Hirobumi Sunayama

Fluorescent protein recognition polymer thin films capable of selective signal transduction of target binding events prepared by molecular imprinting with a post-imprinting treatment.

The functional monomer bearing three functional groups for protein imprinting was designed, which has a structure consisting of a polymerizable methacryloyl group, a secondary amine group for fluorescent dye conjugation by a post-imprinting treatment, and a benzoic acid moiety capable of interacting with a target protein. Lysozyme-imprinted polymer thin films were prepared on the initiator-immobilized glass substrates by radical polymerization in the presence of lysozyme, the designed functional monomer, a co-monomer(s) and a crosslinker. After the removal of lysozyme, fluorescein isothiocyanate was introduced into the secondary amine group of the functional monomer residues in the imprinted thin film as a fluorescent reporter dye (post-imprinting treatment). Lysozyme was selectively bound to the thin film with a binding constant of ca. 10(6) M(-1). Since the reporter dye can be only introduced into the binding cavity, the fluorescent response can be detected only when the guest is bound to the cavity, namely only specific binding events can be transduced as fluorescence spectral change. Compared with the SPR measurement, selective binding to the imprinted cavity can be more precisely detected by the proposed method, enabling us to prepare a new class of protein recognizable materials with the ability of the specific signal transduction of protein binding events.

Fluorescent molecularly imprinted polymer thin films for specific protein detection prepared with dansyl ethylenediamine-conjugated O-acryloyl L-hydroxyproline.

Protein-imprinted polymers, capable of specific transduction of protein binding events into fluorescent signal change, were designed and synthesized by using dansyl ethylenediamine-conjugated O-acryloyl L-hydroxyproline (Hyp-En-Dans). Human serum albumin (HSA) was used as a model target protein and HSA-imprinted polymers (HSA-IP) were prepared on glass substrates. Specific fluorescence change was observed for HSA binding on the imprinted polymer thin film, whereas a weaker response was observed for other proteins, including bovine serum albumin, chymotrypsin, lysozyme, and avidin. The binding specificity was found to derive from the rigid structure of the hydrogen-bondable pyrrolidine moiety. Compared with SPR measurements, the non-specific binding caused by the polymer matrix and/or randomly located fluorescent monomer residues that did not compose specific binding sites did not contribute to the observed fluorescence change. These results revealed that the proposed protein-imprinting technique using Hyp-En-Dans could provide a highly selective protein-sensing platform, in which only specific binding events would be detected by fluorescent measurements.

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.

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.

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.

Regulation of protein-binding activities of molecularly imprinted polymers via post-imprinting modifications to exchange functional groups within the imprinted cavity

We prepared lysozyme‐imprinted polymers bearing modifiable sites within the imprinted cavity to introduce various functional groups via post‐imprinting modifications. For this purpose, ({[2‐(2‐methacrylamido)‐ethyldithio]‐ethylcarbamoyl}‐methoxy)acetic acid (MDTA), which has a carboxy group to interact with the target protein, lysozyme, and a disulfide linkage for post‐imprinting modifications, was used as a functional monomer. A lysozyme‐imprinted polymer film was prepared by copolymerization of MDTA with a cross‐linker, N,N′‐methylenebisacrylamide, in the presence of lysozyme. After removing lysozyme, followed by reducing the disulfide linkage, various functional groups, such as carboxy, amino, sulfonate, and oligo‐ethylene oxide, were introduced to the exposed thiol groups via a disulfide exchange reaction using the pyridyldisulfide derivatives of these functional groups. Various functional groups could be introduced reversibly via this post‐imprinting disulfide exchange reaction after the construction of the lysozyme‐imprinted cavities. The modification regulated the protein‐binding activity. The proposed post‐imprinting modification system, based on a molecular imprinting process, is expected to lead to the development of advanced materials for fine‐tuning and/or introducing desired functions.

Fluorescence signaling molecularly imprinted polymers for antibiotics prepared via site-directed post-imprinting introduction of plural fluorescent reporters within the recognition cavity

An antibiotic-imprinted cavity with two different fluorescent dyes was prepared by molecular imprinting and subsequent post-imprinting modifications (PIMs), for the readout of a specific binding event as a fluorescence signal. The fluorescent dyes were site-specifically introduced into the cavity using an orthogonal reversible bonding reaction, Schiff base formation, and a disulfide exchange reaction. The template molecule, comprising cephalexin connected to a Schiff base monomer and a disulfide monomer, was copolymerized with a crosslinker. This was followed by Schiff base hydrolysis and a disulfide exchange reaction, yielding the APO-type scaffold (PRECURSOR). Two different fluorophores, 4-formylsalicylic acid (FSA) and 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD), were introduced into the PRECURSOR by site-directed PIMs, yielding a fluorescent HOLO polymer. The obtained fluorescent polymer, HOLO(FSA)(Cys-DBD), was able to selectively readout the binding events as a fluorescence signal change. We found that the proposed strategy has the potential to open up a new class of synthetic materials possessing various desirable functions.

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.

Orientationally Fabricated Zwitterionic Molecularly Imprinted Nanocavities for Highly Sensitive Glycoprotein Recognition

Glycoprotein recognition has recently gained a lot of attention, since glycoproteins play important roles in a diverse range of biological processes. Robustly synthesized glycoprotein receptors, such as molecularly imprinted polymers (MIPs), which can be easily and sustainably handled, are highly attractive as antibody substitutes because of the difficulty in obtaining high-affinity antibodies specific for carbohydrate-containing antigens. Herein, molecularly imprinted nanocavities for glycoproteins have been fabricated via a bottom-up molecular imprinting approach using surface-initiated atom transfer radical polymerization (SI-ATRP). As a model glycoprotein, ovalbumin was immobilized in a specific orientation onto a surface plasmon resonance sensor chip by forming a conventional cyclic diester between boronic acid and cis-diol. Biocompatible polymer matrices were formed around the template molecule, ovalbumin, using SI-ATRP via a hydrophilic comonomer, 2-methacryloyloxyethyl phosphorylcholine, in the presence of pyrrolidyl acrylate (PyA), a functional monomer capable of electrostatically interacting with ovalbumin. The removal of ovalbumin left MIPs with binding cavities containing boronic acid and PyA residues located at suitable positions for specifically binding ovalbumin. Careful analysis revealed that strict control over the polymer significantly improved sensitivity and selectivity for ovalbumin recognition, with a limit of detection of 6.41 ng/mL. Successful detection of ovalbumin in an egg white matrix was demonstrated to confirm the practical utility of this approach. Thus, this strategy of using a polymer-based recognition of a glycoprotein through molecularly imprinted nanocavities precisely prepared using a bottom-up approach provides a potentially powerful approach for detection of other glycoproteins.

Oriented, molecularly imprinted cavities with dual binding sites for highly sensitive and selective recognition of cortisol

Novel, molecularly imprinted polymers (MIPs) were developed for the highly sensitive and selective recognition of the stress marker cortisol. Oriented, homogeneous cavities with two binding sites for cortisol were fabricated by surface-initiated atom transfer radical polymerization, using a cortisol motif template molecule (TM1) which consists of a polymerizable moiety attached at the 3-carbonyl group of cortisol via an oxime linkage and an adamantane carboxylate moiety coupled with the 21-hydroxyl group. TM1 was orientationally immobilized on a β-cyclodextrin (β-CD)-grafted gold-coated sensor chip by inclusion of the adamantane moiety of TM1, followed by copolymerization of a hydrophilic comonomer, 2-methacryloyloxyethyl phosphorylcholine, with or without a cross-linker, N,N′-methylenebisacrylamide. Subsequent cleavage of the oxime linkage leaves the imprinted cavities that contain dual binding sites—namely, the aminooxy group and β-CD—capable of oxime formation and hydrophobic interaction, respectively. As an application, MIP-based picomolar level detection of cortisol was demonstrated by a competitive binding assay using a fluorescent competitor. Cross-linking of the MIP imparts rigidity to the binding cavities, and improves the selectivity and sensitivity significantly, reducing the limit of detection to 4.8 pM. In addition, detection of cortisol in saliva samples was demonstrated as a feasibility study.