the field of biosensing.

Precisely controlled molecular imprinting of glutathione-s-transferase by orientated template immobilization using specific interaction with an anchored ligand on a gold substrate

We demonstrate a novel synthetic route for molecularly imprinted polymer (MIP) thin films using a bottom-up approach utilizing protein–ligand specific interactions. The ligand was anchored on a gold substrate and served to (i) orient the immobilized target protein for precise formation of homogeneous binding cavities and (ii) act as a binding site with high affinity and selectivity on the MIP thin films after release of the immobilized protein. The MIP thin films were synthesized by controlled/living radical polymerization (CLRP), which allowed for precise control of the film thickness to optimize binding performance. A mixed self-assembled monolayer comprising anchored maleimide groups and bromoisobutyryl groups was constructed on a gold substrate: the former oriented the immobilization of the target protein and the latter initiated CLRP. The chosen model target protein and ligand were glutathione-s-transferase-π (GST-π) and glutathione (GSH), a protein-specific ligand to GST-π. The obtained MIP thin films of precisely controlled film thickness exhibited high affinity toward the target protein compared to non-imprinted polymer (NIP) thin films. Protein binding selectivity was investigated using a selectivity parameter (α) calculated by surface plasmon resonance response with reference proteins, human serum albumin (HSA) and fibrinogen (FIB). The results indicated that the MIP film thickness affects the protein binding selectivity: a polymer thickness of approximately 15 nm gave more selective protein binding (selectivity parameter for α(HSA) = 0.09 and for α(FIB) = 0.30). Furthermore, we clarified that a more hydrophilic polymer matrix in the presence of NaCl gave more selective binding of GST-π. Our findings show that this bottom-up synthetic route has potential for facilitating the fabrication of highly specific MIPs as artificial protein recognition materials.

Synthesis of Monodispersed Submillimeter-Sized Molecularly Imprinted Particles Selective for Human Serum Albumin Using Inverse Suspension Polymerization in Water-in-Oil Emulsion Prepared Using Microfluidics

We synthesized monodispersed submillimeter-sized (100 μm-1 mm) microgels by inverse suspension polymerization of water-soluble monomer species with a photoinitiator in water-in-oil (W/O) droplets formed by the microchannel. After fundamental investigations of the selection of suitable surfactants, surfactant concentration, and flow rate, we successfully prepared monodispersed submillimeter-sized W/O droplets. Because radical polymerization based on thermal initiation was not appropriated based on colloidal stability, we selected photoinitiation, which resulted in the successful synthesis of monodispersed submillimeter-sized microgels with sufficient colloidal stability. The microgel size was controlled by the flow rate of the oil phase, which maintained the monodispersity. In addition, the submillimeter-sized microgels exhibit high affinity and selective binding toward HSA utilizing molecular imprinting. We believe the monodispersed submillimeter-sized molecularly imprinted microgels can be used as affinity column packing materials without any biomolecules, such as antibodies, for sample pretreatment to remove unwanted proteins without a pump system.

Molecularly Imprinted Polymer Arrays as Synthetic Protein Chips Prepared by Transcription-type Molecular Imprinting by Use of Protein-Immobilized Dots as Stamps.

Molecularly imprinted polymer (MIP) arrays were demonstrated for the recognition of proteins. They were prepared via transcription-type molecular imprinting where patterned dots composed of biotinylated nanoparticles were first immobilized on a glass substrate followed by the immobilization of versatile biotinylated proteins via avidin-biotin interactions, yielding a multiple protein-immobilized stamp as a mold that could be transcribed. MIPs were prepared between the stamp and a methacrylated glass substrate, and after the stamp was peeled off, MIP dots were able to be prepared on the methacrylated glass substrate according to the positions of the immobilized proteins on the stamp. We confirmed that the prepared MIP array showed the expected selective binding toward the corresponding template proteins by conducting competitive binding assays using the fluorescently labeled proteins as corresponding competitors. The binding behaviors were consistent with those obtained by a surface plasmon resonance sensing system. We believe that the proposed platform involving the easily handled nanoparticle-based protein stamps for the preparation of MIP arrays can provide a new type of pattern recognition-based protein chip, which can be adopted as a substitute for the use of conventional protein arrays in various research and industrial fields in the life sciences.

A Programmable Signaling Molecular Recognition Nanocavity Prepared by Molecular Imprinting and Post‐Imprinting Modifications

Inspired by biosystems, a process is proposed for preparing next-generation artificial polymer receptors with molecular recognition abilities capable of programmable site-directed modification following construction of nanocavities to provide multi-functionality. The proposed strategy involves strictly regulated multi-step chemical modifications: 1) fabrication of scaffolds by molecular imprinting for use as molecular recognition fields possessing reactive sites for further modifications at pre-determined positions, and 2) conjugation of appropriate functional groups with the reactive sites by post-imprinting modifications to develop programmed functionalizations designed prior to polymerization, allowing independent introduction of multiple functional groups. The proposed strategy holds promise as a reliable, affordable, and versatile approach, facilitating the emergence of polymer-based artificial antibodies bearing desirable functions that are beyond those of natural antibodies.

Molecularly Imprinted Nanogels Acquire Stealth In Situ by Cloaking Themselves with Native Dysopsonic Proteins

Protein corona formation was regulated on the surface in vivo by molecular imprinting to enable polymeric nanogels to acquire stealth upon intravenous administration. Albumin, the most abundant protein in blood, was selected as a distinct protein component of protein corona for preparing molecularly imprinted nanogels (MIP-NGs) to form an albumin-rich protein corona. Intravital fluorescence resonance energy transfer imaging of rhodamine-labeled albumin and fluorescein-conjugated MIP-NGs showed that albumin was captured by MIP-NGs immediately after injection, forming an albumin-rich protein corona. MIP-NGs circulated in the blood longer than those of non-albumin-imprinted nanogels, with almost no retention in liver tissue. MIP-NGs also passively accumulated in tumor tissue. These data suggest that this strategy, based on regulation of the protein corona in vivo, may significantly influence the development of drug nanocarriers for cancer therapy.

A molecularly imprinted nanocavity-based fluorescence polarization assay platform for cortisol sensing

We prepared core-shell-type molecularly imprinted polymer particles (MIP-NPs) for cortisol using cortisol-21-monomethacrylate as a template molecule, itaconic acid as an additional functional monomer, styrene as a comonomer and divinylbenzene as a crosslinker, and established a fluorescence polarization-based sensing nano-platform for the competitive binding assay of cortisol using dansyl-labeled cortisol (dansyl-cortisol). Before the preparation of MIP-NPs, the binding behavior of bulk MIPs prepared by conventional radical polymerization was preliminarily characterized. NIPs prepared with methacrylic acid instead of cortisol-21-monomethacrylate showed less binding activity than the MIPs, revealing that the molecular imprinting process enhanced the affinity toward cortisol. Since the imprinting effect was confirmed in this system, the fluorescence polarization-based sensing nano-platform for cortisol was constructed using MIP-NPs with dansyl-cortisol, where the binding event of cortisol was transduced into the fluorescence anisotropy change of dansyl-cortisol from the bound-state to the free-state, on the basis of the concentration-dependent competitive replacement of dansyl-cortisol by cortisol added on MIP-NPs. The complex of MIP-NPs with dansyl-cortisol was more effectively formed than that of the reference polymer particles (R-MIP-NPs) prepared without itaconic acid, suggesting that the itaconic acid and cortisol-21-monomethacrylate-derived methacrylic acid residues can work cooperatively. Highly sensitive cortisol detection was achieved by the proposed molecularly imprinted nanocavity-based fluorescence polarization assay for cortisol sensing with dansyl-cortisol, and the apparent limit of detection was estimated to be ca. 80 nM.

Preparation of block copolymer particles by two-step, reversible chain transfer catalyzed polymerization (RTCP) with nitrogen catalyst in miniemulsion systems

We demonstrated a successful preparation of poly(methyl methacrylate) (PMMA)-b-poly(benzyl methacrylate) (PBzMA) particles in aqueous media by two-step reversible chain transfer catalyzed polymerization (RTCP) with N-iodosuccinimide as a catalyst at 70 °C. The polymerization smoothly proceeded, and the number-average molecular weight (Mn) increased linearly with conversion, which agreed with the theoretical molecular weight (Mn,th). The molecular weight distribution at each conversion was narrow (polydispersity index ≈ 1.4). Approximately 88% PMMA chains became PMMA-b-PBzMA at 64% conversion, which was a relatively high value.