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Analytical Biochemistry

Leila Rastegar, Hossein Mighani, Alireza Ghassempour
5-methylcytosine (5mC) is an epigenetic mark which has a profound effect on various fundamental processes in cells. In present study, at first Hydrophilic Interaction Liquid Chromatography (HILIC) was compared with Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) based on their selectivity (α), retention factor (k), and resolution (R) for cytosine (C) and 5mC nucleobases. We tried to justify the separation mechanism on the basis of mobile phase and solute polarity, structural characterization of solute and stationary phases, log Do/w, and pka under both modes...
July 18, 2018: Analytical Biochemistry
Roberto Alan Federico-Perez, Zi-Ling Xue
Indole is a chemical from the decomposition of shrimp and is used extensively to indicate seafood freshness. US Food and Drug Administration (FDA) sets its concentration of <25 μg/100 g shrimp as the threshold for Class I (fresh shrimp). A novel optical probe is reported to quantitatively analyze trace indole in shrimp, including the Class I threshold concentration. Based on an Ehrlich-type reaction, visible spectroscopic analysis of indole in petroleum ether gives a limit of detection (LoD) and quantification (LoQ) of 0...
July 18, 2018: Analytical Biochemistry
Yijia Xiong, Tammie J McQuistan, James W Stanek, James E Summerton, John E Mata, Thomas C Squier
Here we identify a low-cost diagnostic platform using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide (PMO) probe pairs, which upon binding target oligonucleotides undergo fluorescence resonance energy transfer (FRET). Using a target oligonucleotide derived from the Ebola virus (EBOV), we have derivatized PMO probes with either Alexa Fluor488 (donor) or tetramethylrhodamine (acceptor). Upon EBOV target oligonulceotide binding, observed changes in FRET between PMO probe pairs permit a 25 pM lower limit of detection; there is no off-target binding within a complex mixture of nucleic acids and other biomolecules present in human saliva...
July 18, 2018: Analytical Biochemistry
Kaili Li, Henan Xu, Wei Jiang, Jing Li, Wenjun Liu, Tao Wang, Min Fang
Pyrogens are a class of heterogeneous compounds that cause fever and induce inflammatory responses in the host. Lipopolysaccharides (LPS, also known as endotoxin) is the major pyrogen in the category of drug quality control. Accurate and fast quantification of pyrogens is crucial for drug safety. In the present study, we aimed to develop a sensitive and reliable method for rapid detection of pyrogens using luciferase reporter assay. Stable human A549 luciferase reporter cells were constructed under the control of a NF-κB-responsive element or IFN-β promoter...
July 18, 2018: Analytical Biochemistry
Saya Okada, Satoshi Yankawa, Hisato Saitoh
Giant plasma membrane vesicles (GPMVs) are large extracellular vesicles produced by the exposure of cells to paraformaldehyde or other stresses, providing an experimental system to elucidate cell surface dynamics. Here we show that addition of the membrane permeable fluorescent RNA-indicators, acridine orange and thioflavin T, to GPMV-containing solutions prepared from cultured HeLa cells was sufficient for the fluorescent visualization of seemingly all GPMVs. Our findings provide a wash-free instant method using non-lipid-type fluorescent dyes for GPMV detection, which should be useful for researchers interested in studying cell membrane dynamics and biochemistry using GPMVs...
July 18, 2018: Analytical Biochemistry
Hossein Momeneh, Mohammad Bagher Gholivand
Using square wave voltammetry, a carbon paste electrode modified by molecularly imprinted polymer (MIP) as a recognition element of mycophenolate mofetil (MMF) and multi-walled carbon nanotubes was used for MMF monitoring To investigate the electrode during modification, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were utilized. After optimization of the effective parameters, the anodic peak current of MMF was utilized for dynamic range study which was linear in 9.9 nM-87 μM range...
July 17, 2018: Analytical Biochemistry
Aiichiro Muraoka, Yoshinori Matsuura, Hisashi Naitow, Makoto Ihara, Naoki Kunishima
It is known that the crystallizability of protein molecules may be improved by replacing their surface lysine residues with other residue types. Here an experimental method to identify surface lysine residues by NHS-biotin chemical modification combined with MALDI-TOF MS was proposed and was evaluated using PH1033 protein from Pyrococcus horikoshii. Interestingly, the biotinylation experiment with a protein-reagent molar ratio of 1:1 revealed that only seven of twenty-two lysine residues in the protein comprising 144 residues were labeled...
July 17, 2018: Analytical Biochemistry
Pratuyasha Dutta, Tulsidas G Shrivastav, Sonu Chand Thakur
In order to develop ELISA for medroxyprogesterone acetate, medroxyprogesterone acetate-3-carboxymethyloxime (MPA-3-CMO) was coupled to bovine serum albumin (BSA) by N-hydroxysuccinimide mediated carbodiimide reaction to prepare immunogen. The same immunogen was used to raise the antiserum in New Zealand white rabbit. Similarly, MPA-3-CMO was coupled to horseradish peroxidase (HRP) by N-hydroxysuccinimide mediated carbodiimide reaction for the preparation of enzyme conjugate. The MPA-3-CMO-HRP enzyme conjugate and MPA-3-CMO-BSA-antibody were used for further standardization and development of the assay...
July 16, 2018: Analytical Biochemistry
Enrico Monachino, Harshad Ghodke, Richard R Spinks, Ben S Hoatson, Slobodan Jergic, Zhi-Qiang Xu, Nicholas E Dixon, Antoine M van Oijen
Rolling-circle DNA amplification is a powerful tool employed in biotechnology to produce large from small amounts of DNA. This mode of DNA replication proceeds via a DNA topology that resembles a replication fork, thus also providing experimental access to the molecular mechanisms of DNA replication. However, conventional templates do not allow controlled access to multiple fork topologies, which is an important factor in mechanistic studies. Here we present the design and production of a rolling-circle substrate with a tunable length of both the gap and the overhang, and we show its application to the bacterial DNA-replication reaction...
July 14, 2018: Analytical Biochemistry
Francesca Maccari, Fabio Galeotti, Veronica Mantovani, Lucia Zampini, Lucia Padella, Laura Rigon, Daniela Concolino, Agata Fiumara, Elisa Pascale, Annarita Pittalà, Tiziana Galeazzi, Chiara Monachesi, Lucia Marchesiello Rita, Giovanni Coppa, Orazio Gabrielli, Nicola Volpi
Dried blood spot (DBS) technology is a cheap and easy method largely applied in newborn screening. Mucopolysaccharidoses (MPS) are characterized by the deficit of enzymes that degrade glycosaminoglycans (GAGs) characterized by progressive worsening of the conditions. For a possible early diagnosis of MPS, we developed a method of uronic acid (UA)-GAGs determination in DBS of 600 healthy newborns and from a small group of MPS subjects matched for age. Spotted blood UA-GAGs of the normal newborns are composed of 67...
July 13, 2018: Analytical Biochemistry
Sang Joon Lee, Jaehyeon Lee, Kiwoong Kim
In this study, the spontaneous ion concentration polarization phenomenon induced by pressure via a cation-selective membrane was theoretically and experimentally investigated. Unlike conventional electrokinetic ion concentration polarization, which uses electric current as a driving flux of cations through the membrane, advection caused by pressure is used as a transmembrane driving flux of cations to spontaneously and stably form an ion depletion zone in the present ion concentration polarization technique...
July 12, 2018: Analytical Biochemistry
Kawther El-Gendy, Eman Mosallam, Nabila Ahmed, Nagat Aly
A sensitive linker-assisted enzyme-linked immunosorbent assay (L'ELISA) was developed for the analysis of glyphosate in Egyptian soil samples. Polyclonal glyphosate antibodies were produced from rabbits immunized with glyphosate protein conjugate. The conjugate was prepared by activating the carboxylic groups of proteins; thyroglobulin or bovine serum albumin with 1-ethyl-3- (3-diaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide followed by directly coupled to the amino group of glyphosate...
July 9, 2018: Analytical Biochemistry
Shujuan Chen, Yongsheng Li, Zhenzhen Fu, Yue Zeng, Li He, Kang Zhou, Xiaoling Ao, Shuliang Liu, Likou Zou
We have developed a label-free, enzyme-free, modification-free and DNA extraction-free fluorescent aptasensing (LEFA) method for detection of E. coli O157:H7 based on G-quadruplex formation using two ingeniously designed hairpin probes (GHP1 and GHP2). In the presence of E. coli O157:H7, it released the single stranded initiation sequence (IS) resulting in the toehold strand displacement between GHP1 and GHP2, which in turn led to the cyclic reuse of the production of DNA assemblies with numerous G-quadruplex structures and initiation sequences...
July 7, 2018: Analytical Biochemistry
Aliye Ozalp, Begona Barroso, John Meijer, Cas van den Beld
The aim of this work was to assess the influence of preanalytical variables on the stability of two endogenous opioid peptides (methionine-enkephalin and leucine-enkephalin) in human plasma. For this purpose, first a sensitive LC-MS/MS analytical method was developed and validated for the simultaneous quantitative analysis of these two peptides. The methodology consisted of a simple protein precipitation step followed by UPLC separation and MRM quantitative analysis using a stable isotope labelled methionine-enkephalin as internal standard...
July 4, 2018: Analytical Biochemistry
Elisa Rasouli, Zohreh Shahnavaz, Wan Jeffrey Basirun, Majid Rezayi, Amir Avan, Majid Ghayour-Mobarhan, Roshanak Khandanlou, Mohd Rafie Johan
Human papillomavirus (HPV) is one of the most common sexually transmitted disease, transmitted through intimate skin contact or mucosal membrane. The HPV virus consists of a double-stranded circular DNA and the role of HPV virus in cervical cancer has been studied extensively. Thus it is critical to develop rapid identification method for early detection of the virus. A portable biosensing device could give rapid and reliable results for the identification and quantitative determination of the virus. The fabrication of electrochemical biosensors is one of the current techniques utilized to achieve this aim...
July 4, 2018: Analytical Biochemistry
Yanxue Cai, Keren Kang, Yujia Liu, Yu Wang, Xiaowei He
We have developed a reliable and rapid immunoassay based on a facile synthesis of fluorescent nanoparticles integrated in immunochromatography technique to quantitatively detect C-reactive protein (CRP). The method is based on a sandwich immunoassay using the Nile-red doped nanoparticles/CRP monoclonal antibody conjugate. The method is simple and fast, with a detection limit of 0.091 mg/L. It provides quantitative analysis in the range of 0.1-160 mg/L, which is adequate for detecting CRP of acute inflammatory or cardiovascular disease...
June 30, 2018: Analytical Biochemistry
Shuyi Cheng, Ping Wang, Chunxian Tao, Dawei Zhang, Zhenqing Li, Yoshinori Yamaguchi
Effective separation of short DNA fragments is important for the identification of PCR or LAMP products. We investigated the effect of electric field strength, sample plug width, effective length of the capillary, concentration and molecular weight of polymer on the separation performance of small DNA. Results demonstrated that the sample plug played a non-negligible role in the peak broadening. The migration time of DNA was exponentially decreased with the increase of electric field strength. Increasing effective length of capillary, concentration or molecular weight of HEC may improve the separation performance, but it was at the cost of long migration time...
June 30, 2018: Analytical Biochemistry
Tianchuan Zhu, Jian'an He, Wanshan Chen, Ho Pui Ho, Siu Kai Kong, Chenlong Wang, Jun Long, Jacky Fong-Chuen Loo, Dayong Gu
Dengue is the most prevalent mosquito-borne viral disease in tropical and subtropical regions worldwide. Since its clinical symptoms are non-specific and easily mistaken as other kinds of infection, laboratory diagnosis is required to confirm dengue infections. In this study, ten peptides (E1-E10) from the envelope protein of dengue virus (DENV) were first identified using bioinformatic tool. The screened peptides were then synthesized for the peptide-based chemiluminescence enzyme immunoassay (CLEIA). Two peptides, E1 and E7, were found as the best candidate antigen and therefore used as downstream application in the development of low-cost peptide-based anti-DENV immunoglobulin M antibodies (IgM) indirect CLEIA...
June 29, 2018: Analytical Biochemistry
Ronni Eg Sahl, Thomas Morville, Regitze Kraunsøe, Flemming Dela, Jørn Wulff Helge, Steen Larsen
Skeletal muscle is a heterogeneous tissue and it is essential to know the methodological variation and reliability when measuring aspects of muscle function. We assessed the methodological and biological variation when measuring mitochondrial respiratory capacity (MRC), citrate synthase (CS) activity and myosin heavy chain (MHC) composition in muscle biopsies from nine healthy male participants, and in addition we assessed variation in MRC in isolated mitochondria and yeast suspension. We analysed MRC, CS activity and MHC composition in duplicates (intra-biopsy variation) to quantify the methodological variation, as well as the biological variation from multiple muscle biopsies (inter-biopsy variation) obtained at different sites of the same muscle...
June 29, 2018: Analytical Biochemistry
Santosh Kumar Palanirajan, Sathyanarayana N Gummadi
Human phospholipid scramblase 1 (hPLSCR1) is an ATP independent, Ca2+ dependent transmembrane protein mediating bidirectional translocation of phospholipids across the lipid bilayer but the mechanism of scrambling is unknown. Determination of the hPLSCR1 structure would help understand the mechanism and its multi-functional property. Recombinant hPLSCR1 forms inclusion bodies (IBs), when over-expressed in E. coli BL21 (DE3) and recovery of active hPLSCR1 from IBs, were time-consuming and resulted in low protein yield...
June 29, 2018: Analytical Biochemistry
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