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Covalent labeling

Quang Nghia Pham, Kieu The Loan Trinh, Seung Won Jung, Nae Yoon Lee
We demonstrate the integration of DNA amplification and detection functionalities developed on a lab-on-a-chip microdevice utilizing solid-phase polymerase chain reaction (SP-PCR) for point-of-need (PON) DNA analyses. First, the polycarbonate microdevice was fabricated by thermal bonding to contain microchambers as reservoirs for performing SP-PCR. Next, the microchambers were subsequently modified with polyethyleneimine and glutaraldehyde for immobilizing amine-modified forward primers. During SP-PCR, the immobilized forward primers and freely diffusing fluorescence-labeled reverse primers cooperated to generate target amplicons, which remained covalently attached to the microchambers for the fluorescence detection...
May 19, 2018: Biotechnology and Bioengineering
Longyi Zhu, Qihui Liu, Bangyu Yang, Huangxian Ju, Jianping Lei
A pixel counting strategy is designed based on DNA walker-triggered fluorescence spots for ultrasensitive detection of nucleic acid. The two-dimensional DNA walker was constructed by hybridization of the dye-labeled hairpin structure (hDNA) as track and swing strand (sDNA) as DNAzyme with two types of capture DNAs covalently modified by click chemistry on glass slide. Introduction of target DNA unlocked the sDNA via strand displacement to form the activated DNAzyme, and the latter cut nearby hDNA with Mn2+ as cofactor, resulting in fluorescence recovery of dye-labeled hDNA on the substrate due to the separation from the quencher...
May 17, 2018: Analytical Chemistry
Cheng-Han Yu, Chi-Chi Chou, Hsin-Fang Tu, Wei-Chieh Huang, Ya-Yeh Ho, Kay-Hooi Khoo, Ming-Shyue Lee, Geen-Dong Chang
Afatinib, used for the first-line treatment of non-small-cell lung carcinoma (NSCLC) patients with distinct epidermal growth factor receptor (EGFR) mutations, inactivates EGFR by mimicking ATP structure and forming a covalent adduct with EGFR. We developed a method to unravel potential targets of afatinib in NSCLC cells through immunoprecipitation of afatinib-labeling proteins with anti-afatinib antiserum and mass spectrometry analysis. Ribonucleotide reductase (RNR) is one of target proteins of afatinib revealed by this method...
April 20, 2018: Oncotarget
Tomonori Tamura, Tsuyoshi Ueda, Taiki Goto, Taku Tsukidate, Yonatan Shapira, Yuki Nishikawa, Alma Fujisawa, Itaru Hamachi
Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~104  M-1  s-1 , comparable to the fastest bioorthogonal chemistry...
May 14, 2018: Nature Communications
Michael T Henzl
ALG-2 dimerization was studied using Förster resonance-energy-transfer. D162C variants of ALG-2des23 were covalently modified with Alexa Fluor 488 and Alexa Fluor 647. When samples of the two labeled protein-preparations are combined, the sensitized emission from AF647 serves as a sensitive probe of dimer formation. At 25 °C, in the absence of divalent ions, the wild-type homodimer, ΔGF122 homodimer, and heterodimer display dissociation constants of 7.1, 26, and 4.5 nM, respectively. At 35 °C, subunit interaction is weaker, indicating that dimer formation is exothermic...
May 5, 2018: Biophysical Chemistry
Koh K Takita, Kazunori K Fujii, Tetsuya Kadonosono, Ryo Masuda, Takaki Koide
We report here a novel class of collagen-binding peptides, cyclic collagen-mimetic peptides (cCMPs), which efficiently hybridize with triple helix-forming portions of collagen. cCMPs are composed of two parallel collagen-like (Xaa-Yaa-Gly)n strands with both termini tethered by covalent linkages. Enzyme-linked immunosorbent assay and western blotting analysis showed that cCMPs exhibit more potent affinity toward collagen than reported collagen-binding peptides and specifically detect different collagen polypeptides in a mixture of proteins...
May 14, 2018: Chembiochem: a European Journal of Chemical Biology
Vladimíra Datinská, Karel Klepárník, Barbora Belšánová, Marek Minárik, František Foret
The synthesis and determination of the structure of a Förster resonance energy transfer probe intended for the detection of specific nucleic acid sequences are described here. The probe is based on the hybridization of oligonucleotide modified quantum dots with a fluorescently labeled nucleic acid sample resulting in changes of the fluorescence emission due to the energy transfer effect. The stoichiometry distribution of oligonucleotides conjugated to quantum dots was determined by capillary electrophoresis separation...
May 9, 2018: Journal of Separation Science
Ahmed Ali, Eun Young Hwang, Jaebum Choo, Dong Woo Lim
Surface-enhanced Raman scattering (SERS) is an optical spectroscopy technique that can detect a variety of analytes with high sensitivity and selectivity without any labels. Controlled clustering of metallic nanoparticles to prepare a new class of SERS nanotags is crucial for the ultra-sensitive detection of specific biological and chemical moieties because increased plasmonic hotspot junctions produce a greatly enhanced SERS signal. We report herein that controlled clustering of Au nanoparticles (AuNPs) was mediated by PEGylated nano-sized graphene (PNG) and that the PNG-induced AuNP clusters (PNG-AuNPCs) were highly sensitive SERS nanotags with colloidal stability for SERS-based biosensing...
May 9, 2018: Analyst
Huong T H Nguyen, Prokopis C Andrikopoulos, Lubomír Rulíšek, Christopher J Shaffer, František Tureček
We report a gas-phase UV photodissociation study investigating non-covalent interactions between neutral hydrophobic pentapeptides and peptide ions incorporating a diazirine-tagged photoleucine residue. Phenylalanine (Phe) and proline (Pro) were chosen as the conformation-affecting residues that were incorporated into a small library of neutral pentapeptides. Gas-phase ion-molecule complexes of these peptides with photo-labeled pentapeptides were subjected to photodissociation. Selective photocleavage of the diazirine ring at 355 nm formed short-lived carbene intermediates that underwent cross-linking by insertion into H-X bonds of the target peptide...
May 7, 2018: Journal of the American Society for Mass Spectrometry
Esther Sánchez-Tirado, Araceli González-Cortés, Paloma Yáñez-Sedeño, José M Pingarrón
This paper reports the development and performance of an electrochemical immunosensor using magnetic multiwalled carbon nanotubes (m-MWCNTs) as nanocarrier tags for the determination of human fetuin A (HFA), a relevant biomarker of obesity, insulin resistance, and type-2 diabetes as well as for pancreatic and liver cancers and inflammatory processes. Screen-printed carbon electrodes were grafted with p-aminobezoic acid and streptavidin was covalently immobilized on the electrode surface. A biotinylated capture antibody was immobilized through streptavidin-biotin interaction and a sandwich assay configuration was implemented using m-MWCNTs conjugated with HRP and anti-HFA antibodies as the detection label...
April 30, 2018: Biosensors & Bioelectronics
Bedilu Allo, Xudong Lou, Alexandre Bouzekri, Olga I Ornatsky
Mass cytometry is a highly multiplexed single-cell analysis platform that uses metal tagged reagents to identify multiple cellular biomarkers. Current metal-tagged reagent preparation employs thiol-maleimide chemistry to covalently couple maleimide-functionalized metal-chelating polymers (MCPs) with antibodies (Abs), a process that requires partial reduction of the Ab to form reactive thiol groups. However, some classes of Abs (for example, IgM) as well as biomolecules lacking cysteine residues has been challenging to label using this method...
May 7, 2018: Bioconjugate Chemistry
Arindam Mal, Rakesh K Mishra, Vakayil K Praveen, M Abdul Khayum, Rahul Banerjee, Ayyappanpillai Ajayaghosh
Ionic covalent organic nanosheets (iCONs), a member of two-dimensional (2D) nanomaterials family, offer a unique functional platform for wide range of applications. Herein, we explore the potential of an ethidium bromide (EB) based covalent organic framework (EB-TFP) that self-exfoliates in water resulting in 2D ionic covalent organic nanosheets (EB-TFP-iCONs) for the selective detection of double-stranded DNA (dsDNA). In aqueous medium, the self-exfoliated EB-TFP-iCONs reassemble in the presence of dsDNA resulting into hybrid EB-TFP-iCONs-DNA crystalline nanosheets with enhanced fluorescence at 600 nm...
May 1, 2018: Angewandte Chemie
Tariq Ahmad Najar, Shruti Khare, Raghavan Varadarajan
Delineating the precise regions on an antigen that are targeted by antibodies is important for the development of vaccines and antibody therapeutics. X-ray crystallography and NMR are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, these are labor-intensive and require purified protein at high concentration. We have recently described [1] a rapid and reliable method that overcomes these constraints, using a panel of single cysteine mutants of the protein of interest and now provide protocols to facilitate its adoption...
2018: Methods in Molecular Biology
Dhruvitkumar S Sutaria, Bartolome Moya, Kari B Green, Tae Hwan Kim, Xun Tao, Yuanyuan Jiao, Arnold Louie, George L Drusano, Jürgen B Bulitta
Penicillin-binding proteins (PBPs) are the high affinity target sites of all β-lactam antibiotics in bacteria. It is well known that each β-lactam covalently binds to and thereby inactivates different PBPs with varying affinity. Despite β-lactams serving as cornerstone of our therapeutic armamentarium against Klebsiella pneumoniae , PBP binding data are missing for this pathogen. We aimed to generate the first PBP binding data on 13 chemically diverse and clinically relevant β-lactams and β-lactamase inhibitors in K...
April 30, 2018: Antimicrobial Agents and Chemotherapy
Elena Polo, Tadeusz T Nitka, Elsa Neubert, Luise Erpenbeck, Lela Vukovic, Sebastian Kruss
Integrins are transmembrane receptors that mediate cell-adhesion, signaling cascades and platelet-mediated blood clotting. Most integrins bind to the common short peptide Arg-Gly-Asp (RGD). The conformational freedom of the RGD motif determines how strong and to which integrins it binds. Here, we present a novel approach to tune binding constants by confining RGD peptide motifs via non-covalent adsorption of single stranded DNA (ssDNA) anchors onto single-walled carbon nanotubes (SWCNTs). Semiconducting SWCNTs display fluorescence in the near infrared (nIR) region and are versatile fluorescent building blocks for imaging and biosensing...
April 30, 2018: ACS Applied Materials & Interfaces
Jonas Schartner, Jörn Güldenhaupt, Sarah Katharina Gaßmeyer, Katharina Rosga, Robert Kourist, Klaus Gerwert, Carsten Kötting
Immobilizing enzymes for biocatalysis offers many advantages, including easy separation of the enzyme from the product and repeated and continuous use. ATR-FTIR spectroscopy is a versatile tool to monitor immobilized enzymes and has been applied to many proteins. However, while the common and convenient immobilization via oligohistidine on mono-NTA layers is adequate for the measurement of difference spectra induced by ligand binding or photochemistry, it lacks the long term stability that is necessary for monitoring biocatalysis...
April 30, 2018: Analyst
Shaohong Yang, Feifei Zhang, Zonghua Wang, Qionglin Liang
A label-free method for the determination of alpha-fetoprotein (AFP) was successfully developed by graphene oxide (GO)-based electrochemical aptasensor. This aptasensor was constructed by covalently immobilizing NH2 -functionalized AFP-specific aptamer on GO with plenty of carboxylic groups. Cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) analysis were carried out to investigate the practicability of the fabrication procedures. Fourier transform infrared spectra (FTIR), Raman, atomic force microscopy (AFM) and scanning electron microscope (SEM) were performed to indicate the changes of the sensing interface...
April 14, 2018: Biosensors & Bioelectronics
Xinke Yu, Eric Y Hayden, Ming Xia, Owen Liang, Lisa Cheah, David B Teplow, Ya-Hong Xie
Amyloid β-protein (Aβ) self-association is one process linked to the development of Alzheimer's disease (AD). Aβ peptides, including its most abundant forms, Aβ40 and Aβ42, are associated with the two predominant neuropathologic findings in AD, vascular and parenchymal amyloidosis, respectively. Efforts to develop therapies for AD often have focused on understanding and controlling the assembly of these two peptides. An obligate step in these efforts is the monitoring of assembly state. We show here that surface-enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) readily distinguishes Aβ40 and Aβ42...
April 26, 2018: Protein Science: a Publication of the Protein Society
Ruibo Wang, Kaimin Cai, Hua Wang, Chen Yin, Jianjun Cheng
In this study, we report incorporation of a covalent linker at the anomeric position of N-azidoacetylmannosamine (ManNAz) for caging its metabolic process. We synthesized a DT-diaphorase-responsive metabolic precursor, HQ-NN-AAM, using an optimized linker. The caged metabolite showed responsiveness to DT-diaphorase in vitro, resulting in metabolic incorporation of an azido sugar into the cell surface in multiple cell lines.
April 27, 2018: Chemical Communications: Chem Comm
Janet M Paper, Thiya Mukherjee, Kathrin Schrick
Background: Phospholipids are important structural and signaling molecules in plant membranes. Some fluorescent dyes can stain general lipids of membranes, but labeling and visualization of specific lipid classes have yet to be developed for most components of the membrane. New techniques for visualizing membrane lipids are needed to further delineate their dynamic structural and signaling roles in plant cells. In this study we examined whether propargylcholine, a bioortholog of choline, can be used to label the major membrane lipid, phosphatidylcholine, and other choline phospholipids in plants...
2018: Plant Methods
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