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Shiro Kobayashi - One of the best experts on this subject based on the ideXlab platform.
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5.10 – Enzymatic Polymerization
Polymer Science: A Comprehensive Reference, 2020Co-Authors: Shiro KobayashiAbstract:Enzymatic polycondensations and Enzymatic ring-opening polyadditions are described in a short comprehensive way. Hydrolases and oxidoreductases are mainly used as Polymerization catalysts; glycosidases and lipases are used for the synthesis of polysaccharides and polyesters, respectively; and peroxidase and laccase are used for the synthesis of polyaromatics. Enzymatic catalysis is highly selective in all respects, that is, it shows enantio-, regio-, and chemoselectivities, and can be conducted under mild reaction conditions. Noticeably, the glycosidase catalysts enabled for the first time the in vitro synthesis of natural biomacromolecules such as cellulose, amylose, chitin, hyaluronan, and chondroitin. Owing to the characteristics of the catalysis, Enzymatic Polymerization has the potential to serve as a basis for ‘green polymer chemistry’, thereby contributing toward solving environmental problems.
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synthesis of unnatural hybrid polysaccharides via Enzymatic Polymerization
ACS symposium series, 2008Co-Authors: Akira Makino, Shiro KobayashiAbstract:Various natural polysaccharides and their derivatives have been synthesized via in vitro Enzymatic Polymerization. The results indicate that the glycosidic hydrolases can catalyze the reaction of unnatural substrates, in spite of the high substrate specificity of the enzyme in vivo. This paper describes the Enzymatic Polymerization to produce unnatural hybrid polysaccharides having a disaccharide repeating unit consisted from components of different two natural homopolysaccharides. Newly synthesized monomers designed on the basis of the transition-state analogue substrate (TSAS) concept were successfully polymerized, giving rise to unnatural hybrid polysaccharides in a regioselective and stereocontrolled manner. Such unnatural polysaccharides having a well-defined structure are hardly synthesized via a conventional organic method. Thus, they are expected as new functional materials to exhibit various biological activities originated from both natures.
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New developments of polysaccharide synthesis via Enzymatic Polymerization.
Proceedings of the Japan Academy. Series B Physical and biological sciences, 2007Co-Authors: Shiro KobayashiAbstract:This review focuses on the in vitro synthesis of polysaccharides, the method of which is “Enzymatic Polymerization” mainly developed by our group. Polysaccharides are formed by repeated glycosylation reactions between a glycosyl donor and a glycosyl acceptor. A hydrolysis enzyme was found very efficient as catalyst, where the monomer is designed based on the new concept of a “transition-state analogue substrate” (TSAS); sugar fluoride monomers for polycondensation and sugar oxazoline monomers for ring-opening polyaddition. Enzymatic Polymerization enabled the first in vitro synthesis of natural polysaccharides such as cellulose, xylan, chitin, hyaluronan and chondroitin, and also of unnatural polysaccharides such as a cellulose–chitin hybrid, a hyaluronan–chondroitin hybrid, and others. Supercatalysis of hyaluronidase was disclosed as unusual Enzymatic multi-catalyst functions. Mutant enzymes were very useful for synthetic and mechanistic studies. In situ observations of Enzymatic Polymerization by SEM, TEM, and combined SAS methods revealed mechanisms of the Polymerization and of the self-assembling of high-order molecular structure formed by elongating polysaccharide molecules.(Communicated by Hitosi NOZAKI, M.J.A.)
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new developments of polysaccharide synthesis via Enzymatic Polymerization
Proceedings of the Japan Academy. Series B Physical and biological sciences, 2007Co-Authors: Shiro KobayashiAbstract:This review focuses on the in vitro synthesis of polysaccharides, the method of which is "Enzymatic Polymerization" mainly developed by our group. Polysaccharides are formed by repeated glycosylation reactions between a glycosyl donor and a glycosyl acceptor. A hydrolysis enzyme was found very efficient as catalyst, where the monomer is designed based on the new concept of a "transition-state analogue substrate" (TSAS); sugar fluoride monomers for polycondensation and sugar oxazoline monomers for ring-opening polyaddition. Enzymatic Polymerization enabled the first in vitro synthesis of natural polysaccharides such as cellulose, xylan, chitin, hyaluronan and chondroitin, and also of unnatural polysaccharides such as a cellulose-chitin hybrid, a hyaluronan-chondroitin hybrid, and others. Supercatalysis of hyaluronidase was disclosed as unusual Enzymatic multi-catalyst functions. Mutant enzymes were very useful for synthetic and mechanistic studies. In situ observations of Enzymatic Polymerization by SEM, TEM, and combined SAS methods revealed mechanisms of the Polymerization and of the self-assembling of high-order molecular structure formed by elongating polysaccharide molecules.
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self assembly of synthetic cellulose during in vitro Enzymatic Polymerization process as studied by a combined small angle scattering method
Macromolecules, 2007Co-Authors: Hirokazu Tanaka, Takeji Hashimoto, Satoshi Koizumi, Kazuhiro Kurosaki, Shiro KobayashiAbstract:We have investigated the self-assembling process of cellulose artificially synthesized via Enzymatic Polymerization as one of general problems of chemical reactions at specific sites and reaction-induced self-assembling process of reaction products in the context of nonequilibrium phenomenon and pattern formation. The chemical reaction and the self-assembling process were explored at real time and in-situ by a combined small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), ultra-SANS, and ultra-SAXS method, together with wide-angle X-ray diffraction and field-emission scanning electron microscopy. The results revealed the following pieces of new evidence: (i) Even in the aqueous reaction medium free from monomers, enzymes (cellulase) as a catalyst aggregate themselves into associations with characteristic lengths larger than 200 nm. (ii) Cellulose molecules created at each active site of enzymes associate themselves around the enzyme associations into cellulose aggregates having surf...
Jun-ichi Kadokawa - One of the best experts on this subject based on the ideXlab platform.
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Synthesis of an amylose–polymer inclusion complex by Enzymatic Polymerization of glucose 1-phosphate catalyzed by phosphorylase enzyme in the presence of polyTHF: a new method for synthesis of polymer–polymer inclusion complexes
Chemical Communications, 2020Co-Authors: Jun-ichi Kadokawa, Yoshiro Kaneko, Hideyuki Tagaya, Koji ChibaAbstract:The Enzymatic Polymerization of α-D-glucose 1-phosphate (Glc-1-P) with phosphorylase in the presence of polytetrahydrofuran (polyTHF) leads to an amylose–polyTHF (polymer–polymer) inclusion complex; the present reaction system provides a new method for the preparation of polymer–polymer inclusion complexes.
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Dynamic fabrication of amylosic supramolecular composites in an Enzymatic Polymerization field
Hybrid Polymer Composite Materials, 2020Co-Authors: Jun-ichi KadokawaAbstract:Abstract This chapter reviews dynamic fabrication of supramolecular composites by means of inclusion complexation by amylose in a phosphorylase-catalyzed Enzymatic Polymerization field. Amylose is a well-known polysaccharide and forms an inclusion complex with various hydrophobic small molecules. A pure amylose is produced by the Enzymatic Polymerization using α- d -glucose 1-phosphate as a monomer and maltooligosaccharide as a primer catalyzed by phosphorylase. The author has found that a propagating chain of amylose in the Enzymatic Polymerization dynamically twines around hydrophobic polymers present in the reaction system to form supramolecular inclusion composites. The author named this Polymerization system as “vine-twining Polymerization” because the formation process is similar as the way that a vine plant grows twining around a rod. Amylosic supramolecular composite materials such as hydrogel and film were dynamically fabricated by means of the vine-twining Polymerization approach in the presence of copolymers covalently grafting with hydrophobic guest polymers. The Enzymatically produced amyloses induced complexation with guest polymers in intermolecular graft copolymers, which acted as crosslinking points to form a supramolecular composite hydrogel. By using the graft copolymer having a film-formable main-chain, a supramolecular composite film was also obtained through hydrogelation. Supramolecular polymeric composites were dynamically fabricated by the vine-twining Polymerization using primer-guest conjugates. The products in the vine-twining Polymerization system formed a polymeric continuum of an inclusion composite, where the Enzymatically produced amylose chain elongated from the conjugate included the guest segment in the other conjugate.
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Synthesis of Amylosic Supramolecular Materials by Glucan Phosphorylase-Catalyzed Enzymatic Polymerization According to the Vine-Twining Approach
Synlett, 2020Co-Authors: Jun-ichi KadokawaAbstract:This article overviews the synthesis of amylosic supramolecular materials through inclusion complexation in glucan phosphorylase (GP)-catalyzed Enzymatic Polymerization. Amylose is a polysaccharide that is known to form inclusion complexes with a number of hydrophobic small guest molecules. A pure amylose can be synthesized by the Enzymatic Polymerization of α- d -glucose 1-phosphate monomer with a maltooligosaccharide primer catalyzed by GP. The author has reported that the propagating amylosic chain in the Enzymatic Polymerization twines around hydrophobic polymers present in aqueous reaction media to form supramolecular inclusion complexes. As it is similar to the way that vines of a plant grow around a rod, this Polymerization is termed ‘vine-twining Polymerization’. Amylosic supramolecular network materials have been obtained through the vine-twining Polymerization by using copolymers, where hydrophobic guest polymers are covalently grafted on hydrophilic main-chain polymers. The Enzymatically produced amylosic chains form complexes with the guest polymers among graft copolymers, which act as cross-linking points to form supramolecular networks, resulting in the formation of soft materials, such as gels and films. Vine-twining Polymerization using appropriately designed guest polymers has also been performed, which leads to supramolecular products that exhibit new functionality. 1 Introduction 2 Vine-Twining Polymerization to Form Supramolecular Inclusion Complexes 3 Selective Complexation of Amylose toward Guest Polymers in Vine-Twining Polymerization 4 Hierarchical Architecture of Amylosic Supramolecular Network Materials by Vine-Twining Polymerization Approach 5 Hierarchical Fabrication of Amylosic Supramolecular Materials by Vine-Twining Polymerization Using Designed Guest Polymers 6 Conclusions
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Formation of microparticles from amylose-grafted poly(γ-glutamic acid) networks obtained by thermostable phosphorylase-catalyzed Enzymatic Polymerization
RSC Advances, 2019Co-Authors: Jun-ichi Kadokawa, Saya Orio, Kazuya YamamotoAbstract:Amylose is a natural polysaccharide with helical conformation, which spontaneously forms water-insoluble assemblies, such as double helixes and inclusion complexes, at ambient temperatures in aqueous media, whereas it is synthesized as a water-soluble single chain by thermostable phosphorylase-catalyzed Enzymatic Polymerization at elevated temperatures in aqueous buffer solvents. In this study, we investigated the Enzymatic Polymerization at 80 °C using a primer-grafted poly(γ-glutamic acid) (PGA) in the presence or absence of poly(L-lactic acid) (PLLA) as a guest polymer for inclusion by amylose. Consequently, the produced amylose-grafted PGAs formed microparticles by cooling the mixtures at room temperature after the Enzymatic Polymerization in either the presence or the absence of PLLA. The particle sizes, which were evaluated by SEM measurement, were dependent on the feed ratios of PLLA. Based on the characterization results by the powder X-ray diffraction, IR, and dynamic light scattering measurements, a mechanism for the formation of the microparticles in the present system is proposed.
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Preparation of Amylose-Carboxymethyl Cellulose Conjugated Supramolecular Networks by Phosphorylase-Catalyzed Enzymatic Polymerization
Catalysts, 2019Co-Authors: Jun-ichi Kadokawa, Takuya Shoji, Kazuya YamamotoAbstract:Enzymatic Polymerization has been noted as a powerful method to precisely synthesize polymers with complicated structures, such as polysaccharides, which are not commonly prepared by conventional Polymerization. Phosphorylase is one of the enzymes which have been used to practically synthesize well-defined polysaccharides. The phosphorylase-catalyzed Enzymatic Polymerization is conducted using α-d-glucose 1-phosphate as a monomer, and maltooligosaccharide as a primer, respectively, to obtain amylose. Amylose is known to form supramolecules owing to its helical conformation, that is, inclusion complex and double helix, in which the formation is depended on whether a guest molecule is present or not. In this paper, we would like to report the preparation of amylose-carboxymethyl cellulose (CMC) conjugated supramolecular networks, by the phosphorylase-catalyzed Enzymatic Polymerization, using maltoheptaose primer-grafted CMC. When the Enzymatic Polymerization was carried out using the graft copolymer, either in the presence or in the absence of a guest polymer poly (ε-caprolactone) (PCL), the Enzymatically elongated amylose chains from the primers on the CMC main-chain formed double helixes or inclusion complexes, depending on the amounts of PCL, which acted as cross-linking points for the construction of network structures. Accordingly, the reaction mixtures totally turned into hydrogels, regardless of the structures of supramolecular cross-linking points.
Masashi Ohmae - One of the best experts on this subject based on the ideXlab platform.
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Enzymatic Polymerization to Cellulose by Crosslinked Enzyme Immobilized on Gold Solid Surface
Chemistry Letters, 2012Co-Authors: Itsuko Nakamura, Masashi Ohmae, Akira Makino, Shunsaku KimuraAbstract:Mutant endoglucanase II was locally immobilized on a gold substrate by crosslinking, and Enzymatic Polymerization to cellulose was analyzed. The crosslinked enzyme showed an enhanced Polymerization...
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Preparation of fibrous cellulose by Enzymatic Polymerization using cross-linked mutant endoglucanase II
Chemical Communications, 2011Co-Authors: Itsuko Nakamura, Masashi Ohmae, Akira Makino, Junji Sugiyama, Yoshiki Horikawa, Shunsaku KimuraAbstract:A cross-linked mutant endoglucanase II was prepared for Enzymatic Polymerization to cellulose. The cross-linked enzyme is composed of three mutant enzymes showing Polymerization activity. A characteristic feature of the Polymerization with this cross-linked enzyme is formation of cellulose fibrils in contrast to plate-like crystals obtained by using a free enzyme.
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Enzymatic Polymerization to Unnatural Hybrid Polysaccharides
Macromolecular Chemistry and Physics, 2007Co-Authors: Masashi Ohmae, Akira Makino, Shiro KobayashiAbstract:Unnatural hybrid polysaccharides with an alternating structure of different kinds of monosaccharide units have been synthesized by Enzymatic Polymerization catalyzed by a glycoside hydrolase. For the synthesis, various activated monomers with a disaccharide structure were designed on the basis of the concept of transition-state analogue substrate (TSAS). The monomers include two types of TSAS monomer structures: a sugar fluoride type and a sugar oxazoline type. Hydrolase catalysis successfully induces polycondensation or ring-opening polyaddition of these monomers to produce unnatural hybrid polysaccharides in good yield. Characterization of the products, mechanistic features of the Polymerization, and specific properties of the product polysaccharides are discussed.
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Chemical Reaction at Specific Sites and Reaction-Induced Self-Assembly as Observed by in Situ and Real Time SANS: Enzymatic Polymerization to Synthetic Cellulose
Biomacromolecules, 2006Co-Authors: Takeji Hashimoto, Masashi Ohmae, Hirokazu Tanaka, Satoshi Koizumi, Kazuhiro Kurosaki, Shiro KobayashiAbstract:We have investigated the self-assembling process of cellulose artificially synthesized via Enzymatic Polymerization by means of in situ and time-resolved SANS (small-angle neutron scattering). The results elucidated the following: (i) Cellulose molecules synthesized at a special reaction site of the enzyme (cellulase) located on or near the smooth surface of self-assembled enzymes formed in the reaction medium. (ii) The synthesized molecules associated themselves via DLA (diffusion-limited association) and crystallized into fibrils. (iii) The fibrils formed the aggregates, which had surface fractal dimension Ds increasing from 2 to 2.3 with the reaction time, on the smooth surface of the enzyme aggregates.
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Enzymatic Polymerization to novel polysaccharides having a glucose n acetylglucosamine repeating unit a cellulose chitin hybrid polysaccharide
Biomacromolecules, 2006Co-Authors: Shiro Kobayashi, Masashi Ohmae, Akira Makino, Toshitsugu Kiyosada, Hironori Matsumoto, Shigeru Kunii, Ken Makiguchi, Akira Matsumoto, Michinobu Horie, Shin-ichiro ShodaAbstract:A cellulose−chitin hybrid polysaccharide having alternatingly β(1→4)-linked d-glucose (Glc) and N-acetyl-d-glucosamine (GlcNAc) was synthesized via two modes of Enzymatic Polymerization. First, a sugar oxazoline monomer of Glcβ(1→4)GlcNAc (1) was designed as a transition-state analogue substrate (TSAS) monomer for chitinase catalysis. Monomer 1 was recognized by chitinase from Bacillus sp., giving rise to a cellulose−chitin hybrid polysaccharide (2) via ring-opening polyaddition with perfect regioselectivity and stereochemistry. Molecular weight (Mn) of 2 reached 4030, which corresponds to 22 saccharide units. Second, a sugar fluoride monomer of GlcNAcβ(1→4)Glc (3) was synthesized for the catalysis of cellulase from Trichoderma viride. The enzyme catalyzed polycondensation of 3, providing a cellulose−chitin hybrid polysaccharide (4) in regio- and stereoselective manner. Mn of 4 reached 2840, which corresponds to 16 saccharide units. X-ray diffraction measurements revealed that these hybrid polysaccharides...
Ashutosh Chilkoti - One of the best experts on this subject based on the ideXlab platform.
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Enzymatic Polymerization of high molecular weight dna amphiphiles that self assemble into star like micelles
Advanced Materials, 2014Co-Authors: Lei Tang, Ashutosh Chilkoti, Vinalia Tjong, Nan K Li, Yaroslava G Yingling, Stefan ZauscherAbstract:High molecular weight ssDNA amphiphiles are synthesized by Enzymatic Polymerization. These highly asymmetric diblock DNA copolymers self-assemble into "hairy", star-like micelles, shown in the AFM image and the DPD snapshot.
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Enzymatic Polymerization of High Molecular Weight DNA Amphiphiles That Self‐Assemble into Star‐Like Micelles
Advanced Materials, 2014Co-Authors: Lei Tang, Ashutosh Chilkoti, Vinalia Tjong, Nan K Li, Yaroslava G Yingling, Stefan ZauscherAbstract:High molecular weight ssDNA amphiphiles are synthesized by Enzymatic Polymerization. These highly asymmetric diblock DNA copolymers self-assemble into "hairy", star-like micelles, shown in the AFM image and the DPD snapshot.
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Spectroscopic Study of a DNA Brush Synthesized in Situ by Surface Initiated Enzymatic Polymerization
Journal of Physical Chemistry B, 2013Co-Authors: M. Nuruzzaman Khan, Ashutosh Chilkoti, Vinalia Tjong, Michael ZharnikovAbstract:We used a combination of synchrotron-based X-ray photoelectron spectroscopy (XPS) and angle-resolved near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to study the chemical integrity, purity, and possible internal alignment of single-strand (ss) adenine deoxynucleotide (poly(A)) DNA brushes. The brushes were synthesized by surface-initiated Enzymatic Polymerization (SIEP) on a 25-mer of adenine self-assembled monolayer (SAM) on gold (A25-SH), wherein the terminal 3′-OH of the A25-SH serve as the initiation sites for SIEP of poly(A). XPS and NEXAFS spectra of poly(A) brushes were found to be almost identical to those of A25-SH initiator, with no unambiguous traces of contamination. Apart from the well-defined chemical integrity and contamination-free character, the brushes were found to have a high degree of orientational order, with an upright orientation of individual strands, despite their large thickness up to ∼55 nm, that corresponds to a chain length of at least several hundred nucleoti...
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direct fluorescence detection of rna on microarrays by surface initiated Enzymatic Polymerization
Analytical Chemistry, 2013Co-Authors: Vinalia Tjong, Hua Yu, Angus Hucknall, Ashutosh ChilkotiAbstract:We report the first demonstration of surface-initiated Enzymatic Polymerization (SIEP) for the direct detection of RNA in a fluorescence microarray format. This new method incorporates multiple fluorophores into an RNA strand using the two-step sequential and complementary reactions catalyzed by yeast poly(A) polymerase (PaP) to incorporate deoxyadenosine triphosphate (dATP) at the 3′–OH of an RNA molecule, followed by terminal deoxynucleotidyl transferase (TdT) to catalyze the sequential addition of a mixture of natural and fluorescent deoxynucleotides (dNTPs) at the 3′–OH of an RNA–DNA hybrid. We found that the 3′-end of RNA can be efficiently converted into DNA (∼50% conversion) by Polymerization of dATP using yeast PaP, and the short DNA strand appended to the end of the RNA by PaP acts as the initiator for the TdT-catalyzed Polymerization of longer DNA strands from a mixture of natural and fluorescent dNTPs that contain up to ∼45 Cy3 fluorophores per 1 kb DNA. We obtained an ∼2 pM limit of detection ...
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amplified on chip fluorescence detection of dna hybridization by surface initiated Enzymatic Polymerization
Analytical Chemistry, 2011Co-Authors: Vinalia Tjong, Hua Yu, Angus Hucknall, Srinath Rangarajan, Ashutosh ChilkotiAbstract:We describe the incorporation of multiple fluorophores into a single stranded DNA (ssDNA) chain using terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3′–OH group of an oligonucleotide primer; we term this methodology surface initiated Enzymatic Polymerization (SIEP) of DNA. We found that long (>1 Kb) ssDNA homopolymer can be grown by SIEP, and that the length of the ssDNA product is determined by the monomer to oligonucleotide initiator ratio. We observed efficient initiation (≥50%) and narrow polydispersity of the extended product when fluorescently labeled nucleotides are incorporated. TdT’s ability to incorporate fluorescent dNTPs into a ssDNA chain was characterized by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the degree of fluorophore incorporation and the length of the polymerized DNA strand. These experiments allowed us to optimize the Polymerization con...
Shulin Yang - One of the best experts on this subject based on the ideXlab platform.
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aptamer initiated on particle template independent Enzymatic Polymerization aptamer otep for electrochemical analysis of tumor biomarkers
Biosensors and Bioelectronics, 2016Co-Authors: Pengjuan Wang, Yan Su, Shengyuan Deng, Shulin Yang, Ali AldalbahiAbstract:Herein, an aptamer-initiated on-particle template-independent Enzymatic Polymerization (aptamer-OTEP) strategy for electrochemical aptasensor (E-aptasensor) is developed for analysis of cancer biomarker carcino-embryonic antigen (CEA). A pair of DNA aptamers is employed which can be specifically bond with CEA simultaneously. One of the aptamer is thiolated at 3'-terminal and immobilized onto the gold electrode as a capture probe, while the other one has a thiol group at its 5'-terminal and is modified onto the gold nanoparticles surface to form a nanoprobe. In the present of target, the two aptamers can "sandwich" the target, thus the nanoprobe is attached to the electrode. Then terminal deoxynucleotidyl transferase (TdT) is employed to catalyze the incorporation of biotin labeled dNTPs into the 3'-OH terminals of the DNA aptamer on the nanoprobe. The as-generated long DNA oligo tentacles allow specific binding of numerous avidin modified horseradish peroxidase (Av-HRP), resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. Taking advantage of the enzyme based nucleic acid amplification and nanoprobe, this strategy is demonstrated to possess the outstanding amplification efficiency.
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Ultrasensitive Electrochemical Aptasensor Based on Surface‐Initiated Enzymatic Polymerization
Chinese Journal of Chemistry, 2016Co-Authors: Pengjuan Wang, Yan Su, Shengyuan Deng, Shulin YangAbstract:Coupled with the ability of fast and quantitative response, electrochemical aptasensors (EA) have great potential in the application of early diagnosis of cancer biomarker. In order to determine the rare biomarkers in the complicate clinical samples, scientists are making unremitting efforts towards improving the sensitivity and selectivity of EA. Herein, a "sandwich"-structure electrochemical aptasensor (SEA) is developed for analysis of cancer biomarker carcino-embryonic antigen (CEA). Two DNA aptamers are employed, one of which is thiolated at 3′-terminal and immobilized onto the gold electrode as a capture probe, while the other one is served as signal probe. The two aptamers could "sandwich" the target and signal probe is then subjected to the terminal deoxynucleotidyl transferase (TdT)-catalyzed incorporation of biotin labeled dNTPs into its 3′-terminal. Thus the as-generated long DNA oligo tails allow specific binding of numerous avidin modified horseradish peroxidase (Av-HRP), resulting in enhanced peroxidase catalyzed electrochemical signals. This signal amplification strategy is termed as surface initiated Enzymatic Polymerization (SIEP). This SIEP amplified SEA has a detection limit of 10 pg·mL−1, indicating the outstanding amplification efficiency.
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ultrasensitive electrochemical aptasensor based on surface initiated Enzymatic Polymerization
Chinese Journal of Chemistry, 2016Co-Authors: Pengjuan Wang, Yan Su, Shengyuan Deng, Shulin YangAbstract:Coupled with the ability of fast and quantitative response, electrochemical aptasensors (EA) have great potential in the application of early diagnosis of cancer biomarker. In order to determine the rare biomarkers in the complicate clinical samples, scientists are making unremitting efforts towards improving the sensitivity and selectivity of EA. Herein, a "sandwich"-structure electrochemical aptasensor (SEA) is developed for analysis of cancer biomarker carcino-embryonic antigen (CEA). Two DNA aptamers are employed, one of which is thiolated at 3′-terminal and immobilized onto the gold electrode as a capture probe, while the other one is served as signal probe. The two aptamers could "sandwich" the target and signal probe is then subjected to the terminal deoxynucleotidyl transferase (TdT)-catalyzed incorporation of biotin labeled dNTPs into its 3′-terminal. Thus the as-generated long DNA oligo tails allow specific binding of numerous avidin modified horseradish peroxidase (Av-HRP), resulting in enhanced peroxidase catalyzed electrochemical signals. This signal amplification strategy is termed as surface initiated Enzymatic Polymerization (SIEP). This SIEP amplified SEA has a detection limit of 10 pg·mL−1, indicating the outstanding amplification efficiency.
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nanoprobe initiated Enzymatic Polymerization for highly sensitive electrochemical dna detection
ACS Applied Materials & Interfaces, 2015Co-Authors: Pengjuan Wang, Yan Su, Lihua Wang, Ali Aldalbahi, Shulin YangAbstract:Electrochemical DNA (E-DNA) sensors have been greatly developed and play an important role in early diagnosis of different diseases. To determine the extremely low abundance of DNA biomarkers in clinical samples, scientists are making unremitting efforts toward achieving highly sensitive and selective E-DNA sensors. Here, a novel E-DNA sensor was developed taking advantage of the signal amplification efficiency of nanoprobe-initiated Enzymatic Polymerization (NIEP). In the NIEP based E-DNA sensor, the capture probe DNA was thiolated at its 3'-terminal to be immobilized onto gold electrode, and the nanoprobe was fabricated by 5'-thiol-terminated signal probe DNA conjugated gold nanoparticles (AuNPs). Both of the probes could simultaneously hybridize with the target DNA to form a sandwich structure followed by the terminal deoxynucleotidyl transferase (TdT)-catalyzed elongation of the free 3'-terminal of DNA on the nanoprobe. During the DNA elongation, biotin labels were incorporated into the NIEP-generated long single-stranded DNA (ssDNA) tentacles, leading to specific binding of avidin modified horseradish peroxidase (Av-HRP). Since there are hundreds of DNA probes on the nanoprobe, one hybridization event would generate hundreds of long ssDNA tentacles, resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. By employing nanoprobe and TdT, it is demonstrated that the NIEP amplified E-DNA sensor has a detection limit of 10 fM and excellent differentiation ability for even single-base mismatch.
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ultrasensitive electrochemical dna sensor based on the target induced structural switching and surface initiated Enzymatic Polymerization
Biosensors and Bioelectronics, 2014Co-Authors: Pengjuan Wang, Yan Su, Shulin Yang, Jianxin Lu, Qing HuangAbstract:In this work, two electrochemical DNA sensors was developed based on the target induced structural switching of stem-loop probe (SLP) and surface initiated Enzymatic Polymerization (SIEP). Both of the electrochemical DNA sensors employed SLPs with the same sequence. However, one had a thiol label at its 3' terminal (the probe was named 3-SLP and the sensor was named 3-SLP-SENS) and the other at its 5' terminal (the probe was named 5-SLP and the sensor was named 5-SLP-SENS). In the initial state of the sensors, both of the probes adopted the stem-loop structure, which shielded the unlabeled terminals of capture probes from being approached. When the loop regions of the capture probes hybridized with the target DNA the conformation of the SLPs was changed to a rigid double-strand, as a result, the 5-SLP released a 3'-OH terminal for SIEP which could be catalyzed by terminal deoxynudeolidyl transferase (TdT). And the 3-SLP released a 5' phosphate terminal which is not suit for SIEP. Thus a signal probe was employed to hybridize with the 5 terminal of 3-SLP and provide a 3'-OH. Both of the sensors were then submitted to the TdT-mediated SIEP. By using biotinylated 2'-deoxyadenosine 5'-triphosphate (biotin-dATP), biotin labels are incorporated into the SIEP-generated long single-stranded DNA. Then avidin-horseradish peroxidases (Av-HRPs) were employed for specific binding to the biotin labels to produce electrochemical signals. The detection performances of two electrochemical DNA sensors were investigated and compared. It was demonstrated that though the 3-SLP-SENS employed extra signal probes, the background current was lower leading to a better detection limit By taking advantage of SLP and SIEP, this 3-SLP-SENS has been able to detect as low as 0:1 pM DNA targets with excellent differentiation ability for even single mismatches. (c) 2013 Published by Elsevier B.V. All rights reserved.