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Analytical Biochemistry
Journal Prestige (SJR): 0.633
Citation Impact (citeScore): 2
Number of Followers: 170  
  Hybrid Journal Hybrid journal (It can contain Open Access articles)
ISSN (Print) 0003-2697 - ISSN (Online) 1096-0309
Published by Elsevier Homepage  [3162 journals]
  • Mycophenolate mofetil sensor based on molecularly imprinted
           polymer/multi-walled carbon nanotubes modified carbon paste electrode
    • Abstract: Publication date: Available online 17 July 2018Source: Analytical BiochemistryAuthor(s): 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. The detection limit of the sensor was 7.0 nM. The capture ability of MIP to target was compared with that of non-imprinted polymer (NIP). The practical application of the sensor in biological fluid samples analysis demonstrates its selectivity, sensitivity, and stability.
  • Homologous ELISA for measurement of medroxyprogesterone acetate in serum
    • Abstract: Publication date: Available online 17 July 2018Source: Analytical BiochemistryAuthor(s): 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. Sensitivity and ED50 of the assay were found to be 0.114 ng/mL and 2.75 ng/mL respectively. Slope (m), intercept (c) and affinity of the assay were 2.17, 0.956 and 9.9 × 10⁻⁸ L/mol respectively. The % cross-reaction of all analogous steroids with MPA-3-CMO-BSA-antibody was less than 0.025%. The recovery of the exogenously spiked MPA serum pools were in the range of 96.83–105.47%. The intra- and inter-assay coefficients of variation ranged from 2.35 to 7.02% and 1.42–4.58%, respectively. The correlation coefficient of the MPA level in serum measured by the developed method with the commercially available kit was found to be 0.95 (n = 37). This developed ELISA was further used for measuring serum level of MPA in rat after administering them different doses of MPA.
  • Availability of NHS-biotin labeling to identify free protein lysine
           revealed by experiment and MD simulation
    • Abstract: Publication date: Available online 17 July 2018Source: Analytical BiochemistryAuthor(s): 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. To investigate the result, we analyzed structures from a molecular-dynamics simulation mimicking the experiment. A logistic regression analysis revealed that the biotinylation was significantly correlated with four factors relevant to the local environment of lysine residues: the solvent accessibility, the electrostatic energy, the number of hydrogen bonds, and the estimated pKa value. This result is overall in agreement with that from the same analysis on the crystal structure. However, reflecting the flexibility of the protein molecule in solution state, the factors except for the electrostatic energy were highly variable in the MD structures depending upon the protonation state of Tyr87. The present procedure of biotin-labeling can avoid lysine residues with extensive intramolecular interactions that are incompatible with the rational design of protein crystals.Graphical abstractImage 1
  • Pressure-driven spontaneous ion concentration polarization using an
           ion-selective membrane
    • Abstract: Publication date: 15 September 2018Source: Analytical Biochemistry, Volume 557Author(s): 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. The ion depletion zone produced in a simple experimental setup was used to filter electrolyte and preconcentrate ions and microparticles. Different from the general assumption of the negligible thickness of the electric double layer in microchannels, the low concentration in the ion depletion zone considerably increased the length of the electric double layer. This enhanced the formation of the ion depletion zone. The present results can improve the understanding on ion transport in the ion concentration polarization system and can be utilized to develop a portable water desalination device for rural/remote areas and for preconcentrating biomolecules.
  • Editorial for Analytical Biochemistry special issue on RPA
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Ciara K. O' Sullivan, Luis Antonio Tortajada-Genaro, Olaf Piepenburg, Ioanis Katakis
  • The effect of electrophoretic parameters on separation performance of
           short DNA fragments
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): 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.
  • Label-free and enzyme-free sensitive fluorescent method for detection of
           viable Escherichia coli O157:H7
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): 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. Then these G-quadruplex structures can be recognized quickly by N-methyl mesoporphyrin IX (NMM) resulting in significantly enhanced fluorescence. The LEFA method was successfully implemented for detecting E. coli O157:H7 with a detection limit of 66 CFU/mL in pure culture, 10 CFU/mL and 1 CFU/mL after pre-incubation of the milk and tap water for 4 and 8 h, respectively. Moreover, the strategy could distinguish viable E. coli O157:H7 from dead E. coli O157:H7 and other species of pathogen cells. Furthermore, the whole process of the strategy is accomplished within 100 min. The results indicated that the approach may be used to effectively control potential microbial hazards in human health, food safety, and animal husbandry.Graphical abstractImage 1
  • Development of a lateral flow immunoassay of C-reactive protein detection
           based on red fluorescent nanoparticles
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): 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. This strategy displayed a good reproducibility and stability to straightforwardly analyze the plasma samples without complicated washing steps, thereby reducing the operating procedures for non-professionals and promoting the detection efficiency and the whole detection process can be completed in 3 min. This approach for carrying out immunoassays can be applied to the detection of CRP in the point-of-care tests.Graphical abstractImage 1Schematic presentation of lateral flow immunoassays (LFIA) based on the use of red fluorescent nanoparticles (RPNs) as signaling labels for the rapid determination of C-reactive protein (CRP) in plasma samples and the core-shell structure of the RPNs.
  • Variation in mitochondrial respiratory capacity and myosin heavy chain
           composition in repeated muscle biopsies
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): 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. Three muscle biopsies (B1, B2 and B3) were obtained from each subject in m. vastus lateralis. Two of the biopsies were from the same leg and one from the other leg.For MRC, intra-biopsy coefficient of variation (CV) was 8.4% and inter-biopsy CV was 13.3%. For MHC type I, IIa and IIx intra-biopsy CV was 8.3, 6.0 and 22.3%, respectively. Inter-biopsy CV for these MHC types were 21.5, 15.4 and 42.0%, respectively. For CS activity intra-biopsy CV was 0.6% and inter-biopsy CV was 15.3%. No differences between B1, B2 and B3 were detected for MRC, CS activity or MHC composition.
  • Development of peptide-based chemiluminescence enzyme immunoassay (CLEIA)
           for diagnosis of dengue virus infection in human
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): 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. 176 serum samples were used to study the presence of anti-DENV IgM antibodies to evaluate the diagnostic ability of IgM-CLEIA. Receiver operating characteristic curve (ROC) was used to estimate the diagnostic cut-off value. The sensitivity and the specificity reached 82.5% and 94.6% respectively when peptide E1 was used, but declined to 79.2% and 92.9% respectively when peptide E7 was used. Therefore, the combination of E1 and E7 was used to improve the sensitivity and the specificity to 85.0% and 96.4% respectively in 1.5 h assay time, providing a potentially practical use for the diagnosis of DENV infections in patients' serum.
  • Rapid method for an enhanced recovery of biologically active human
           phospholipid scramblase1 from inclusion bodies
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): 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. This study aims to optimize and enhance the expression and purification of active recombinant hPLSCR1 by various strategies. Additives including stabilizers and detergents were added during cell lysis to improve the recovery of soluble hPLSCR1. Five E. coli strains, BL21 (DE3), C43 (DE3), Rosetta, BL21-CodonPlus-RP, and BL21 (DE3) pLysS were screened for maximum yield of soluble protein but localized in IBs. To recover hPLSCR1 from IBs, different additives were added of which, 0.3% N-lauroyl sarcosine (NLS) recovered ∼50% of bioactive hPLSCR1 from IBs. E. coli C43 (DE3) gave higher yields of purified protein (7.76 mg/g cell) followed by E. coli BL21 (DE3) pLysS (5.87 mg/g cell). This report describes a rapid and efficient method for solubilizing membrane proteins from inclusion bodies with a higher recovery.
  • Selection of DNA aptamers to Streptococcus pneumonia and fabrication of
           graphene oxide based fluorescent assay
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Abdullah Tahir Bayraç, Sultan Ilayda Donmez Pneumococci are one of the leading causes of infections throughout the world causing problems mainly in children, elderly, and immune-deficient patients. In recent years antibiotic resistant Streptococcus pneumoniae strains become widespread. Therefore simple, rapid, and specific detection methods are needed for public health. In this study, DNA aptamer probes against S. pneumoniae were selected using bacterial Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and these probes were integrated in to a graphene oxide (GO) based fluorescent assay. Among the tested aptamers three candidates Lyd-1, Lyd-2 and Lyd-3 showed Kd values of 844.7 ± 123.6, 1984.8 ± 347.5, and 661.8 ± 111.3 nM, respectively. These candidates showed binding affinity to S. pneumoniae and no specific binding to the bacteria used in negative selection. The binding of aptamers were showed by fluorescence spectroscopy and flow cytometry. GO based label-free fluorescent assay developed using Lyd-3 aptamer had a unique detection limit of 15 cfu mL−1. Thus we believe that the selected aptamers and fabricated GO based assay has potential to be used in the detection of S. pneumoniae. Selected aptamers selectively bind to S. pneumonia with anti-pneumococcal potential and holds great potential to be used as molecular probes for identifying and targeting.
  • Development of a mass spectrometry-based tryptophan 2, 3-dioxygenase assay
           using liver cytosol from multiple species
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Jing Wang, Ryan H. Takahashi, Kevin DeMent, Amy Gustafson, Jane R. Kenny, Simon G. Wong, Zhengyin Yan A novel and rapid method to determine the potency of inhibitors for tryptophan 2, 3-dioxygenase (TDO2) activities in human and preclinical species was successfully developed and validated utilizing LC-MS/MS. Previously reported TDO2 activity assays are resource intensive, requiring cloning and overexpression of TDO2. Here, we demonstrated that liver cytosol contained sufficient active TDO2 for evaluating the potency of TDO2 inhibitors across multiple species. TDO2 expression in human cytosol was estimated by LC-MS/MS to be 41 pmoL/mg cytosolic protein, with similar levels in dogs and monkeys, whereas mice and rats had 9.6 and 5.0-fold greater expression, respectively. Reaction conditions for TDO2-mediated conversion of l-tryptophan to kynurenine were optimized. Marked differences in kinetic parameters and inhibition potency were observed in TDO2 across species, with different Km values in dog (0.055 mM), monkey (0.070 mM), human (0.19 mM), mouse (0.32 mM) and rat (0.36 mM). Subsequently, IC50 values were determined for a series of TDO2 inhibitors in liver cytosol of five species, and good agreement with the literature values was observed for human enzyme. Taken together, these data indicate that TDO2 inhibition can be rapidly determined in readily available hepatic cytosol to assess potential species differences in potency.
  • Expression and purification of nuclease-free protocatechuate
           3,4-dioxygenase for prolonged single-molecule fluorescence imaging
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Gayan Senavirathne, Miguel A. Lopez, Ryan Messer, Richard Fishel, Kristine E. Yoder Single-molecule (SM) microscopy is a powerful tool capable of visualizing individual molecules and events in real time. SM imaging may rely on proteins or nucleic acids labelled with a fluorophore. Unfortunately photobleaching of fluorophores leads to irreversible loss of signal, impacting the collection of data from SM experiments. Trace amounts of dissolved oxygen (O2) are the main cause of photobleaching. Oxygen scavenging systems (OSS) have been developed that decrease dissolved O2. Commercial OSS enzyme preparations are frequently contaminated with nucleases that damage nucleic acid substrates. In this protocol, we purify highly active Pseudomonas putida protocatechuate 3,4-dioxygenase (PCD) without nuclease contaminations. Quantitation of Cy3 photostability revealed that PCD with its substrate protocatechuic acid (PCA) increased the fluorophore half-life 100-fold. This low cost purification method of recombinant PCD yields an enzyme superior to commercially available OSS that is effectively free of nuclease activity.
  • On-chip membrane protein cell-free expression enables development of a
           direct binding assay: A curious case of potassium channel KcsA-Kv1.3
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Amit Vaish, Shaobin Guo, Richard M. Murray, Peter J. Grandsard, Qing Chen Despite the significant role integral membrane proteins (IMPs) play in the drug discovery process, it remains extremely challenging to express, purify, and in vitro stabilize them for detailed biophysical analyses. Cell-free transcription-translation systems have emerged as a promising alternative for producing complex proteins, but they are still not a viable option for expressing IMPs due to improper post-translational folding of these proteins. We have studied key factors influencing in vitro folding of cell-free-expressed IMPs, particularly oligomeric proteins (i.e., ion channels). Using a chimeric ion channel, KcsA-Kv1.3 (K-K), as a model IMP, we have investigated several physiochemical determinants including artificial bilayer environments (i.e., lipid, detergent) for K-K in vitro stabilization. We observed that fusion of a ‘superfolder’ green fluorescent protein (sfGFP) to K-K as a protein expression reporter not only improves the protein yield, but surprisingly facilitates the K-K tetramer formation, probably by enhancing the solubility of monomeric K-K. Additionally, anionic lipids (i.e., DMPG) were found to be essential for the correct folding of cell-free-expressed monomeric K-K into tetramer, underscoring the importance of lipid-protein interaction in maintaining structural-functional integrity of ion channels. We further developed methods to integrate cell-free-expressed IMPs directly onto a biosensor chip. We employed a solid-supported lipid bilayer onto the surface plasmon resonance (SPR) chip to insert nascent K-K in a membrane. In a different approach, an anti-GFP-functionalized surface was used to capture in situ expressed K-K via its sfGFP tag. Interestingly, only the K-K-functionalized capture surface prepared by the latter strategy was able to interact with K-K's small binding partners. This generalizable approach can be further extended to other membrane proteins for developing direct binding assays involving small ligands.Graphical abstractImage 1
  • Partially 13C-labeled mouse tissue as reference for LC-MS based
           untargeted metabolomics
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Frederik Dethloff, Christoph Bueschl, Hermann Heumann, Rainer Schuhmacher, Christoph W. Turck The inclusion of stable isotope-labeled reference standards in the sample is an established method for the detection and relative quantification of metabolic features in untargeted metabolomics. In order to quantify as many metabolites as possible, the reference should ideally include the same metabolites in their stable isotope-labeled form as the sample under investigation. We present here an attempt to use partially 13C-labeled mouse material as internal standard for relative metabolite quantification of mouse and human samples in untargeted metabolomics.We fed mice for 14 days with a13C-labeled Ralstonia eutropha based diet. Tissue and blood amino acids from these mice showed 13C enrichment levels that ranged from 6% to 75%. We used MetExtract II software to automatically detect native and labeled peak pairs in an untargeted manner. In a dilution series and with the implementation of a correction factor, partially 13C-labeled mouse plasma resulted in accurate relative quantification of human plasma amino acids using liquid chromatography coupled to mass spectrometry, The coefficient of variation for the relative quantification is reduced from 27% without internal standard to 10% with inclusion of partially 13C-labeled internal standard.We anticipate the method to be of general use for the relative metabolite quantification of human specimens.Graphical abstractImage 1
  • Optimization of the ninhydrin reaction and development of a multiwell
           plate-based high-throughput proline detection assay
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Mi Rha Lee, Cheol Soo Kim, Tusan Park, Young-Soo Choi, Kyeong-Hwan Lee We developed a high-throughput technique for highly sensitive measurement of trace amounts of proline, an indicator of drought stress in plants, using an optimized proline-ninhydrin reaction. In order to do this, proline detection time was minimized by omitting phosphoric acid from the ninhydrin reagent. Chromophore extraction using toluene was also omitted, thus lowering the risks to environment and human health, and allowing the use of readily available polystyrene plates. Proline detection sensitivity was assessed based on the concentration of sulfosalicylic acid in the solution, which indicated that 1% sulfosalicylic acid yielded the best sensitivity and linearity. These findings were applied to a multiwell plate-based multiplex analysis using a dry oven for the simultaneous analysis of a large number of drought-stressed plant samples with trace amounts of proline. The results showed that proline could be effectively detected in plants grown in soil with water content under 5%, demonstrating its potential for diagnosing drought early. The proposed multiwell plate-based multiplex assay is expected to be useful in manifold agricultural applications.
  • Advancements in electrochemical DNA sensor for detection of human
           papilloma virus - A review
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Elisa Rasouli, Zohreh Shahnavaz, Wan Jefrey 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. In such electrochemical biosensors, a single-strand DNA is immobilized onto an electrically conducting surface and the changes in electrical parameters due to the hybridization on the electrode surface are measured. This review covers the recent developments in electrochemical DNA biosensors for the detection of HPV virus. Due to the several advantages of electrochemical DNA biosensors, their applications have witnessed an increased interest and research focus nowadays.Graphical abstractImage 1
  • Isothermal amplification using modified primers for rapid electrochemical
           analysis of coeliac disease associated DQB1*02 HLA allele
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Sallam Al-Madhagi, Hamdi Joda, Miriam Jauset-Rubio, Mayreli Ortiz, Ioanis Katakis, Ciara K. O'Sullivan DNA biosensors are attractive tools for genetic analysis as there is an increasing need for rapid and low-cost DNA analysis, primarily driven by applications in personalized pharmacogenomics, clinical diagnostics, rapid pathogen detection, food traceability and forensics. A rapid electrochemical genosensor detection methodology exploiting a combination of modified primers for solution-phase isothermal amplification, followed by rapid detection via hybridization on gold electrodes is reported. Modified reverse primers, exploiting a C18 spacer between the primer-binding site and an engineered single stranded tail, are used in a recombinase polymerase amplification reaction to produce an amplicon with a central duplex flanked by two single stranded tails. These tails are designed to be complementary to a gold electrode tethered capture oligo probe as well as a horseradish peroxidase labelled reporter oligo probe. The time required for hybridization of the isothermally generated amplicons with each of the immobilized and reporter probes was optimised to be 2 min, in both cases. The effect of amplification time and the limit of detection were evaluated using these hybridization times for both single stranded and double stranded DNA templates. The best detection limit of 70 fM for a ssDNA template was achieved using 45 min amplification, whilst for a dsDNA template, just 30 min amplification resulted in a slightly lower detection limit of 14 fM, whilst both 20 and 45 min amplification times were observed to provide detection limits of 71 and 72 fM, respectively, but 30 and 45 min amplification resulted in a much higher signal and sensitivity. The genosensor was applied to genomic DNA and real patient and control blood samples for detection of the coeliac disease associated DQB1*02 HLA allele, as a model system, demonstrating the possibility to carry out molecular diagnostics, combining amplification and detection in a rapid and facile manner.
  • l-arginine+methylation+index&rft.title=Analytical+Biochemistry&rft.issn=0003-2697&">GC-MS quantification of urinary symmetric dimethylarginine (SDMA), a
           whole-body symmetric l-arginine methylation index
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Alexander Bollenbach, Erik Hanff, Bibiana Beckmann, Ruan Kruger, Dimitrios Tsikas Circulating and excretory NG,N´G-dimethyl-l-arginine (symmetric dimethylarginine, SDMA) and NG,NG-dimethyl-l-arginine (asymmetric dimethylarginine, ADMA) are cardiovascular risk factors. Despite close chemical structures, the gas chromatography-mass spectrometry (GC-MS) measurement of SDMA is remarkably more difficult than that of ADMA for as yet unknown reasons. Here, we describe an improved GC-MS method for the quantitative determination of SDMA in human urine using commercially available NG,N´G-di-[2H3]methyl-l-arginine (d6-SDMA) as internal standard. The method is based on a single derivatization step with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) to N,N,N,O-tetrakis-pentafluoropropionyl derivatives, electron-capture negative-ion chemical ionization and selected-ion monitoring of the mass-to-charge (m/z) ions of m/z 456 for SDMA and m/z 462 for d6-SDMA.
  • Modified parallel strategies for preparation of heteroduplex plasmids for
           in vitro mismatch repair assays
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Gaurav G. Shimpi, Sergio Vargas, Gert Wörheide We present efficient and reproducible parallel strategies for preparing large quantities of pure heteroduplex plasmids containing defined mismatches. The strategies described involve the use of synthetic oligonucleotides, the commercially available pGEM-T plasmid, and nicking enzymes to prepare prerequisite ssDNA. Alternatively, bacterial packaging cell lines containing an engineered phagemid construct to produce ssDNA without the need of a helper phage were utilized, hence providing added flexibility and choice. These integrated approaches help to construct different mismatch substrates of choice in large quantities, thus enhancing the usability of mismatch repair assays and extending their range and accessibility to wider research groups.
  • Two-dimensional liquid chromatography-mass spectrometry for the
           characterization of modified oligonucleotide impurities
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Stilianos G. Roussis, Isaiah Cedillo, Claus Rentel A 2D-LC system coupled with a TOF mass spectrometer has been evaluated for its capabilities to provide enhanced characterization of oligonucleotide impurities. To address loop limitations in the total volume (40 μL) sampled across 1D peaks, a column trap was incorporated between the 1D and 2D columns. The main advantages of the column trap include reduction of the total number of sequential 2D runs required to fully sample broad 1D peaks, and most importantly, reduction of the error in quantitative determination of the components in broad 1D peaks by avoiding the numerical stitching of data from several 2D runs. Comprehensive RP x IP provided orthogonal separation despite its lower 1D resolution. In contrast, IP x IP did not provide orthogonal separation. RP x IP using the direct on-line extended heart-cutting system with the column trap showed additional benefits, in the elimination of off-line fractionation and sample handling errors and was successfully applied in a pH stability study of a crude oligonucleotide. SAX x IP successfully separated the isobaric “n+16” doublet of the “n + O” and “[n + S-O]” impurity species, a feat not currently possible by mass spectrometry alone or 1D-LC, demonstrating the importance of the added capabilities of the 2D-LC approach.Graphical abstractImage 1
  • Surveying GPCR solubilisation conditions using surface plasmon resonance
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Iva Hopkins Navratilova, Tonia Aristotelous, Louise E. Bird, Andrew L. Hopkins Biophysical screening techniques, such as surface plasmon resonance, enable detailed kinetic analysis of ligands binding to solubilised G-protein coupled receptors. The activity of a receptor solubilised out of the membrane is crucially dependent on the environment in which it is suspended. Finding the right conditions is challenging due to the number of variables to investigate in order to determine the optimum solubilisation buffer for any given receptor. In this study we used surface plasmon resonance technology to screen a variety of solubilisation conditions including buffers and detergents for two model receptors: CXCR4 and CCR5. We tested 950 different combinations of solubilisation conditions for both receptors. The activity of both receptors was monitored by using conformation dependent monoclonal antibodies and the binding of small molecule ligands. Despite both receptors belonging to the chemokine receptor family they show some differences in their preference for solubilisation conditions that provide the highest level of binding for both the conformation dependent antibodies and small molecules. The study described here is focused not only on finding the best solubilisation conditions for each receptor, but also on factors that determine the sensitivity of the assay for each receptor. We also suggest how these data about different buffers and detergents can be used as a guide for selecting solubilisation conditions for other membrane proteins.
  • An efficient method to evaluate experimental factor influence on in vitro
           binding of aptamers
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Donglin Diao, Na Qiao, Xiao Wu, Jiyuan Li, Xinhui Lou Nucleic acid-based aptamers are promising alternative to antibodies, however, their selection process (SELEX) is challenging. A number of simulations and few experiments have been reported offering insights into experimental factors (EFs) that govern the effectiveness of the selection process. Though useful, these previous studied were either lack of experimental confirmation, or considered limited EFs. A more efficient experimental method is highly desired. In this study, we developed a fast method that is capable to quantitatively probe the influence of multiple EFs. Based on the fact that the aptamer enrichment efficiency is highly affected by background binding, the binding ratio between the numbers of specific aptamer binders and nonspecific (unselected library) binders per bead was used to quantitatively evaluate EF effects. Taking thrombin and streptavidin as models, three previously studied EFs (surface coverage, buffer composition, and DNA concentration) and four never-studied ones (surface chemistry, heat treatment, elution methodology and pool purity) were investigated. The EFs greatly affected binding ratio (ranging from 0.03 ± 0.03 to 14.60 ± 2.30). The results were in good agreement with the literature, suggesting the good feasibility of our method. Our study provides guidance for the choice of EFs not only for aptamer selection, but also for binding evaluation of aptamers.
  • Imaging DNA single-strand breaks generated by reactive oxygen species
           using a liquid crystal-based sensor
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Hyeong Jin Kim, Chang-Hyun Jang DNA single-strand breaks (SSBs) have attracted much interest since they are highly related to carcinogenesis and ageing. Herein, we report a new liquid crystal (LC)-based sensor for the detection of DNA SSBs generated by reactive oxygen species (ROS) created from the Fenton reaction. The adsorption of single-stranded DNA (ssDNA) onto the cationic surfactant-laden aqueous/LC interface interferes with the surfactant layer, inducing a planar orientation of the LCs. However, the DNA SSBs generated by the Fenton reaction-produced ROS result in a decrease of the electrostatic interactions between the ssDNA and cationic surfactant molecules, causing rearrangement of the surfactant layer and reorientation of the LCs back to a homeotropic alignment. The changes in orientation of the LCs caused by the DNA SSBs are simply converted and observed as a shift from a bright optical image of the LCs to a dark one under a polarized light microscope. With this simple LC-based approach, the DNA SSBs could be detected more effectively and rapidly without any complex instrumentation or intricate processes. Therefore, our research provides a novel strategy for the detection of DNA damage as well as better insight into the DNA-damaging process.Graphical abstractImage 1
  • Coomassie does it (better): A Robin Hood approach to total protein
    • Abstract: Publication date: 1 September 2018Source: Analytical Biochemistry, Volume 556Author(s): Nour Noaman, Jens R. Coorssen Quantitative comparative proteomics require accurate and reproducible assessments of total protein concentration. We report a straightforward, cost-effective adaptation of an established commercial method for total protein quantification (EZQ™), utilising non-proprietary materials and colloidal Coomassie Brilliant Blue (cCBB) staining to achieve greater reproducibility, equal sensitivity, and optimal linearity of signal within a practical concentration range for proteins in common solubilisation buffers (i.e. for isoelectric focussing and/or SDS-PAGE). This method provided more accurate and precise determinations of total protein concentration in human serum prepared for two-dimensional gel electrophoresis, indicating it may be better suited as the lead-in to most quantitative proteomic analyses.
  • Determination of glyphosate residues in Egyptian soil samples
    • Abstract: Publication date: Available online 10 July 2018Source: Analytical BiochemistryAuthor(s): 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. The L'ELISA used succinic anhydride to derivatize glyphosate, which mimics the epitopic attachment of glyphosate to thyroglobulin. L'ELISA recognized the derivatized glyphosate with a limit of detection (LOD) of 0.8 ng g−1 and sensitivity (IC50 value) of 0.018 μg g−1. The recovery values of the spiked soil samples with different concentrations of glyphosate were in the range of 87.4–97.2%. Good correlation was achieved between L'ELISA and conventional high-pressure liquid chromatography (HPLC) with fluorescence detection. This study demonstrated the utility and convenience of the sensitive, simple, practical and cost-effective L'ELISA method for glyphosate analysis in soil samples. Also, it is ideal for rapid screening of a large number of environmental samples.
  • Pre-analytical stability of methionine-enkephalin and leucine-enkephalin
           in human plasma
    • Abstract: Publication date: Available online 4 July 2018Source: Analytical BiochemistryAuthor(s): 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.The method with a limit of quantitation of 10 pg/mL showed good reproducibility with excellent accuracy and precision, and was linear up to 2000 pg/mL. An extensive evaluation of the pre-analytical stability of these peptides in human blood was carried out to ensure an adequate sample collection procedure to obtain reliable results in the analysis of clinical samples.
  • A surface plasmon resonance based inhibition immunoassay for measurement
           of steroid hormones
    • Abstract: Publication date: Available online 28 June 2018Source: Analytical BiochemistryAuthor(s): Yong Cao, Mark T. McDermott Quantitative measurement of small-molecule metabolites is now emerging as an effective way to link the metabolite profile to disease state. Surface plasmon resonance (SPR) is a sensing platform that has demonstrated applicability for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. Herein, we utilized an indirect detection format and developed an inhibition immunoassay for the quantitative measurement of 17β-estradiol (E2) using SPR. One competitor, BSA-E2 conjugate, was immobilized to the SPR chip via the reaction between the primary amino group of the conjugate and the succinimide group (NHS) introduced by the formation of a thiol-NHS monolayer on gold surface. Free E2 molecules compete with BSA-E2 on chip surface for binding sites provided by a monoclonal anti-E2 antibody. It was found the binding affinity of the antibody to BSA-E2 conjugate increases with decreasing surface coverage of BSA-E2 conjugate. Under optimal conditions, a sigmoidal calibration curve with a negative slope and a dynamic range from 10 pM to 2 nM was generated. The detection limit of the immunoassay is estimated to be 0.3 pM. Moreover, the immunoassay exhibits high specificity for E2 detection using estrone (E1) as a potential interference.Graphical abstractImage 1
  • A novel electrochemical DNA biosensor for Ebola virus detection
    • Abstract: Publication date: Available online 14 June 2018Source: Analytical BiochemistryAuthor(s): Hoda Ilkhani, Siamak Farhad The aim of this study was to fabricate a novel electrochemical-based DNA-sensing device for Ebola virus DNA diagnostic by an enzyme-amplified detection, which improves the sensitivity and selectivity of the sensor. A thiolated DNA capture probe sequence was immobilized on the screen printed electrode surface and hybridized with biotinylated target strand DNA for the fabrication of Ebola DNA-sensing devices. Prior to the electrochemical detection of the enzymatic product by differential pulse voltammetry (DPV) method, the biotinylated hybrid was labelled with a streptavidin-alkaline phosphate conjugate on the surface of the working electrode. All the experiment steps were optimized using electrochemical impedance Spectroscopy (EIS) and the optimum condition for biosensor fabrication was achieved. The last step, the selectivity, reproducibility and sensitivity of fabricated electrochemical DNA biosensor was obtained.
  • Amplified FRET based CA15-3 immunosensor using antibody functionalized
           luminescent carbon-dots and AuNPs-dendrimer aptamer as donor-acceptor
    • Abstract: Publication date: Available online 14 June 2018Source: Analytical BiochemistryAuthor(s): Somayeh Mohammadi, Abdollah Salimi, Somayeh Hamde Qaddareh We proposed an amplified FRET immunosensing for detection of CA15-3 tumor marker by highly biospecific interactions between CA 15–3 antigen and the corresponding antibody and aptamer. In this sandwich type immunoassay, CA15-3 antibody-functionalized carbon dots and AuNPs labeled PAMAM-Dendrimer/aptamer were used as donor/acceptor, respectively. When CA 15-3 Ag was added to homogenous immunoassay, the strong complex interaction between CA15-3 Ab-CA15-3 Ag- aptamer caused in more coming closer carbon dot and AuNPs and more decreasing fluorescence signal. The decreased fluorescence intensity was linear at three ranges including in concentration range 1.1 μUmL−1 to 16 μU mL−1 with regression of R2 = 0.9879, at the concentration range 16 μU mL−1 to 0.163 mU mL−1 with regression of R2 = 0.9944 and at the concentration range 0.163 mU mL−1 to 5.0 mU mL−1 with regression of R2 = 0.9805. The detection limit of the FRET immunoassay was 0.9 μU mL−1. In addition, this FRET immunosensing is applicable in diluted human serum. The recovery values were in the range of 95.86–96.97% for CA 15-3 Ag in spiked serum sample with RSD lower than 7.3%. The proposed immunoassay could be a valid model for establishing other immunoassays for detection of different cancer tumor markers with relevant antigens and antibodies.Graphical abstractImage 1
  • Investigation of binding characteristics of immobilized toll-like receptor
           3 with poly(I:C) for potential biosensor application
    • Abstract: Publication date: Available online 26 May 2018Source: Analytical BiochemistryAuthor(s): Kristin D. Topping, David G. Kelly Toll-like receptor 3 (TLR3), a pathogen recognition receptor of the innate immune response, recognizes and is activated by double-stranded RNA (dsRNA), which is indicative of viral exposure. A sensor design exercise was conducted, using surface plasmon resonance detection, through the examination of several immobilization approaches for TLR3 as a biorecognition element (BRE) onto a modified gold surface. To examine the TLR3-dsRNA interaction a synthetic analogue mimic, poly (I:C), was used. The interaction binding characteristics were determined and compared to literature data to establish the optimal immobilization method for the TLR3 BRE. A preliminary evaluation of the efficacy of the selected TLR3 surface as a broad-spectrum viral biosensor was also performed. Amine-coupling was found to be the most reliable method for manufacturing repeatable and consistent TLR3 BRE sensor surfaces, although this immobilization schema is not tailored to place the receptor in a spatially-specific orientation. The equilibrium dissociation constant (KD) measured for this immobilized TLR3-poly (I:C) interaction was 117 ± 3.30 pM. This evaluation included a cross-reactivity study using a selection of purified E. coli and synthetic double- and single-stranded nucleic acids. The results of this design exercise and ligand binding study will inform future work towards the development of a broad-spectrum viral sensor device.Graphical abstractImage 1
  • Quantification of Müllerian Inhibiting Substance/Anti-Müllerian Hormone
           polypeptide by isotope dilution mass spectrometry
    • Abstract: Publication date: Available online 6 May 2018Source: Analytical BiochemistryAuthor(s): Gail Whiting, Jackie Ferguson, Min Fang, David Pepin, Patricia Donahoe, Paul Matejtschuk, Chris Burns, Jun X. Wheeler Measurement of serum concentrations of Müllerian inhibiting substance (MIS), also known as anti-Müllerian Hormone (AMH) by immunoassay is gaining clinical acceptance and widespread use for the diagnosis of ovarian conditions and for prediction of the response to ovarian stimulation protocols as part of assisted reproductive therapies. Provision of an International Standard to harmonize immunoassay methods is required. It is desirable for the content of a future International Standard to be assigned in mass units for consistency with the units reported by current methods. Isotope dilution mass spectrometry (IDMS), a physicochemical method with traceability to the SI (Système International d'Unités) unit of mass, is a candidate approach to provide orthogonal data to support this mass assignment.
  • Use of DNA aptamer for sandwich type detection of Listeria
    • Abstract: Publication date: Available online 10 April 2018Source: Analytical BiochemistryAuthor(s): Soo Hwan Suh, Soo Jung Choi, Hari P. Dwivedi, Matthew D. Moore, Blanca I. Escudero-Abarca, Lee-Ann Jaykus A single stranded (ss) DNA aptamer, specific to members of Listeria genus, was used to develop a two-site binding sandwich assay for capture and detection of L. monocytogenes. Antibody-immobilized immunomagnetic beads were used to capture L. monocytogenes, followed by their exposure to the aptamer detector. Detection was achieved by amplification of the cell-bound aptamer by qPCR. The lower limit of detection for the combined assay was 2.5 CFU L. monocytogenes in 500 μl buffer. This is juxtaposed to a detection limit of 2.4 log10 CFU in 500 μl buffer for immunomagnetic separation coupled with qPCR detection of L. monocytogenes targeting the hly gene. When applied to turkey deli meat, after 24 h of non-selective enrichment, the two-site binding sandwich assay showed positive results at initial inoculum levels of 1–2 log10 CFU per 25 g sample. The assay reported here is a novel approach for detection of L. monocytogenes in foods and environmental samples.
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