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Analytical Biochemistry
Journal Prestige (SJR): 0.633
Citation Impact (citeScore): 2
Number of Followers: 177  
  Hybrid Journal Hybrid journal (It can contain Open Access articles)
ISSN (Print) 0003-2697 - ISSN (Online) 1096-0309
Published by Elsevier Homepage  [3162 journals]
  • Crystal violet stains proteins in SDS-PAGE gels and zymograms
    • Abstract: Publication date: Available online 17 November 2018Source: Analytical BiochemistryAuthor(s): Robert G.E. Krause, J.P. Dean Goldring Coomassie brilliant blue R250, an anionic dye is the most popular stain to detect proteins resolved in SDS-PAGE gels. Crystal violet, a cationic dye was found to be versatile and stained proteins in SDS-PAGE gels and in zymograms. Stained proteins can be transferred to nitrocellulose and the stained proteins on the western detected with enzyme coupled antibodies. Staining can be reversed. Staining takes 3 h at RT or 30 min at 60 °C. Crystal violet stained some E. coli high and low molecular weight proteins not stained by Coomassie blue R250. Crystal violet stained down to 16 ng of protein, some five-fold lower than Coomassie blue, though the two stains had a similar linear dynamic range. The staining sensitivity could be increased to 2 ng when crystal violet and Coomassie blue were combined in a double staining/counterion dye formulation. The low concentrations of the dye without a destaining step reduces the costs of the technique and results in a more environmentally friendly stain compared to traditional staining methods.Graphical abstractImage 1
  • Simplified LC/MS assay for the measurement of isolevuglandin protein
           adducts in plasma and tissue samples
    • Abstract: Publication date: Available online 17 November 2018Source: Analytical BiochemistryAuthor(s): Valery N. Yermalitsky, Elena Matafonova, Keri Tallman, Zhuoheng Li, William Zackert, L. Jackson Roberts, Venkataraman Amarnath, Sean S. Davies Isolevuglandins (IsoLGs) are a family of highly reactive 4-ketoaldehydes formed by lipid peroxidation that modify the lysyl residues of cellular proteins. Modification of proteins by IsoLGs have been shown to contribute to disease processes such as the development of hypertension. Accurate quantitation of the extent of protein modification by IsoLGs is essential for understanding the mechanisms whereby these modifications contribute to disease and the efficacy of interventions designed to prevent this modification. The previously described LC/MS assay to quantitate IsoLG protein adducts was extremely labor-intensive and time consuming, and while it offered reasonably low intra-day variation for replicate samples, variation when replicate samples were processed on separate days was significant. These limitations significantly restricted utilization of this approach. We therefore performed a series of studies to optimize the assay. We now report a significantly simplified LC/MS assay for measurement of IsoLG protein adducts with increased sensitivity and lower intra-day and inter-day variability.
  • Label-free drug screening assay multiplexed with an orthogonal
           time-resolved fluorescence labeled assay
    • Abstract: Publication date: Available online 16 November 2018Source: Analytical BiochemistryAuthor(s): László Kiss, Attila Cselenyák, András Visegrády Cell-based assays against cell surface receptor targets are essential in vitro models of target-based drug discovery. At the lead generation phase large-scale functional screening assays monitoring individual cellular readouts detect interactions between the compounds and the predefined pathways but might lack sufficient sensitivity owing to the complexity of downstream signaling pathways. Cellular label-free assays offer advantages over labeled detection approaches as they reflect whole-cell responses without the prerequisite of detecting only a single cellular analyte and introducing additional genetic manipulations in favor of the chosen detection method. The combination of a label-free assay and labeled assays might integrate the advantageous characteristics of both approaches with regards to added pharmacological information and a bigger pool of chemical starting material. Here we report multiplexing of dynamic mass redistribution label-free technology with HTRF-based cAMP detection on an alpha2c adrenergic receptor expressing cell line. Besides describing the challenging assay development work associated with the set goal, a pilot screening campaign on ca. 1600 compounds is also presented. The combined assay demonstrated the ability to detect relevant activities in both readouts. Interpretation of the results as well as an outlook for further possible opportunities and applications are also discussed.
  • Straightforward and rapid method for detection of cyclin-dependent
           kinase-like 5 activity
    • Abstract: Publication date: Available online 14 November 2018Source: Analytical BiochemistryAuthor(s): Syouichi Katayama, Tetsuya Inazu Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase, with its gene mutation leading to a neurodevelopmental disorder. Pathogenic point mutations are mostly observed within the catalytic domain of CDKL5, therefore loss of catalytic activity may be related to disease onset. However, this hypothesis has rarely been demonstrated. Here, we report an efficient method for detecting CDKL5 activity. Appropriately, CDKL5 underwent autophosphorylation following expression in Escherichia coli, with autophosphorylated CDKL5 detected as a band shift by phos-tag SDS–PAGE, without enzyme purification. Thus, this protocol is useful for examining the relationship between disease-causing mutations and their activity.
  • Enzyme kinetics in crowded solutions from isothermal titration calorimetry
    • Abstract: Publication date: Available online 12 November 2018Source: Analytical BiochemistryAuthor(s): Ksenia Maximova, Jakub Wojtczak, Joanna Trylska Isothermal titration calorimetry (ITC) is a universal technique that directly measures the heat absorbed or released in a process. ITC is typically used to determine thermodynamic parameters of association of molecules without the need to label them. However, ITC is still rarely applied to study chemical reactions catalyzed by enzymes. In addition, these few studies of enzyme kinetic measurements that have been performed were in diluted solutions. Yet, to estimate realistic kinetic parameters, we have to account for the fact that enzymatic reactions in cells occur in a crowded environment because cells contain 200–400 g/L of macromolecular crowders such as proteins, ribosomes and lipids. Thus we expanded the ITC application for solutions mimicking the cellular environment by adding various macromolecular crowders. We investigated how these crowders affect the kinetics of trypsin-catalyzed reactions and determined the Michaelis-Menten parameters for hydrolysis of two trypsin substrates: Nα-benzoyl-l-arginine ethyl ester (BAEE) and Nα-benzoyl-dl-arginine β-naphthylamide (BANA). Since ITC enables investigations of complex and turbid solutions with label-free reagents, it seems a perfect technique for kinetic analyses in crowded solutions. ITC also offers the opportunity to control enzyme-crowder and substrate-crowder interactions.
  • Electrochemiluminescent immunosensor for prostate specific antigen based
           upon luminol functionalized platinum nanoparticles loaded on graphene
    • Abstract: Publication date: Available online 12 November 2018Source: Analytical BiochemistryAuthor(s): Malik Saddam Khan, Wenjuan Zhu, Asghar Ali, Shah Masood Ahmad, Xiaojian Li, Lei Yang, Yaoguang Wang, Huan Wang, Qin Wei A novel label-free electrochemiluminescent (ECL) immunosensor based upon luminol functionalized platinum nanoparticles loaded on graphene sheets (Lu-Pt@GS) as sensing platform was fabricated for highly sensitive and selective determination of prostate specific antigen (PSA). In this work, for the first time luminol was employed as both ECL luminescence reagent and reductants to in-situ reduce H2PtCl6 forming Pt NPs on surface of GS. A great deal of luminol could be attached onto the surface of Pt NPs within the reduction process, which can generate strong ECL emission. Pt NPs not only could enhance ECL signals of luminol but supply active sites for the immobilization of PSA antibodies with micro friendly environment. For preventing the consecutive reaction among luminol and H2O2, single-step cycle pulse was adopted, resulting in stable and strong ECL signals. Under optimized experimental conditions, the proposed ECL immunosensor acquired a wide linear range of 1 pg/mL to 10 ng/mL with a relatively low detection limit of 0.3 pg/mL for PSA. Furthermore, due to high sensitivity, simplicity and cost-effectiveness, the designed immunosensor provides a new method for detecting other important biomarkers in clinical analysis.
  • A fluorescent probe for hypochlorite with colorimetric and fluorometric
           characteristics and imaging in living cells
    • Abstract: Publication date: Available online 12 November 2018Source: Analytical BiochemistryAuthor(s): Cunjie Hu, Jianping Li, Liqiang Yan An excellent fluorescent probe with “turn on” phenomenon for sensitive monitoring hypochlorite (ClO−) was prepared using the mild condensation reaction between coumarin and hydroxylamine (NH2OH). The probe possessed potent selectivity to hypochlorite (ClO−) with obvious color changes from yellow to light yellow and green to blue fluorescence emission, which could be noticed by the naked eye. Moreover, the probe has been succeeded in imaging ClO− in living A549 cells and thus has the potential prospect in the visual detection of intracellular ClO−.Graphical abstractImage 1
  • A cyclometalated iridium(III) complex-based fluorescence probe for
           hypochlorite detection and its application by test strips
    • Abstract: Publication date: Available online 11 November 2018Source: Analytical BiochemistryAuthor(s): Linxi Hou, Mingqin Shangguan, Zhen Lu, Sili Yi, Xingzong Jiang, Heng Jiang A new cyclometalated iridium(III) complex-based fluorescence probe (IrCN) for hypochlorite (ClO−) has been synthesized and characterized. The probe displayed nonfluorescent around 577 nm, while a 54-fold enhancement in fluorescence emission intensity was observed after the addition of ClO− due to the removal of C=N isomerization effect. Such “turn-on” fluorescence probe worked excellently in wide pH range (5–12) with short response time (
  • Analysis of butterfly reproductive proteins using capillary
           electrophoresis and mass spectrometry
    • Abstract: Publication date: Available online 10 November 2018Source: Analytical BiochemistryAuthor(s): Maria Khihon Rokhas, Johanna Liljestrand Rönn, Christer Wiklund, Åsa Emmer A method for analysis of proteins from spermatophores transferred from male to female Pieris napi butterflies during mating has been developed. The proteins were solubilized from the dissected spermatophores using different solubilization agents (water, methanol, acetonitrile and hexafluoroisopropanol). Capillary electrophoresis (CE) analysis was performed using an acidic background electrolyte containing a fluorosurfactant to avoid protein-wall adsorption, and to increase separation performance. The samples were also analyzed with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), in a lower m/z range (1000–6000) and a higher m/z range (6000–12000). Solubilization with different solvents and the use of alternative matrices gave partly complementary profiles.
  • MIonSite: Ligand-specific prediction of metal ion-binding sites via
           enhanced AdaBoost algorithm with protein sequence information
    • Abstract: Publication date: Available online 9 November 2018Source: Analytical BiochemistryAuthor(s): Liang Qiao, Dongqing Xie Accurately targeting metal ion-binding sites solely from protein sequences is valuable for both basic experimental biology and drug discovery studies. Although considerable progress has been made, metal ion-binding site prediction is still a challenging problem due to the small size and high versatility of the metal ions. In this paper, we develop a ligand-specific predictor called MIonSite for predicting metal ion-binding sites from protein sequences. MIonSite first employs protein evolutionary information, predicted secondary structure, predicted solvent accessibility, and conservation information calculated by Jensen-Shannon Divergence score to extract the discriminative feature of each residue. An enhanced AdaBoost algorithm is then designed to cope with the serious imbalance problem buried in the metal ion-binding site prediction, where the number of non-binding sites is far more than that of metal ion-binding sites. A new gold-standard benchmark dataset, consisting of training and independent validation subsets of Zn2+, Ca2+, Mg2+, Mn2+, Fe3+, Cu2+, Fe2+, Co2+, Na+, K+, Cd2+, and Ni2+, is constructed to evaluate the proposed MIonSite with other existing predictors. Experimental results demonstrate that the proposed MIonSite achieves high prediction performance and outperforms other state-of-the-art sequence-based predictors. The standalone program of MIonSite and corresponding datasets can be freely downloaded at for academic use.
  • Analysis of oxygen-18 labeled phosphate to study positional isotope
           experiments using LC-QTOF-MS
    • Abstract: Publication date: Available online 9 November 2018Source: Analytical BiochemistryAuthor(s): Sujatha Chilakala, Iteen Cheng, Ireen Lee, Yan Xu A method is proposed in this paper for the determination of oxygen-18 labeled phosphate so that positional isotope experiments using sensitive and rapid liquid chromatography–QTOF–mass spectrometry (LC-QTOF-MS) experiments can be carried out. The positional isotope exchange technique is a useful tool in understanding the mechanisms and kinetics of many enzyme-catalyzed reactions. Detection of the positions and concentration of these exchanged isotopes is the key. Gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance imaging are commonly used analytical techniques for measurement of 18O/16O, 31P and 15N isotope enrichment. Since these techniques either require a time-consuming derivatization step or have a limited sensitivity, an LC and accurate mass-based method for monitoring 18O/16O exchange was developed and compared with a standard GC-MS method. Our results showed that the LC-QTOF-MS method developed was not only as accurate as the standard GC-MS method, but also a sensitive and robust analytical platform for the simultaneous determination of isotope enrichment and the analysis of positional isotopes without chemical derivation. The LC-QTOF-MS method developed was successfully applied to the measurement of 18O/16O in the reversibility study of ATP hydrolysis by Lon proteases.
  • A modified method for plasmid extraction from Lactobacillus plantarum
           contained lysozyme removal step
    • Abstract: Publication date: Available online 6 November 2018Source: Analytical BiochemistryAuthor(s): F. Yao, X.Y. Xu, Q. Pan Plasmids of Lactobacillus plantarum PC518 cannot be effectively extracted by existing methods. It was studied that the effect of lysozyme treatment and removal on plasmid extraction by 7 protocols. The modified method was compared with a commercial kit using L. plantarum PC518, 410, 9L15, and JS193 and Weissella cibaria M2 as the tested strains. The results suggested that the step of lysozyme removal is the key to improve the efficiency of plasmid extraction. The concentrations of plasmid DNA isolated from the 5 tested strains were increased by 10.6, 9.5, 6, 5.6 and 1.5 times respectively compared with the commercial kit.
  • Development of a quantification method for adenosine in tumors by LC-MS/MS
           with dansyl chloride derivatization
    • Abstract: Publication date: Available online 5 November 2018Source: Analytical BiochemistryAuthor(s): Kelly J. Goodwin, Eric Gangl, Ujjal Sarkar, Petar Pop-Damkov, Natalie Jones, Alexandra Borodovsky, Richard Woessner, Adrian J. Fretland Adenosine is known to be an important signaling molecule in many physiological processes and has recently been shown to be an important molecule in oncology. A fit for purpose method has been developed for the quantification of adenosine in murine tumor samples using pre-column derivatization and liquid chromatography-mass spectrometry (LC-MS/MS). To overcome adenosine quantification challenges, derivatization with dansyl chloride was employed. This derivatization technique, following protein precipitation and liquid-liquid extraction, improved the sensitivity and selectivity of adenosine in tumor samples through the reduction of endogenous interference and matrix effects. This method utilizes a mouse plasma calibration curve, qualified over a range of 0.019 μM–37 μM. The 15 min derivatization incubation time and 1 min chromatographic run time allow for higher throughput. The following established method overcomes challenges associated with the quantification of low molecular weight, polar, endogenous molecules, such as adenosine, using derivatization and LC-MS/MS. With the additional analysis of murine tumors, this method will contribute to the understanding of the impact adenosine plays in the tumor microenvironment and the bearing it has on targeted cancer therapies.
  • Distribution of three isoforms of antimicrobial peptide, chrysophsin-1, -2
           and -3, in the red sea bream, Pagrus (Chrysophrys) major
    • Abstract: Publication date: Available online 4 November 2018Source: Analytical BiochemistryAuthor(s): Takayuki Saitoh, Yasumitsu Seto, Yukichi Fujikawa, Noriaki Iijima We report here a liquid chromatography/electrospray ionization-tandem mass spectrometry assay for the quantification of three isoforms of antimicrobial peptide (AMP), chrysophsin-1, -2 and -3, in the red sea bream, Pagrus (Chrysophrys) major. Chrysophsin-1 was mainly distributed in the pyloric caeca and gills, followed by intestine and stomach. Chrysophsin-2 was detected in the gills and stomach, but chrysophsin-3 was only in the gills. The present procedure is valuable as a general method for simultaneous determination of the level of multiple AMP isoforms in fish tissues, and the data give important information for understanding the significance of each AMP isoform in host defense.
  • Development and comparative analysis of yeast protein extraction protocols
           for mass spectrometry
    • Abstract: Publication date: Available online 2 November 2018Source: Analytical BiochemistryAuthor(s): Lauana Fogaça de Almeida, Leonardo Nazário de Moraes, Lucilene dos Santos Delazari, Guilherme Targino Valente Mass spectrometry is the most used method for protein identification and quantification. Here we developed four protein extraction protocols precisely for mass spectrometry, and we compared with other ones already published. The best protocol developed by us consists on a simple extraction solution, a heat-shock step, and does not use protease inhibitor; moreover, it is the most efficient and uniform among replicates, besides to be safe, cheap and fast. That method also provided the highest number of proteins uniquely identified and allows finding a diversity of protein classes, which their absence is a problem to be avoided.
  • Optimisation of a high-throughput fluorescamine assay for detection of
           N-acyl-L-homoserine lactone acylase activity
    • Abstract: Publication date: Available online 2 November 2018Source: Analytical BiochemistryAuthor(s): Shereen A. Murugayah, Suzanne L. Warring, Monica L. Gerth N-acyl-L-homoserine lactone (AHL) acylases are a well-known group of enzymes that disrupt quorum sensing in Gram-negative bacteria by degrading AHL signalling molecules. This degradation of signalling molecules (termed 'quorum quenching') has potential uses in the prevention or reduction of biofilm formation and/or bacterial infections. Therefore, there is a great deal of interest in the identification and characterisation of quorum quenching enzymes. Here, we present an optimised fluorescamine-based assay for the detection of AHL acylase activity and demonstrate it can be used in a high-throughput screening format.
  • Serological diagnosis of Toxoplasmosis disease using a fluorescent
           immunosensor with chitosan-ZnO-nanoparticles
    • Abstract: Publication date: Available online 28 October 2018Source: Analytical BiochemistryAuthor(s): Victoria Medawar-Aguilar, Claudio F. Jofré, Martín A. Fernández-Baldo, Andrés Alonso, Sergio Angel, Julio Raba, Sirley V. Pereira, Germán A. Messina This article describes a microfluidic LIF immunosensor for the quantitative determination of anti-Toxoplasma gondii IgG (anti-T. gondii) specific antibodies. The serological detection of these antibodies plays a crucial role in the clinical diagnosis of toxoplasmosis. Zinc oxide nanoparticles (ZnO-NPs) obtained by wet chemical procedure were covered with chitosan and then used to conjugate T-gondii antigens into the central microfluidic channel. Serum samples containing anti-T-gondii IgG antibodies were injected into the immunosensor where they interact immunologically with T. gondii antigens. Bound antibodies were quantified by the addition of anti-IgG antibodies labeled whit alkaline phosphatase (ALP). ALP enzymatically converts the non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to soluble fluorescent methylumbelliferone that was measured using excitation at 355 nm and emission at 440 nm. The relative fluorescent response of methylumbelliferone is proportional to the concentration of anti-T. gondii IgG antibodies. The coefficients of variation are less than 4.73% for within-day assays and less than 6.34% for between-day assays. Results acquired by LIF immunosensor agree with those obtained by enzyme-linked immunosorbent assay method, suggesting that the designed sensor represents a promising tool for the quantitative determination of anti-T. gondii IgG antibodies of clinical samples.Graphical abstractImage 1
  • A chemiluminescent dual-aptasensor capable of simultaneously quantifying
           prostate specific antigen and vascular endothelial growth factor
    • Abstract: Publication date: 1 January 2019Source: Analytical Biochemistry, Volumes 564–565Author(s): Junhyun Chong, Hayun Chong, Ji Hoon Lee An aptasensor with guanine chemiluminescence detection was developed for the early diagnosis of prostate cancer with the simultaneous quantifications of prostate specific antigen (PSA) and vascular endothelial growth factor (VEGF). A PSA DNA aptamer conjugated with Texas Red emits red light in guanine chemiluminescence reaction, whereas a VEGF DNA aptamer conjugated with 6-FAM emits green light. The PSA and VEGF aptamers immobilized on the surface of paramagnetic beads mixed with a sample were incubated for 30 min. After the incubation, PSA and VEGF bound with PSA and VEGF aptamers were simultaneously quantified for 20 s using guanine chemiluminescence detections operated with two photomultiplier tubes and two optical filters capable of selectively collecting green or red light. With the increase of PSA concentration, the strength of the red light emitted from Texas Red dropped exponentially, whereas with the increase of VEGF concentration, the green light emitted from 6-FAM was enhanced. The limits of detection were as low as 0.6 ng/ml for PSA and 0.4 ng/ml for VEGF. We confirmed that the dual-aptasensor can be applied as an advanced and new medical device capable of simultaneously quantifying PSA and VEGF to early diagnose prostate cancer with good accuracy, precision, and reproducibility.Graphical abstractImage 1
  • Prediction of membrane protein types by exploring local discriminative
           information from evolutionary profiles
    • Abstract: Publication date: Available online 27 October 2018Source: Analytical BiochemistryAuthor(s): Muhammad Kabir, Muhammad Arif, Farman Ali, Saeed Ahmad, Zar Nawab Khan Swati, Dong-Jun Yu Membrane protein is a pivotal constituent of a cell that exerts a crucial influence on diverse biological processes. The accurate identification of membrane protein types is deeply essential for revealing molecular mechanisms and drug development. Primarily, several traditional methods were exploited to classify these types. However, experimental methods are laborious, time-consuming, and costly due to rapid exploration of uncharacterized protein sequences generated in the postgenomic era. Hence, machine learning-based methods are more indispensable for reliable and fast identification of membrane protein types. A variety of state-of-the-art investigations have been elucidated to improve prediction performance, but predictive validity is still insufficient. Motivated by this, we designed a promising sequential support vector machine based predictor called TargetHMP to predict types of membrane proteins. We captured the local informative features by exploring evolutionary profiles through a novel method called the segmentation-based pseudo position specific scoring matrix (Seg-PsePSSM). TargetHMP attained high accuracy of 94.99%, 93.48%, and 90.36% on the S1, S2, and S3 datasets, respectively, using a vigorous leave-one-out cross-validation test. The results indicate that the performance of the proposed method outperformed prior predictors. We expect that the proposed approach will help research academia in general and pharmaceutical drug discovery in particular.
  • Analysis of glucose-derived amino acids involved in one-carbon and cancer
           metabolism by stable-isotope labeling and gas chromatography mass
    • Abstract: Publication date: Available online 26 October 2018Source: Analytical BiochemistryAuthor(s): Mark L. Sowers, Jason Herring, William Zhang, Hui Tang, Yang Ou, Wei Gu, Kangling Zhang A major hallmark of cancer is a perturbed metabolism resulting in high demand for various metabolites, glucose being the most well studied. While glucose can be converted into pyruvate for ATP production, the serine synthesis pathway (SSP) can divert glucose to generate serine, glycine, and methionine. In the process, the carbon unit from serine is incorporated into the one-carbon pool which makes methionine and maintains S-adenosylmethionine levels, which are needed to maintain the epigenetic landscape and ultimately controlling what genes are available for transcription. Alternatively, the carbon unit can be used for purine and thymidylate synthesis. We present here an approach to follow the flux through this pathway in cultured human cells using stable isotope enriched glucose and gas chromatography mass spectrometry analysis of serine, glycine, and methionine. We demonstrate that in three different cell lines this pathway contributes only 1–2% of total intracellular methionine. This suggests under high extracellular methionine conditions, the predominance of carbon units from this pathway are used to synthesize nucleic acids.
  • An integrative UHPLC-MS/MS untargeted metabonomics combined with
           quantitative analysis of the therapeutic mechanism of Si-Ni-San
    • Abstract: Publication date: Available online 24 October 2018Source: Analytical BiochemistryAuthor(s): Jing Wen, Lina Yang, Feng Qin, Longshan Zhao, Zhili Xiong A UHPLC-MS/MS untargeted serum metabonomic method combined with quantitative analysis of five potential biomarkers in rat serum was developed and validated, to further understand the anti-liver injury effect of Si-Ni-San and its mechanism on liver injury rats in this study. The metabolites were separated and identified on BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using the ACQUITY UHPLC-MS system (Waters Corp., Milford, MA, USA). Principal component analysis (PCA) was used to identify potential biomarkers. Primary potential biomarkers including phenylalanine, tryptophan, Glycochenodeoxycholic acid (GCDCA) and hysophosphatidylcholine (LPC), which were related to amino acid metabolism, lipid metabolism, bile acid biosynthesis and oxidation-antioxidation balance, were found in the untargeted metabonomic research. Moreover, these targeted biomarkers were further separated and quantified in multiple-reaction monitoring (MRM) with positive ionization mode. The proposed method was linear for each analyte with correlation coefficients over 0.99. The intra- and inter-day precision values (relative standard deviation, RSD) were less than 13.1% and accuracy (relative error, RE) was from −9.5% to 10.3% at all quality control (QC) levels. The validated method was successfully applied to study the serum samples of control group, model group, positive control group (silymarin group) and Si-Ni-San group in rats.Graphical abstractImage 1
  • Characterization of residue-specific glutathionylation of CSF proteins in
           multiple sclerosis – A MS-based approach
    • Abstract: Publication date: Available online 24 October 2018Source: Analytical BiochemistryAuthor(s): Deepsikha Srivastava, Gosala Raja Kukkuta Sarma, Delon Snehal Dsouza, Monita Muralidharan, Krishnamachari Srinivasan, Amit Kumar Mandal Reduction of disulfide linkage between cysteine residues in proteins, a standard step in the preanalytical preparation of samples in conventional proteomics approach, presents a challenge to characterize S-glutathionylation of proteins. S-glutathionylation of proteins has been reported in medical conditions associated with high oxidative stress. In the present study, we attempted to characterize glutathionylation of CSF proteins in patients with multiple sclerosis which is associated with high oxidative stress. Using nano-LC/ESI-MS platform, we adopted a modified proteomics approach and a targeted database search to investigate glutathionylation at the residue level of CSF proteins. Compared to patients with Intracranial hypertension, the following CSF proteins: Extracellular Superoxide dismutase (ECSOD) at Cys195, α1-antitrypsin (A1AT) at Cys232, Phospholipid transfer protein (PLTP) at Cys318, Alpha-2-HS-glycoprotein at Cys340, Ectonucleotide pyrophosphate (ENPP-2) at Cys773, Gelsolin at Cys304, Interleukin-18 (IL-18) at Cys38 and Ig heavy chain V III region POM at Cys22 were found to be glutathionylated in patients with multiple sclerosis during a relapse. ECSOD, A1AT, and PLTP were observed to be glutathionylated at the functionally important cysteine residues. In conclusion, in the present study using a modified proteomics approach we have identified and characterized glutathionylation of CSF proteins in patients with multiple sclerosis.Graphical abstractImage 1
  • An accessible and high-throughput strategy of continuously monitoring
           apoptosis by fluorescent detection of caspase activation
    • Abstract: Publication date: Available online 24 October 2018Source: Analytical BiochemistryAuthor(s): Kelly M. Hanson, Jacob N. Finkelstein We present a real-time, high-throughput, and cost-effective method of detecting apoptosis in vitro using a previously developed reagent that detects caspase activation by fluorescence. Current methods of assessing apoptosis fail to account for the dimension of time, and thus are limited in data yielded per sample. This reagent allows real-time detection of apoptosis, but until now has been restricted to a costly automated detection system. Here, we describe apoptosis detection with the Essen Bioscience IncuCyte® Caspase-3/7 Reagent using a multimode microplate reader, a common instrument in biological laboratories, which may be used prior to or in lieu of the automated system. This modified microplate reader apoptosis assay was validated against the established automated system, and was shown to detect a strong dose-response relationship (automated system r2 = 0.9968, microplate reader r2 = 0.9924). We also propose a quick and reliable method of quantifying cell density by Hoechst 33342 nuclear staining in microplates (r2 = 0.8812 between Hoechst signal and cell density). We assert that the dimension of time should not be overlooked, and that the method presented here is an accessible strategy for many researchers due to low startup cost and precise detection of apoptosis in real time.
  • A novel aptamer-based test for the rapid and accurate diagnosis of pleural
    • Abstract: Publication date: Available online 22 October 2018Source: Analytical BiochemistryAuthor(s): Pooja Kumari, Surabhi Lavania, Shaifali Tyagi, Abhijeet Dhiman, Deepak Rath, Divya Anthwal, Rakesh Kumar Gupta, Neera Sharma, A.K. Gadpayle, R.S. Taneja, Lokesh Sharma, Yusra Ahmad, Tarun Kumar Sharma, Sagarika Haldar, Jaya Sivaswami Tyagi Pleural tuberculosis (pTB) is diagnosed by using a composite reference standard (CRS) since microbiological methods are grossly inadequate and an accurate diagnostic test remains an unmet need. The present study aimed to evaluate the utility of Mycobacterium tuberculosis (Mtb) antigen and DNA-based tests for pTB diagnosis. Patients were classified as ‘Definite TB’, ‘Probable TB’ and ‘Non-TB’ disease according to the CRS. We assessed the performance of in-house antigen detection assays, namely antibody-based Enzyme-Linked ImmunoSorbent Assay (ELISA) and aptamer-based Aptamer-Linked Immobilized Sorbent Assay (ALISA), targeting Mtb HspX protein and DNA-based tests namely, Xpert MTB/RIF and in-house devR-qPCR. ROC curves were generated for the combined group of ‘Definite TB’ and ‘Probable TB’ vs. ‘Non-TB’ disease group and cut-off values were derived to provide specificity of ≥98%. The sensitivity of ALISA was ∼93% vs. ∼24% of ELISA (p-value ≤0.0001). devR-qPCR exhibited a sensitivity of 50% vs. ∼22% of Xpert (p-value ≤0.01). This novel aptamer-based ALISA test surpasses the sensitivity criterion and matches the specificity requirement spelt out in the ‘Target product profile’ for extrapulmonary tuberculosis samples by WHO (Sensitivity ≥80%, Specificity 98%). The superior performance of the aptamer-based ALISA test indicates its translational potential to bridge the existing gap in pTB diagnosis.
  • Development of a homogeneous time-resolved fluorescence assay for
           detection of viral double-stranded RNA
    • Abstract: Publication date: Available online 21 October 2018Source: Analytical BiochemistryAuthor(s): Motomichi Fujita, Koji Adachi, Michiaki Nagasawa The group of positive-sense single-stranded RNA ((+) ssRNA) viruses includes many important human pathogens. However, specific antiviral agents are not currently available for many RNA viruses. For screening of antiviral agents, methods that are simple, rapid, and compatible with high-throughput are required. Here, we describe a novel method for measurement of double-stranded RNA using a homogeneous time-resolved fluorescence assay. This method allowed detection of human rhinovirus (HRV), enterovirus, coxsackievirus, and murine norovirus. Furthermore, this method detected antiviral activity of a HRV 3C protease inhibitor. The assay may be useful for discovery of antiviral agents against (+) ssRNA viruses.Graphical abstractImage 1
  • Does vacuum centrifugal concentration reduce yield or quality of nucleic
           acids extracted from FFPE biospecimens'
    • Abstract: Publication date: Available online 19 October 2018Source: Analytical BiochemistryAuthor(s): Ignacio Sánchez, Fay Betsou, William Mathieson Vacuum centrifugal (SpeedVac) concentration is commonly applied to nucleic acids extracted from formalin-fixed paraffin-embedded (FFPE) sections, but with an unknown impact. We concentrated DNA and RNA from FFPE biospecimens using different time/temperature SpeedVac combinations of up to 30 min concentration at 45 °C, then used spectrophotometry, spectrofluorometry, RIN, PERM, DV200, qRT-PCR, DIN and the Illumina FFPE QC Assay to assess the changes in quantity, purity and integrity induced by the concentration process. We found the effects of SpeedVac concentration to be inconsequential, but an aliquot of elution buffer should be concentrated for use as the blank in spectrophotometry assays.
  • pDHS-DSET: Prediction of DNase I hypersensitive sites in plant genome
           using DS evidence theory
    • Abstract: Publication date: Available online 17 October 2018Source: Analytical BiochemistryAuthor(s): Shanxin Zhang, Jinhe Lin, Lei Su, Zhiping Zhou Predicting DNase I hypersensitive sites (DHSs) is an essential topic in the field of transcriptional regulatory elements, which provides clues for deciphering the function of noncoding genomic regions. To the best of our knowledge, several computational approaches are currently available for prediction of DHSs in the plant genome, but there is still room for improvement. In the present work, a DS evidence theory-based method was proposed. At first, four sequence-derived feature representation methods, i.e., kmer, reverse complement kmers, mismatch profile, and pseudo dinucleotide composition, were utilized to encode the sequences. Then, four support vector machine based sub-classifiers was built with these sequence-derived features. Finally, the DS evidence theory was applied to obtain the final results by fusing the outputs of these four base learners. In this work, to solve the data imbalance problem, a bidirectional synthetic sampling algorithm was proposed to obtain balanced dataset during training the models. In the computational experiments, the proposed method achieved accuracy up to 88.85%, and 88.60% in Arabidopsis thaliana and rice genome, respectively. Compared with existing DHSs prediction methods, the proposed method can achieve comparable or better performances, suggesting the usefulness of the method for DHSs prediction.
  • Development and validation of analytical methodologies for the
           quantification of PCK3145 and PEG-PCK3145 in mice
    • Abstract: Publication date: Available online 17 October 2018Source: Analytical BiochemistryAuthor(s): Charikleia Danika, Mohamed A. El Mubarak, Iliana Leontari, Gregory B. Sivolapenko PCK3145 is an anti-metastatic synthetic peptide against prostate cancer. The objective of the study is to develop and validate novel and sensitive methods for the determination of PCK3145 and Pegylated PCK3145 in mouse plasma. An LC–MS/MS method was developed and validated for the determination of PCK3145 giving high sensitivity and linearity in the range of 0.125–4.0 μg/mL. PCK3145 characterised by short half-life, therefore, it was conjugated with the poly ethylene glycol (PEG). However, LC-MS/MS has been more difficult to apply for the quantitative analysis of PEGylated peptides due to the large size. A UHPLC-UV method was developed and validated for the determination of PEG-PCK3145, with linearity of 0.05–2.0 mg/mL. In order to further improve the sensitivity for the detection of PEG-PCK3145, an indirect ELISA method was used. It was found that this method was capable of detecting PCK3145 through the quantification of PEG with excellent sensitivity found at 0.132 ng/mL. The in vitro proteolytic stability of PCK3145 and PEG-PCK3145 in mouse plasma and whole blood was studied by LC-MS/MS and UHPLC, respectively. The LC–MS/MS and ELISA methods can be applied for monitoring levels of PCK3145 in mouse plasma for in vivo pharmacokinetic and bioavailability animal studies.
  • A receptor-based chemiluminescence enzyme linked immunosorbent assay for
           determination of tetracyclines in milk
    • Abstract: Publication date: Available online 16 October 2018Source: Analytical BiochemistryAuthor(s): Ge Wang, Hui Cai Zhang, Jing Liu, Jian Ping Wang This study for the first time reported a receptor based chemiluminescence immunoassay for analysis of tetracyclines. During the experiments, the gene of TetR protein was transformed into Rosetta-gami(DE3) to express a receptor, and its recognition mechanism for 5 tetracyclines was studied by using molecular docking technique. Results showed that hydrophobic interaction and Pi-Pi bond were the main intermolecular forces responsible for TetR-tetracyclines binding, and the D ring was the main receptor binding position in tetracyclines molecules. Then the receptor was used as recognition reagent to develop a direct competitive enzyme linked immunosorbent assay for determination of the 5 drugs in milk, and the light signal was induced with 4-(imidazol-1-yl)phenol enhanced luminol-H2O2 system. The limits of detection for the 5 drugs in milk were in the range of 5–16 pg/mL, and the recoveries from the standards fortified blank milk were in the range of 71.7%–95.8%. Therefore, this method could be used as a simple, rapid, and ultra-sensitive tool to monitor the residues of tetracyclines in milk.Graphical abstractMolecular docking of TetR-tetracycline and the assay procedures of the receptor based chemiluminescence sensor for determination of tetracyclines.Image 1
  • Use of capillary electrophoresis to select a DNA aptamer that recognizes
           swine anaphylatoxin C5a
    • Abstract: Publication date: Available online 16 October 2018Source: Analytical BiochemistryAuthor(s): Zhiping Li, Xiwen Wang, Man Chen, Qianxue Li, Han Qu, Ting Shao, Rui Sun, Yandong Zhang, Zhiping Xia Complement factor 5a is a potent proinflammatory mediator that contributes to the pathogenesis of numerous inflammatory diseases. Protein-based C5a inhibitors have proven to be clinically valuable. Aptamers, which are oligonucleic acid chains or polypeptides, can bind to target molecules and hence have the potential to be used for detection and blockade of targets. Here, we describe the discovery that the single-stranded DNA aptamer S1 can bind specifically to swine C5a, which can then be quickly selected for with capillary electrophoresis for high-throughput sequencing. Aptamer S1 bound specifically to swine C5a with a dissociation constant of 4 μM as measured by surface plasmon resonance (SPR). Moreover, aptamer S1 inhibited C5a-induced chemotaxis of neutrophils in vitro. Our study suggests that the S1 aptamer has great potential to be a key structure in the development of effective therapeutic agents against inflammatory diseases.
  • Evaluation of mycoplasma removal reagents using qPCR-based quantification
    • Abstract: Publication date: Available online 15 October 2018Source: Analytical BiochemistryAuthor(s): Xuebo Xu, Yueyang Lai, Ruiying Yan, Zichun Hua In this study, we evaluated the efficacy of various mycoplasma removal reagents using nuclear staining, DNA gel electrophoresis, and qPCR-based quantification. Our results showed Plasmocure and Plasmocin are two effective anti-mycoplasma reagents whose effects can be observed within a week. However, prolonged treatment with Plasmocin led to development of resistance. Withdrawal of anti-mycoplasma reagents led to reoccurrence of mycoplasma contamination, but addition of prevention reagent, such as Primocin, prevented recontamination. Therefore, sequential treatment by Plasmocure and Primocin is the best course of action against mycoplasma contamination. Lastly, we developed methods based on qPCR to estimate the average number of mycoplasma associated with a single contaminated cell. We have shown, for the first time, that untreated contaminated BEAS-2B cells have approximately 300–400 mycoplasma contaminants per cell in the cytoplasm or attached to the cell membrane. Furthermore, withdrawal of anti-mycoplasma reagents led to reoccurrence of mycoplasma contamination within two days, and therefore continued use of prevention reagent is imperative.
  • Rapid detection of Escherichia coli O157:H7 by a fluorescent
           microsphere-based immunochromatographic assay and immunomagnetic
    • Abstract: Publication date: Available online 11 October 2018Source: Analytical BiochemistryAuthor(s): Qianru Li, Yuexi Yang, Fei Hu, Yanxue Cai, Xiaoyun Liu, Xiaowei He To ensure food safety and avoid infections by Escherichia coli O157:H7 (E. coli O157:H7), we developed a novel fluorescent microsphere (FM)-based immunochromatography assay (FM-ICA). FMs were conjugated to anti-E. coli O157:H7 monoclonal antibody (MAb) as an ICA probe, Immunomagnetic beads (IM-beads) were prepared by conjugating functionalized magnetic microspheres with the antibody for enrichment and separation of pathogenic bacteria from complex food matrices. Under selected conditions, a standard curve of FM-ICA measurement of E. coli O157:H7 was developed with a linear range from 3 × 105 to 6 × 107 colony-forming units (CFU)/mL in PBS buffer. The recoveries of intra- and inter assay ranged from 101.64% to 107.03% and 95.62%–110.2% respectively, with CV below 10%. The FM-ICA showed good sensitivity, accuracy and precision. When IM-beads separation plus FM-ICA (IMS-FICA) were used to assay raw food samples, sensitivity was 3 × 103 CFU/mL, a 33-fold improvement compared with FM-ICA only. Moreover, this method had high specificity for E. coli O157:H7 and can be used to assay E. coli O157:H7 in beef, milk and water samples. This assay can be completed within 2 h and has great potential for on-site quantitation of E. coli O157:H7 with simplicity, rapidity, sensitivity, and cost-effectiveness.
  • Preparation of stable tau oligomers for cellular and biochemical studies
    • Abstract: Publication date: Available online 11 October 2018Source: Analytical BiochemistryAuthor(s): Thomas K. Karikari, David A. Nagel, Alastair Grainger, Charlotte Clarke-Bland, Eric J. Hill, Kevin G. Moffat Increasing evidence suggests that small oligomers are the principal neurotoxic species of tau in Alzheimer's disease and other tauopathies. However, mechanisms of tau oligomer-mediated neurodegeneration are poorly understood. The transience of oligomers due to aggregation can compromise the stability of oligomers prepared in vitro. Consequently, we sought to develop an efficient method which maintains the stability and globular conformation of preformed oligomers. This study demonstrates that labeling a single-cysteine form of the pro-aggregant tau four-repeat region (K18) with either Alexa Fluor 488-C5-maleimide or N-ethylmaleimide in reducing conditions stabilizes oligomers by impeding their further aggregation. Furthermore, the use of this approach to study the propagation of labeled extracellular tau K18 oligomers into human neuroblastoma cells and human stem cell-derived neurons is described. This method is potentially applicable for preparing stabilized oligomers of tau for diagnostic and biomarker tests, as well as for in vitro structure-activity relationship assays.
  • Urea can inhibit efficient reduction and alkylation of protein dimers in
           solution demonstrated by the beta subunit of alpha glucosidase II
    • Abstract: Publication date: Available online 10 October 2018Source: Analytical BiochemistryAuthor(s): Meena Kumari, Antje Anji Protein reduction and alkylation is routinely used for analysis of protein dimers and protein complexes in cell fractions using two dimensional gel electrophoresis and mass spectrometry. To resolve the heterogeneity of a high molecular weight protein band that is highlighted by an antibody to the beta subunit of alpha glucosidase II (GIIβ), we performed reduction and alkylation of cytosolic proteins extracted from mouse brain. The presence of urea in the reduction/alkylation buffer inhibited the chemical processes. It is thus recommended that protein reduction/alkylation be performed both in the presence and absence of urea for the separation of mono-/hetero-mers.
  • Arrayed microfluidic chip for detection of circulating tumor cells and
           evaluation of drug potency
    • Abstract: Publication date: Available online 9 October 2018Source: Analytical BiochemistryAuthor(s): Xuejia Huang, Jianlin Tang, Lanlan Hu, Ruyu Bian, Ming Liu, Wenxuan Cao, Huijing Zhang Circulating tumor cells (CTCs) from peripheral blood of cancer patients are considered as one of the most promising pharmacodynamic (PD) biomarkers due to its non-invasive property in disease diagnosis and prognosis. However, the detection of extremely low number of CTCs in patient blood requires methods with high sensitivity and accuracy. We fabricated an arrayed geometrically enhanced mixing (GEM) chip with a "dislocation herringbone" layout based on cell immunoaffinity. By optimizing the injection and rinsing flow rate, an average cell capture rate of 87.02% and an average capture purity of 99.58% were achieved using the human lung adenocarcinoma cell lines H1975. In addition, we determined the specificity, precision, accuracy, and detection limit of our chip. The results demonstrated the chip was stable, accurate and reliable for the “liquid biopsy” of lung cancer cells using the peripheral blood of patients. Our chip can also be used to evaluate the potency of different drugs against tumor cells in parallel due to the presence of four independent microchannels.
  • Dual cycle amplification and dual signal enhancement assisted sensitive
           SERS assay of MicroRNA
    • Abstract: Publication date: Available online 9 October 2018Source: Analytical BiochemistryAuthor(s): Yingdi Wu, Ying Li, Huixia Han, Caisheng Zhao, Xiaoru Zhang A sensitive surface-enhanced Raman scattering (SERS) approach has been developed for detection of microRNA (miRNA) based on target-triggered dual signal amplification including strand displancement amplification (SDA) and hybridization chain reaction (HCR). With the assistant of polymerase and nicking endonuclease (NEase), target miRNA combines with the single stranded template DNA to generate a great amount of trigger DNA which can induce HCR. Coupled the dual cycle amplification of SDA and HCR with the dual enhancement of gold nanoparticles (AuNPs), a low detection limit of 0.5 fM for miRNA is obtained using the proposed strategy. With high sensitivity, universality, rapid analysis, and high selectivity, this method has a great potential for detecting biomolecules with trace amounts in bioanalysis and clinical biomedicine.
  • Skin secretion transcriptome remains in chromatographic fractions suitable
           for molecular cloning
    • Abstract: Publication date: Available online 6 October 2018Source: Analytical BiochemistryAuthor(s): Carolina Proaño-Bolaños, Mei Zhou, Tianbao Chen, Chris Shaw Traditional sources of mRNA for molecular cloning on amphibian skin secretion studies have been the frog's skin and skin secretions. Here, we demonstrate that mRNA isolated from chromatographic fractions of skin secretions is amenable for molecular cloning assays. We identified precursor sequences of the Arg0, Trp5, Leu8-bradykinin and six antimicrobial peptides of Pelophylax esculentus (Ranidae). These results show that both transcriptomic and peptidomic analyses can be performed with a single sample reducing in half the amount of starting skin secretion required. This is a significant advantage when working with endangered or very rare amphibian species, where minimal samples are available.
  • Characterization of receptor binding kinetics for vascular endothelial
           growth factor-A using SPR
    • Abstract: Publication date: Available online 5 October 2018Source: Analytical BiochemistryAuthor(s): Madelane Teran, Matthew A. Nugent Angiogenesis is a highly regulated process orchestrated, in large part, by the vascular endothelial growth factor-A (VEGF-A) system of ligands and receptors. Considerable effort has been invested in finding optimal ways to modulate VEGF-A activity to treat disease, however, the mechanisms by which the various components interact remain poorly understood. This is in part because of the difficulty of analyzing the various interactions in an intercomparable manner. In the present study, we established conditions to allow for the detailed characterization of the molecular interactions between VEGF and its receptors and the co-receptor NRP-1 using surface plasmon resonance (SPR). We found that VEGF dissociated 25-times faster from its major signaling receptor, VEGF receptor-2 (VEGFR-2) than from its “decoy” receptor, VEGF receptor-1 (VEGFR-1). Using a systematic approach, we obtained kinetic parameters for each individual interaction under a consistent set of experimental conditions allowing for comparison between various receptors. The set of quantitative kinetic parameters and experimental conditions reported herein will provide valuable tools for developing comprehensive models of the VEGF system.
  • Characterization of recombinant monoclonal antibody charge variants using
           WCX chromatography, icIEF and LC-MS/MS
    • Abstract: Publication date: Available online 3 October 2018Source: Analytical BiochemistryAuthor(s): Hailong Liu, Weicheng Ren, Li Zong, Jinliang Zhang, Yingwu Wang Charge heterogeneity is an important aspect of research into the development of monoclonal antibody drugs. In the present study, charge variants were separated into four fractions using weak cation exchange chromatography and were thoroughly analyzed using liquid chromatography–mass spectrometry at multiple levels. Molecular weight analysis of intact antibody and subunits confirmed the presence of heavy-chain leader sequences, light-chain leader sequences, dehydration, and cysteinylation. Peptide mapping of the fractions using different enzymes further localized the modified sites. Modified proportions identified at peptide level were compared with the purity detected by imaged capillary isoelectric focusing, the results showed that basic variant 1 consisted of cysteinylation and dehydration of asparagine, and basic variant 2 fully accounted for the N-terminal leader sequence of the heavy chain. About 14.8% of the acidic variant can be explained by N-terminal leader sequences in the light chain, and 18% of the acidic variant was demonstrated to be deamidation of asparagine in the heavy chain. There was approximately 54.2% of the acidic variant still cannot be explained. It was hypothesized that those acidic variants that have not yet been identified are an ensemble of molecules with slight molecular weight differences or the same molecular weight but different structures.
  • Detection of amyloid β oligomers toward early diagnosis of
           Alzheimer's disease
    • Abstract: Publication date: Available online 26 September 2018Source: Analytical BiochemistryAuthor(s): Soyoon Sarah Hwang, Hon Chan, Mirco Sorci, James Van Deventer, Dane Wittrup, Georges Belfort, David Walt Amyloid β (Aβ) peptide accumulation in the brain is considered to be one of the hallmarks of Alzheimer's disease. Here, we compare two analytical techniques for detecting neurotoxic Aβ1-42 oligomers - Quartz Crystal Microbalance with Dissipation (QCM-D) and Single Molecule Array (Simoa). Both detection methods exploit a feature of the monoclonal antibody bapineuzumab, which targets N-terminal residues 1–5 of Aβ with high affinity and use it as both a capture and detection reagent. Assays developed with the two methods allow us to specifically recognize neurotoxic Aβ1-42 oligomers and higher aggregates such as fibrils but discriminate against Aβ1-42 monomer species. We find that for detection of Aβ1-42 oligomers, Simoa was roughly 500 times more sensitive than the QCM-D technique with limits of detection of 0.22 nM and 125 nM, respectively.
  • Label-free and sensitive detection assay for terminal deoxynucleotidyl
           transferase via polyadenosine-coralyne fluorescence enhancement strategy
    • Abstract: Publication date: Available online 26 August 2018Source: Analytical BiochemistryAuthor(s): Yuanyuan Wang, Xu Sun, Jianxiong Zeng, Minggang Deng, Nan Li, Qiutong Chen, Hua Zhu, Fenyong Liu, Xiwen Xing Terminal deoxynucleotidyl transferase (TdT) is a unique template-free polymerase that randomly adds multiple deoxyribonucleoside triphosphates (dNTPs) to the 3′-OH terminus of ssDNA. This characteristic makes TdT a versatile enzymatic tool in many fields. Moreover, aberrant TdT expression is a well-recognized biomarker of several leukemic diseases and is related to carcinogenesis. In this study, we developed a facile, rapid, label-free, and convenient assay for TdT detection. TdT-generated poly A tails formed a fluorescent enhancement complex in the presence of coralyne. To achieve a better signal-to-noise ratio, we used potassium thiocyanate (KSCN), instead of other halogen anions (KCl, KBr, KI, NaI) as the quenching agent of dissociate coralyne. Our results demonstrate that this assay is extremely facile, rapid, and label-free; at levels as low as 0.025 U/mL, TdT was distinctly detected within 55 min. And the determination of TdT activity in RBL-2H3 and Reh cells lysates exhibited a good sensing performance, demonstrating its potential applications in biochemical research and clinical diagnosis.Graphical abstractImage 1
  • Development of an immunochromatographic assay for the specific detection
           of Bacillus thuringiensis (Bt) Cry1Ab toxin
    • Abstract: Publication date: Available online 18 August 2018Source: Analytical BiochemistryAuthor(s): Sa Dong, Yuan Liu, Xiao Zhang, Chongxin Xu, Xianjin Liu, Cunzheng Zhang Cry1Ab has been widely used in genetically modified (GM) crops and its amino acid sequence had high identity to Cry1Ac toxin. Existing nanogold immunochromatographic strips cannot distinguish Cry1Ab from Cry1Ac toxin. In this study, a rapid (5–6 min), qualitative nanogold immunochromatographic strip was successfully developed for the specific detection of Cry1Ab toxin. The assay was based on double antibody sandwich format with the visual detection limit (vLOD) of 0.1 μg mL−1. The results of immunochromatographic assay were all positive validated against the DAS-ELISA (recoveries between 109.6 and 111.8%). In addition, 10%, 5% and 0% error probability results were found in 20 times repeated tests for Cry1Ab concentration of 0.1, 0.2, 0.5 and 1 μg mL−1, respectively, demonstrating the reproducibility of the test strip. Furthermore, the test strip could be stored for 3 months under dry conditions without significant loss of sensitivity. Furthermore, the practical sample analysis results showed that the test strip was able to detect the presence of Cry1Ab in GM materials containing as low as 0.5% MON 810 Bt maize which indicated the practical value of the test strip. To our knowledge, this is the first report on the detection of Cry1Ab by immunochromatographic assay without interference from Cry1Ac toxin.
  • 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
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