Eri Takano

SPR sensing of bisphenol A using molecularly imprinted nanoparticles immobilized on slab optical waveguide with consecutive parallel Au and Ag deposition bands coexistent with bisphenol A-immobilized Au nanoparticles.

A slab-type optical waveguide (s_OWG)-based microfluidic SPR measurement system for bisphenol A was developed. This s_OWG possesses consecutive parallel gold and silver deposition bands in the line of plasmon flow, allowing two individual SPR signals to be independently obtained as a result of the difference in resonant reflection spectra of these metals. As a molecular recognition element, molecularly imprinted polymer nanoparticles (MIP-Np) were employed and immobilized on the surface of each of the gold and silver deposition bands. The resonant reflection spectra were measured on the MIP-Np-immobilized consecutive parallel gold and silver deposition bands coexistent with BPA-AuNp. The Ag-based SPR spectra showed a red shift (0.7 nm) when free BPA (0.1 mM) was passed over the BPA-AuNp/immobilized MIP-Np complexes formed on the s_OWG, unlike the case for the Au deposition band, while a large excess of BPA induced a blue shift due to the competitive desorption of BPA-AuNp from the immobilized MIP-Np on the s_OWG. By using the proposed detection system, binding events of other small molecules could be monitored in conjunction with the use of MIP-Np and labeled-AuNp.

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.

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.

Post-imprinting and In-Cavity Functionalization

Molecularly imprinted polymers (MIPs) are artificial materials capable of molecular recognition for target molecules. Currently MIPs have been prepared without further modification after polymerization, and used for predetermined single purposes. Post-imprinting modifications (PIMs) presented here can provide site-specific modifications within the molecularly imprinted binding cavities after polymerization, enabling MIPs to become more complex functional materials as were the cases of naturally occurring conjugated proteins. We present an overview of the research on MIPs involving PIMs, including transformation of binding sites, on/off switching of binding activity, introduction of desirable functions such as fluorescent signalling functions, catalytic activity, and so on. The combination of PIMs with molecular imprinting appears to be a powerful tool for preparing a diverse range of biomimetic functional materials.

Dummy Template-Imprinted Polymers for Bisphenol A Prepared Using a Schiff Base-Type Template Molecule with Post-Imprinting Oxidation

A new dummy template molecule, N,N′-bis(3-vinylbenzylidene)-4,4′-diaminodiphenylmethane (VB-DADPM) was designed to synthesize dummy template-imprinted polymer (DIP) for Bisphenol A (BPA). Since the Schiff-base part of VB-DADPM is easily cleaved by a weak acid treatment, the DADPM moiety can be removed after the co-polymerization with cross-linkers. After the post-imprinting oxidation was carried out to transform the residual aldehyde to carboxylic acid, binding sites toward BPA were generated only inside the binding cavity, which had an estimated binding constant of 1.3 × 106 M−1, and could recognize the difference between similar molecules BPA and BPB, the latter of which has an ethyl group instead of a methyl group at the central part of BPA.

Pipette tip biosensors for bacterial double-stranded DNA using bioluminescence induced by zinc finger luciferase

AbstractThe authors describe a pipette type of biosensor for detecting target genes and using a zinc finger protein fused to luciferase (ZF luciferase). The ZF protein binds to a specific DNA sequence, and the target double-stranded (ds) DNA can be detected by monitoring the enzymatic activity of ZF luciferase. A small avidin-immobilized reaction plate is placed on a plastic pipette tip (referred to as Biologi tip). The dsDNA detection procedures are carried out by using a programmable dispensing robot equipped with a photodetector. These procedures include (a) the aspiration of an analyte to capture the biotinylated target dsDNA (a product of a polymerase chain reaction) on the small reaction plate inside the pipette tip, (b) the introduction of ZF luciferase and luciferin into the pipette tip, and (c) migration of the pipette tip to the detection port to measure bioluminescence on the small reaction plate. The emission originating from luciferase activity is observed on the reaction plate containing immobilized biotin-tagged target dsDNA, whereas plates containing non-target or biotinylated single-stranded DNA only do not yield a signal. The intensity of emission increases proportionally to the concentration of dsDNA, and the detection limit of the target dsDNA is as low as 62 pM. An actual genomic DNA sample from Escherichia coli O157 was successfully detected by this automatic analyzer using the Biologi tip equipped with a reaction plate. This indicates that this system has a large potential for practical applications, including in particular point-of-care analyses in hygiene control, food safety testing, and clinical diagnosis. Graphical abstractA pipette-type biosensor was developed to detect target genes using a luciferase-fused zinc finger protein, where a small NeutrAvidin-immobilized reaction plate was placed on the tip, and the biotinylated target double-stranded DNA was detected by monitoring the bound luciferase activity.

Transcription-Type Protein Imprinted Polymers for SPR Sensing Prepared Using Target-immobilized Stamps based on Submicrometer-Sized Particles via Biotin-Avidin Linkage

Abstract Transcribed molecularly imprinted polymers (MIPs), prepared by using the biotinylated submicrometersized particles-immobilized stamp on which the biotinconjugated protein was hierarchically immobilized via avidin, were used successfully in the recognition of cytochrome c (Cyt) as a model protein. The transcribed MIP for Cyt was prepared on the gold-coated surface plasmon resonance (SPR) sensor chip, and the binding behavior of Cyt, myoglobin, ribonuclease A, lysozyme, and avidin was evaluated to confirm the selectivity for Cyt. The imprint effect of the transcribed MIP was revealed by comparing the MIP and the corresponding non-imprinted polymer prepared using the stamp without the Cyt immobilization.