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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  [3161 journals]
  • An electrochemical biosensor to distinguish between normal and cancer
           cells based on monitoring their acidosis using gold-coated silicon
           Nano-roughened electrode
    • Abstract: Publication date: Available online 13 September 2018Source: Analytical BiochemistryAuthor(s): Alireza Alikhani, Milad Gharooni, Hassan Moghtaderi, Fatemeh Farokhmanesh, Hamed Abiri, Mona Salimi, Farnoosh Attari, Mohammad AbdolahadAbstractOne of the most interesting fields of research in cancer diagnosis is tracing the relation between extracellular media and cancer progression. Detecting the secreting contents of the cells and translating these molecular identifications into label-free recognizable patterns would open new opportunities in cancer research. Electrochemical responses are in the range of most attractive sensing mechanisms especially in biochemical approaches. Perturbed ionic exchanges as a known biochemical function of cancer cells presented a strong correlation with the pH of the tumor microenvironment. Different ionic activities detected by an electrochemical bio-sensing system in the malignant and normal cells in the presence of acidic ambient were our main results presented in this research. Herein, silicon Nano-roughened substrate as a well-known electrochemical interface was applied in the construction of the biosensor. Viability rate as well as apoptotic factors involving in cancer progression were assessed by biochemical assays in normal (MCF10A) and cancer (MCF7 and MDA-MB468) breast cells. Our findings demonstrated that pH-based electrochemical responses were matched with the results obtained from the biological analyses of both normal and malignant cells. Induction of acidosis in the cells followed by monitoring their electrochemical responses would be a new trend in microenvironment based cancer investigation.
  • Predicting lysine lipoylation sites using bi-profile bayes feature
           extraction and fuzzy support vector machine algorithm
    • Abstract: Publication date: Available online 12 September 2018Source: Analytical BiochemistryAuthor(s): Zhe Ju, Shi-Yun WangLipoylation is a highly conserved post-translational modification which has been found to be involved in many biological processes and closely associated with various metabolic diseases. The accurate identification of lipoylation sites is necessary to elucidate the underlying molecular mechanisms of lipoylation. As the traditional experimental methods are time consuming and expensive, it is desired to develop computational methods to predict lipoylation sites. In this study, a novel predictor named LipoPred is proposed to predict lysine lipoylation sites. On the one hand, an effective feature extraction method, bi-profile bayes encoding, is employed to encode lipoylation sites. On the other hand, a fuzzy support vector machine algorithm is proposed to solve the class imbalance and noise problem in the prediction of lipoylation sites. As illustrated by 10-fold cross-validation, LipoPred achieves an excellent performance with a Matthew's correlation coefficient of 0.9930. Therefore, LipoPred can be a useful bioinformatics tool for the prediction of lipoylation sites. Feature analysis shows that some residues around lipoylation sites may play an important role in the prediction. The results of analysis and prediction could offer useful information for elucidating the molecular mechanisms of lipoylation. A user-friendly web-server for LipoPred is established at abstractImage 1
  • Purification of equine IgG3 by lectin affinity and an interaction analysis
           via microscale thermophoresis
    • Abstract: Publication date: Available online 12 September 2018Source: Analytical BiochemistryAuthor(s): Salvatore G. De-Simone, Hilton J. Nascimento, Isis C. Prado, Aniesse S. Aguiar, Anibal R. Melgarejo, Jorge L. Pina, Patricia F. Ferreira, David W. ProvanceAbstractThe availability of purified antibodies is a prerequisite for many applications and the appropriate choice(s) for antibody-purification is crucial. Numerous methods have been developed for the purification of antibodies from different sources with affinity chromatography-based methods being the most extensively utilized. These methods are based on high specificity, easy reversibility and biological interactions between two molecules (e.g., between receptor and ligand or antibody and antigen). However, no simple techniques have yet been described to characterize and purify subclasses of immunoglobulins (Ig) from some animals of biotechnology importance such as equines, which are frequently used to produce biotherapeutic antibodies. The sera of these animals present a large number of Ig classes that have a greater complexity than other animals. The implementation of an effective protocol to purify the desired antibody class/subclasses requires meticulous planning to achieve yields at a high purity. The IgG3 subclass of equine-Ig has recently been used as antigen in a new diagnostic test for allergic responses to horse sera-based therapies. Here, we defined a simple method using Jacalin lectin immobilized on Sepharose beads to prepare highly pure equine IgG3 antibodies with a determination of the affinity constants for Jacalin lectin and horse IgG3.
  • Isothermal DNA amplification combined with lateral flow dipsticks for
           detection of biothreat agents
    • Abstract: Publication date: Available online 11 September 2018Source: Analytical BiochemistryAuthor(s): Aleksandra A. Zasada, Katarzyna Zacharczuk, Kamila Formińska, Aldona Wiatrzyk, Robert Ziółkowski, Elżbieta MalinowskaAbstractThe recently developed methods of nucleic acids isothermal amplification are promising tools for point-of-care diagnostics and in the field detection of pathogenic microorganisms. However, application of these methods outside a laboratory faces some challenges such as the rapid and sensitive detection of amplified products and the absence of cross-reactivity with genetically related microorganisms. In the presented study we compared three methods of isothermal DNA amplification loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and thermophilic helicase-dependent isothermal DNA amplification (tHDA), for detection of highly dangerous pathogens, such as Bacillus anthracis, Francisella tularensis and Yersinia pestis, and combined them with lateral flow dipsticks for the rapid visualization of amplified products. We observed low specificity of the three methods for B. antharcis, medium for Y. pestis and high for F. tularensis detection. Sensitivity and the detection limit were high and comparable for all the methods. We concluded that the lateral flow dipsticks have been a very useful tool for product detection of the isothermal amplification methods and enable reading the results without the use of any equipment. However, our results showed that the use of isothermal amplification methods is strongly related to the risk of false positive results.
  • Validation of a quick and low-cost DNA extraction protocol applicable to
           long-stored blood samples
    • Abstract: Publication date: Available online 11 September 2018Source: Analytical BiochemistryAuthor(s): Gabriel Tassi Mousquer, Lila Partichelli Maciel, Ana Carolina Pompeu Saraiva, Elis Regina Dalla Costa, Maria Lucia Rosa Rossetti
  • iRNA(m6A)-PseDNC: Identifying N6-methyladenosine sites using pseudo
           dinucleotide composition
    • Abstract: Publication date: Available online 8 September 2018Source: Analytical BiochemistryAuthor(s): Wei Chen, Hui Ding, Xu Zhou, Hao Lin, Kuo-Chen ChouAbstractAs a prevalent post-transcriptional modification, N6-methyladenosine (m6A) plays key roles in a series of biological processes. Although experimental technologies have been developed and applied to identify m6A sites, they are still cost-ineffective for transcriptome-wide detections of m6A. As good complements to the experimental techniques, some computational methods have been proposed to identify m6A sites. However, their performance remains unsatisfactory. In this study, we firstly proposed an Euclidean distance based method was proposed to construct a high quality benchmark dataset. By encoding the RNA sequences using pseudo nucleotide composition, a new predictor called iRNA(m6A)-PseDNC was developed to identify m6A sites in the Saccharomyces cerevisiae genome. It has been demonstrated by the 10-fold cross validation tests that the performance of iRNA(m6A)-PseDNC is superior to the existing methods. Meanwhile, for the convenience of most experimental scientists, established at the site is its web-server, by which user can easily get their desired results without need to go through the detailed mathematics. It is anticipated that iRNA(m6A)-PseDNC will become a useful high throughput tool for identifying m6A sites in the S. cerevisiae genome.
  • Trypsin enhances aptamer screening: A novel method for targeting proteins
    • Abstract: Publication date: Available online 6 September 2018Source: Analytical BiochemistryAuthor(s): Wanli Yan, Lide Gu, Shu Liu, Wei Ren, Mingsheng Lyu, Shujun WangAbstractA novel screening method for protein aptamer selection was developed in this study. Aptamers with high affinity and specificity to the surface recombinant antigen of Helicobacter pylori (HP-Ag) and to tumor markers carcinoembryonic antigen (CEA), cancer antigen 125 (CA125) and cancer antigen 19-9(CA19-9) were screened using trypsin enhanced screening method. Briefly, the target proteins above were immobilized onto 96-well polystyrene plates and incubated with a single-stranded DNA (ssDNA) library for aptamer selection. Then, trypsin was introduced to digest the proteins and obtain ssDNA that bound to the target proteins with high specificity. The concentration of ssDNA that shed from protein-ssDNA complexes was detected. After sequencing, the enrichment of target-specific aptamers was monitored and the affinity of each aptamer was analyzed. Urea, which has been reported in other article, was used to compare with trypsin. The results revealed that trypsin was more effective than urea for protein aptamer selection. The protocol used in this study provided a novel method for generating aptamers.
  • Enrichment of midsized RNAs with manganese chloride precipitation
    • Abstract: Publication date: Available online 5 September 2018Source: Analytical BiochemistryAuthor(s): Dillon Friday, Paul Freidhoff, Tilman Baumstark, Michael F. BruistAbstractEnrichment of specific RNAs is important for RNA analysis. MnCl2 has been used previously to enrich viroid RNA fractions from total RNA from infected plants. We have expanded this method to show that MnCl2 can enrich single-stranded as well as structured RNAs of 450 nt and below from a total RNA preparation. We have applied this method to map the transcription start sites of a PSTVd transcript from total RNA from yeast under conditions where the RNA was previously undetectable.
  • A cautionary note on the use of cap analogue affinity resins
    • Abstract: Publication date: Available online 4 September 2018Source: Analytical BiochemistryAuthor(s): Regina Cencic, Jerry PelletierAbstractAll cellular cytoplasmic mRNAs are capped at their 5’ ends with an m7GpppN group. Several proteins that mediate cap function have been identified by cap affinity purification, enabling their characterization in a number of biological processes. Among these, eukaryotic initiation factor (eIF) 4E is the best characterized and plays a critical role in regulating ribosome recruitment to mRNAs during translation initiation. Cap affinity chromatography is often used to identify eIF4E-interacting proteins, which could play critical roles in molding the eIF4E-interactome and impacting on eIF4E-directed translation. Here we address how improper implementation of this technology can lead to false conclusions and provide recommendations to ensure correct interpretation of data obtained by this approach.
  • An efficient colorimetric high-throughput screening method for synthetic
           activity of tyrosine phenol-lyase
    • Abstract: Publication date: Available online 31 August 2018Source: Analytical BiochemistryAuthor(s): Xiao-Ling Tang, Hui Suo, Ren-Chao Zheng, Yu-Guo ZhengAbstractTyrosine phenol-lyase (TPL) naturally catalyzes the reversible β-elimination of l-tyrosine to phenol, pyruvate and ammonium. With its reverse reaction (synthetic activity), l-tyrosine and its derivatives could be synthesized with high atom economy, which are widely used in pharmaceutical industries. In this study, a high-throughput screening method for synthetic activity of TPL was developed. One of the substrate, sodium pyruvate was found to react with salicylaldehyde under alkali condition, forming a yellow color compound. The concentration of sodium pyruvate can be quantified according to the absorbance of the colorimetric compound at wavelength of 465 nm and the activity of TPL could be screened according to the decrease of the absorbance. After optimization of the colorimetric reaction conditions, the established high-throughput screening method was successfully used for screening of TPL with enhanced activity for l-DOPA synthesis. The confirmed sensitivity and accuracy demonstrated the feasibility and application potential of this screening method.
  • Acetylcholinesterase biosensor based on functionalized surface of carbon
           nanotubes for monocrotophos detection
    • Abstract: Publication date: Available online 31 August 2018Source: Analytical BiochemistryAuthor(s): Zou Bin, Chu Yanhong, Xia Jiaojiao, Yao JingAbstractMonocrotophos (Ops) has been widely used as pesticide in crop production but simultaneously could accumulate in the nature and seriously impact food safety and human health. It is necessary to develop a high sensitivity biosensor for accurate and fast detection of OPs. In this study, multi-walled carbon nanotubes (MWCNTs) were selected as acetylcholinesterase (AChE) carrier and their surface was modified by introducing different functional groups (-SH, -NH2, -Cl, -OH), hydrophobic alkyl groups (-CH3, -(CH2)2CH3, -(CH2)7CH3, -(CH2)15CH3) and ionic liquids (-IL1, -IL2). The interaction mechanism of MWCNTs functionalized surface and AChE has been revealed by studying characteristics of AChE immobilized on different carrier surface. Finally, compared with reported references and above other modifiers, we found that MWCNTs surface modified by -IL1 was the best carrier for AChE and the detection limit of IL1-MWCNTs/AChE/GCE was 3.3 × 10−11 M. At optimum reaction condition (pH 7.0, AChE loading 0.25 U, Inhibition time 14 min), storability test indicated reactivity of IL1-MWCNTs/AChE/GCE remained above 98.5% within two weeks. For real vegetable sample detection, the recoveries of IL1-MWCNTs/AChE/GCE were found to be between 90.0% and 104%. These results demonstrated novel biosensors could act as device of high sensitivity for accurate and fast detection of OPs.
  • Detection of catalase activity with aldehyde-doped liquid crystals
           confined in microcapillaries
    • Abstract: Publication date: Available online 31 August 2018Source: Analytical BiochemistryAuthor(s): Jinseob Rim, Chang-Hyun JangAbstractIn this study, a simple, rapid, and label-free sensor was developed for detecting the enzymatic activity of catalase (CAT) with liquid crystals (LCs) confined in microcapillaries. Inside a microcapillary functionalized with n-octyltrichlorosilane, aldehyde-doped LCs anchored radially so that a pattern of straight lines was observed under a polarized optical microscope (POM). However, once hydrogen peroxide (HP) oxidized the aldehyde into carboxylic acid, which has surface activity, the orientation of the LCs at the interface changed, resulting in a distinct pattern change, from straight to crossed. In this system, the enzymatic activity of CAT could be detected as it inhibits the oxidation by decomposing HP; as a result, the pattern changed back to the straight one. From the orientational and optical shift, the enzymatic activity of CAT was detected up to a concentration of 0.8 fM under mild experimental conditions and 8 aM at pH 9.0. This result suggests the need for further study of microcapillary systems to develop simple and sensitive sensors for biochemical interactions.
  • Re-evaluation of the rabbit myosin protein standard used to create the
           empirical statistical model for decoy library searching
    • Abstract: Publication date: Available online 29 August 2018Source: Analytical BiochemistryAuthor(s): Jaimie Dufresne, Angelique Florentinus-Mefailoski, Pei-Hong Zhu, Peter Bowden, John G. MarshallAbstractA Rabbit myosin standard, like that used to create the empirical statistical model, was randomly and independently sampled by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The rabbit myosin protein standard appeared pure by SDS-PAGE and CBBR staining but showed many other proteins by silver staining. The LC-MS intensity from the myosin and IgG samples were above the 99% safe limit of detection and quantification computed from 36 blank LC-ESI-MS/MS runs. The myosin contained ≤406 Gene Symbols, open reading frames or loci where 79 protein types showed ≥3 peptides from X!TANDEM. Myosins, actin, troponin, other proteins showed 95%–100% homology between the rabbit versus the human decoy library. The myosin protein complex from STRING was true positive compared to random or noise spectra MS/MS with a low type I error (p-value) and low FDR (q-value) computed in R. SDS-PAGE, Western blot, comparison to random and noise MS/MS spectra, X!TANDEM p-values, FDR corrected q-values, and STRING all agreed that the error rate of LC-ESI-MS/MS with a quadrupole ion trap is far below that assumed a priori by the design of the empirical statistical model for decoy library searching.
  • Electrochemical study of a glassy carbon electrode modified by
           poly-4-nitroaniline murexide and its sensitivity for metal ions
    • Abstract: Publication date: Available online 29 August 2018Source: Analytical BiochemistryAuthor(s): C. Ben Ali Hassine, H. BarhoumiThe electrochemical modification of a glassy carbon electrode using reduced poly-4 nitroaniline (P-4NA) and it's applicability for determination of metallic ions was performed in this study. The electrode modification was performed by cyclic voltammetry in the potential range between 0.9 V and 1.4 V vs Ag/Ag+ (in 10 mM AgNO3) at the scan rate of 100 mV/s by 50 cycles in non-aqueous media. The reduction of nitro groups on the P-4NA modified glassy carbon electrode surface was performed in the potential range between −0.1 V and −0.8 V vs Ag/AgCl(Sat. KCl) at a scan rate of 100 mV/s in 100 mM aqueous HCl solution. The reduced P-4NA glassy carbon surface was modified with the murexide. The affinity of the modified glassy carbon electrode with some metallic ions was investigated by electrochemical impedance spectroscopy method in phosphate buffer solution (pH = 5).Graphical abstractIllustration of the modification steps of the GC electrode.Image 1
  • High-throughput investigation of transglutaminase 2 kinase regulation
           using a novel cysteine-modified peptide array
    • Abstract: Publication date: Available online 27 August 2018Source: Analytical BiochemistryAuthor(s): Se-Hui Jung, Mi-Hye Kwon, Seong-Hyeon Lee, Eun-Taek Han, Won Sun Park, Seok-Ho Hong, Young-Myeong Kim, Kwon-Soo HaTransglutaminase 2 (TGase2) kinase has emerged as an important regulator of apoptosis as well as chromatin structure and function; however, details about the pathophysiological functions of TGase2 kinase have been limited because of the lack of a suitable activity assay for systematic investigation of TGase2 kinase regulation in a high-throughput manner. Thus, we developed a novel on-chip TGase2 kinase activity assay using a cysteine-modified insulin-like growth factor-binding protein-3-derived peptide (CMI peptide) on an array platform. This peptide array-based activity assay was reproducible, with a detection limit of 2.127 μg/ml. We successfully applied this assay to investigate the effects of thiol-reactive compounds and divalent cations on TGase2 kinase by determining the half maximal inhibitory concentrations (IC50). Thiol-reactive compounds inhibited TGase2 kinase activity in a concentration-dependent manner, with IC50 values ranging from 0.125 to 5.550 mM. Divalent metal cations also showed a concentration-dependent inhibition, with IC50 values ranging from 0.005 to 1.937 mM; however, Ca2+ had no effect on TGase2 kinase activity. Thus, this novel kinase activity assay using the CMI peptide array described here is suitable for systematic investigation of TGase2 kinase regulation and may be useful for investigating the roles of TGase2 kinase in pathogenesis of kinase-mediated diseases.Graphical abstractImage 1
  • 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 XingTerminal 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
  • Mass spectrometry evaluation of a neuroblastoma SH-SY5Y cell culture
    • Abstract: Publication date: 15 October 2018Source: Analytical Biochemistry, Volume 559Author(s): Jimmy Rodriguez Murillo, Indira Pla, Livia Goto-Silva, Fábio C.S. Nogueira, Gilberto B. Domont, Yasset Perez-Riverol, Aniel Sánchez, Magno JunqueiraCell line-based proteomics studies are susceptible to intrinsic biological variation that contributes to increasing false positive claims; most of the methods that follow these changes offer a limited understanding of the biological system. We applied a quantitative proteomic strategy (iTRAQ) to detect intrinsic protein variation across SH-SY5Y cell culture replicates. More than 95% of the quantified proteins presented a coefficient of variation (CV) 
  • Fabrication of a highly sensitive electrochemical sensor based on
           electropolymerized molecularly imprinted polymer hybrid nanocomposites for
           the determination of 4-nonylphenol in packaged milk samples
    • Abstract: Publication date: 15 October 2018Source: Analytical Biochemistry, Volume 559Author(s): Lufei Zheng, Chao Zhang, Jun Ma, Sihui Hong, Yongxin She, A.M. Abd EI-Aty, Yahui He, Hailong Yu, Haijin Liu, Jing WangAbstractHerein, an imprinted electrochemical sensor based on graphene-Au nanoparticles incorporated with molecularly imprinted polymer (MIP) was fabricated for determination of 4-nonylphenol (4-NP). Grafted MIP electropolymerized on nanoscale multilayer films electrode was achieved using 4-NP as a template and P-aminothiophenol as a functional monomer. The electrochemical properties of the MIP nanoscale multilayer membrances were characterized and measured by cyclic voltammetry and differential pulse voltammetry techniques; using ferrocyanide/ferricyanide-redox marker. Several important parameters were optimized and investigated to improve the performance of the sensor. Under the optimized conditions, the developed sensor showed an excellent linear response over the concentration ranges of 50–500 ng mL−1 (4-NP) with a detection limit of 0.01 ng mL−1(S/N = 3). The developed sensor showed a good selective recognition of 4-NP compared with structural analogue, exhibited a good reproducibility and accuracy with long-term stability. At last, the feasibility of the proposed methodology was successfully applied fordetection of 4-NP in milk and its packaging materials.
  • A non-enzymatic electrochemical immunoassay for quantitative detection of
           Escherichia coli O157:H7 using Au@Pt and graphene
    • Abstract: Publication date: 15 October 2018Source: Analytical Biochemistry, Volume 559Author(s): Fanjun Zhu, Guangying Zhao, Wenchao DouHerein, a non-enzymatic sandwich-type electrochemical immunoassay was fabricated for quantitative monitoring of Escherichia coli O157:H7 (E. coli O157:H7). Silica coated Fe3O4 magnetic nanoparticles (Fe3O4@SiO2) were modified with mouse anti-E. coli O157:H7 monoclonal antibody (Ab1) to act as capture probes to reduce detection time and increase the sensitivity of the immunoassay. The Au@Pt nanoparticles were loaded on neutral red (NR) functionalized graphene to form composite complex rGO-NR-Au@Pt. rGO-NR-Au@Pt has high specific surface area and good biocompatibility. rGO-NR-Au@Pt was used as the carriers of detection antibodies (Ab2). Au@Pt catalyzed the reduction of hydrogen peroxide (H2O2) to detection of E. coli O157:H7 with the thionine (TH) as electron mediator to effectually amply the current signal. Under the optimized conditions, a linear relationship between the reduction peak current change (ΔIpc) and the logarithm of the E. coli O157:H7 concentration is obtained in the range from 4.0 × 103 to 4.0 × 108 CFU mL−1 and the limit of detection (LOD) is 4.5 × 102 CFU mL-1 at a signal-to-noise ratio of 3. The immunoassay exhibits acceptable specificity, reproducibility and stability on the detection of E. coli O157:H7. Furthermore, the immunoassay showed good performance in pork and milk samples. The results suggest that this immunoassay will be promising in the food safety area.Graphical abstractSchematic illustration of (A) the process for preparation of Fe3O4@SiO2-Ab1, (B) the process for preparation of rGO-NR-Au@Pt-Ab2, and (C) the detection process of the immunoassay.Image 1
  • Metabolomics analysis reveals that elevated atmospheric CO2 alleviates
           drought stress in cucumber seedling leaves
    • Abstract: Publication date: Available online 25 August 2018Source: Analytical BiochemistryAuthor(s): Man Li, Yiman Li, Wendong Zhang, Shuhao Li, Yong Gao, Xizhen Ai, Dalong Zhang, Binbin Liu, Qingming LiAbstractElevated atmospheric CO2 alleviates moderate to severe drought stresses at physiological level in cucumber. To investigate the underlying metabolic mechanisms, cucumber seedlings were treated with two [CO2] and three water treatments combinations, and their leaves were analyzed using a non-targeted metabolomics approach. The results showed that elevated [CO2] changed 79 differential metabolites which were mainly associated with alanine, aspartate and glutamate metabolism; arginine and proline metabolism; TCA cycle; and glycerophospholipid metabolism under moderate drought stress. Moreover, elevated [CO2] promoted the accumulation of secondary metabolites; including isoferulic acid, m-coumaric acid and salicyluric acid. Under severe drought stress, elevated [CO2] changed 26 differential metabolites which mainly involved in alanine, aspartate and glutamate metabolism; pyruvate metabolism; arginine and proline metabolism; glyoxylate and dicarboxylate metabolism; cysteine and methionine metabolism; starch and sucrose metabolism; glycolysis or gluconeogenesis; and pyrimidine metabolism. In addition, elevated [CO2] accumulated carbohydrates, 1,2,3-trihydroxybenzene, pyrocatechol, glutamate, and L-gulonolactone, to allow adaption to severe drought. In conclusion, the metabolites and metabolic pathways associated with the alleviation of drought stresses by elevated [CO2] were different according to the level of drought stress. Our results may provide a theoretical basis for CO2 fertilization and application of exogenous metabolites to enhance drought tolerance of cucumber.
  • Colorimetric determination of β-1,2-glucooligosaccharides for an
           enzymatic assay using 3-methyl-2-benzothiazolinonehydrazone
    • Abstract: Publication date: Available online 25 August 2018Source: Analytical BiochemistryAuthor(s): Kaito Kobayashi, Hiroki Aramasa, Hiroyuki Nakai, Masahiro Nakajima, Hayao TaguchiAbstractA colorimetric determination method measuring the reducing ends of sugars is usually used for quantitative evaluation of polysaccharide-degrading activity of endo-type enzymes. However, no appropriate colorimetric method has been established for enzymatic assay of β-1,2-glucanases, which produce β-1,2-glucooligosaccharides from β-1,2-glucans. The Anthon-MBTH method has been potentially the most adaptable for color development of β-1,2-glucooligosaccharides among various known colorimetric methods for detecting the reducing power of oligosaccharides, since the difference between sophorose and other β-1,2-glucooligosaccharides in absorbance is relatively small. Almost the same color development was obtained for β-1,2-glucooligosaccharides when the heating time with the Anthon-MBTH method was changed. The kind of base and concentration of dithiothreitol did not markedly affect the color development. Most buffer components, salts and a chelating reagent used for usual enzymatic experiments did not inhibit color development. Furthermore, assay was performed successfully for a β-1,2-glucanase using the modified MBTH method.
  • Critique of methods for estimating heats in isothermal titration
    • Abstract: Publication date: Available online 24 August 2018Source: Analytical BiochemistryAuthor(s): Joel TellinghuisenAbstractIsothermal titration calorimetry data recorded on a MicroCal/Malvern VP-ITC instrument for water-water blanks and for dilution of aqueous solutions of BaCl2 and Ba(NO3)2 are analyzed using Origin software, the freeware NITPIC program, and in-house algorithms, to compare precisions for estimating the heat per injection q. The data cover temperatures 6–47 °C, injection volumes 4–40 μL, and average heats 0–200 μcal. For water-water blanks, where baseline noise limits precision, NITPIC and the in-house algorithm achieve precisions of 0.05 μcal, which is better than Origin by a factor of 4. The precision differences decrease with increasing q , becoming insignificant for q > 200 μcal. In its default mode, NITPIC underestimates q for peaks with incomplete return to baseline, but the shortfall can be largely corrected by overriding the default injection time parameter. The variance estimates from 26 dilution experiments are used to assess the data variance function. The results determine the conditions under which weighted least squares should be used to estimate thermodynamic parameters from ITC data.
  • Paper-based chemiluminescence enzyme-linked immunosorbent assay enhanced
           by biotin-streptavidin system for high-sensitivity C-reactive protein
    • Abstract: Publication date: Available online 22 August 2018Source: Analytical BiochemistryAuthor(s): Zheng Li, Ming Li, Fei Li, Meizi ZhangHigh-sensitivity C-reactive protein (hs-CRP) has been regarded as a risk predictor of cardiovascular disease (CVD). Despite there are many methods to detect hs-CRP, quantitative, rapid, convenient, multiplex and highly sensitive measurement of it is still a challenge for point-of-care applications. In this study, we developed a paper-based ELISA to detect hs-CRP and the sensitive chemiluminescence was applied as detection signal. In this developed assay method, CRP concentration and chemiluminescence intensity were linearly correlated (r = 0.999) with a limit of detection (LOD) as low as 0.49 ng mL−1, which was comparable to that of conventional ELISA and superior to most of the current reported POCT methods for detection of hs-CRP. The precision of the assay was confirmed for low coefficient of variations, less than 7% for intra-assay and less than 10% for inter-assay. In clinical sample analysis, the results of hs-CRP detected by this assay were in good accordance with which obtained by commercial high sensitivity ELISA kit for in vitro diagnosis (r = 0.975). This assay required only 4 μL of sample and could be finished in less than 30 min. It may therefore be employed as a rapid pre-screening tool to identify patients with elevated risk of CVD.Graphical abstractImage 1
  • Development of a high-content imaging assay for screening compound
    • Abstract: Publication date: Available online 21 August 2018Source: Analytical BiochemistryAuthor(s): Yong Jiang, Ann Hoffman, Geoffrey Quinque, William G. Bonnette, Lawrence M. Szewczuk, Marc A. Holbert, Jeffrey GrossAbstractAggregated compounds can promiscuously and nonspecifically associate with proteins resulting in either false inhibition or activation of many different protein target classes. We developed a high-content imaging assay in a 384-well format using fluorescently labeled target proteins and an Operetta cell imager to screen for compound aggregates that interact with target proteins. The high-throughput assay can not only directly detect the interaction between compound aggregators and the target of interest, but also determine the critical aggregation concentration (CAC) of a given promiscuous small molecule.
  • Peptide ligand-based ELISA reagents for antibody detection
    • Abstract: Publication date: Available online 18 August 2018Source: Analytical BiochemistryAuthor(s): Ewa Heyduk, Rachel Hickey, Nicola Pozzi, Tomasz HeydukAbstractDetection of specific antibodies has numerous research, therapeutic and diagnostic applications. Short peptide ligands that bind specifically to antibodies with continuous epitopes can be derived from epitope mapping experiments. Short peptide ligands (mimotopes) specific to antibodies with discontinuous epitopes can be identified by screening complex peptide libraries. In an effort to enhance practical utility of such peptide ligands, we describe here a simple approach to turn such target antibody-specific peptide ligands into specific ELISA detection reagents. We show that a simple addition of biotinylated peptide ligands to commonly available horseradish peroxidase (HRP)-labeled streptavidin (or HRP-anti-biotin antibody), or digoxigenin-labeled peptides to HRP-anti-digoxigenin antibody detection reagents transformed these generic detection reagents into sensitive target antibody-specific reagents. ELISA assays performed using these reagents exhibited excellent analytical properties indicating their practical utility for antibody detection. One generic detection reagent can be readily transformed into many different specific ELISA reagents by a simple mix and match design using an appropriate target-specific peptide ligand. Simplicity of preparation of these ELISA reagents for detecting antibodies should facilitate their practical applications.
  • 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 ZhangAbstractCry1Ab 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.
  • Fluorescent copper nanoclusters as a nano-dye for DNA methyltransferase
           activity analysis and inhibitor screening
    • Abstract: Publication date: Available online 16 August 2018Source: Analytical BiochemistryAuthor(s): Dengpeng Gao, Hongyue Zhang, Yafei Xu, Yun Liu, Huiying Xu, Jianguo CuiFluorescent copper nanoslusters (CuNCs) as a new class of fluorophores have attracted more and more attention due to their ease of synthesis, excellent optical properties, and low cost. In this study, a novel label-free fluorescent method was developed for the detection of DNA methyltransferases based on template length-dependent of dsDNA-CuNCs. In the absence of DNA adenine methylation methyltransferase (Dam MTase), the dsDNA containing the methylation-responsive sequence could effectively template the formation of fluorescent CuNCs with bright fluorescence. When the dsDNA substrate is methylated by Dam MTase, the methylation-sensitive restriction endonuclease Dpn I cleaves the methylated dsDNA and produces shorter dsDNA product, which fails to template fluorescent CuNCs. So, the Dam MTase activity could be identified by the changes of CuNCs' fluorescence. Based on this method, a linear range of 0.5–10 U/mL was achieved with high sensitivity and selectivity. Moreover, we also demonstrate the proposed method can be applied to evaluation and screening of inhibitors for Dam MTase.Graphical abstractIn this work, we developed a simple homogeneous fluorescence strategy for the detection of Dam MTase activity based on dsDNA templated CuNCs.Image 1
  • Crystallization of Human Erythrocyte Band 3, the anion exchanger, at the
           International Space Station "KIBO″
    • Abstract: Publication date: Available online 15 August 2018Source: Analytical BiochemistryAuthor(s): Hinako Hatae, Koji Inaka, Ryo Okamura, Naoki Furubayashi, Masayuki Kamo, Takuya Kobayashi, Yoshito Abe, So Iwata, Naotaka HamasakiAbstractBand 3 mediates the Cl− and HCO3− exchange across the red blood cell membrane and plays a pivotal role for delivering oxygen appropriately to metabolically active tissues. For understanding molecular mechanisms, it is essential to know the structure and function relationship. In terrestrial environments, however, nobody could make good quality crystals of Band 3 for the X-ray crystallographic study.In this study, we purified the transmembrane domain of Band 3 from human red blood cells and crystallized the purified Band 3 without the Fab fragment at the International Space Station “KIBO” under microgravity environments.
  • Synthesis and purification of linkage-specific polyubiquitin chains of
           distinct length for structural studies
    • Abstract: Publication date: Available online 11 August 2018Source: Analytical BiochemistryAuthor(s): Anshumali Mittal, Binita ShakyaAbstractPolyubiquitylation is one of the most versatile post-translational modifications involved in the regulation of numerous intracellular signaling processes. An assembly procedure that is simple, robust, and efficient to synthesize and purify linkage-specific polyubiquitin chains of defined length at a preparative scale is required in biophysical and structural studies. Here, we have optimized known enzymatic procedures in the form of a protocol to obtain multi-milligrams of Lys48-and Lys63-linked polyubiquitin chain types with more than 99% purity. Mass spectrometry (ESI/MS) analysis of K48- and K63-linked diubiquitin confirmed that the enzymes used in the preparation generated homogeneous linkages with no promiscuity.
  • Continuous assays for meprin alpha and beta using prolyl tripeptidyl
           aminopeptidase (PtP) from Porphyromonas gingivalis
    • Abstract: Publication date: Available online 9 August 2018Source: Analytical BiochemistryAuthor(s): Anja Schulze, Michael Wermann, Hans-Ulrich Demuth, Tadashi Yoshimoto, Daniel Ramsbeck, Dagmar Schlenzig, Stephan SchillingAbstractCommon assays for endoprotease activity of meprin α and β are based on cleavage of internally quenched substrates. Although direct and convenient, for meprins these assays bear disadvantages such as, e.g., significant substrate inhibition or potential fluorescence quenching by compounds applied in inhibitor analysis. Here, we present a novel continuous assay by introducing an auxiliary enzyme, prolyl tripeptidyl aminopeptidase (PtP) and the chromogenic substrate KKGYVADAP-p-nitroanilide. We provide a quick strategy for expression and one-step-purification of the auxiliary enzyme. The enzyme kinetic data for meprin α and β suggest hyperbolic v/S-characteristics, the kinetic parameters of substrate conversion by meprin β were Km = 184 ± 32 μM and kcat = 20 ± 4 s−1. We also present conditions for the use of the fluorogenic substrate KKGYVADAP-AMC to assess meprin β activity. The assays were applied for determination of inhibitory parameters of the natural inhibitor actinonin and two recently published hydroxamates. Hence, we present two novel methods, which can be applied to assess inhibitory mechanism and potency with the attractive current drug targets meprin α and β. Furthermore, the assay might also provide implications for analysis of other endoproteases as well as their inhibitors.
  • Label-free aptasensors based on fluorescent screening assays for the
           detection of Salmonella typhimurium
    • Abstract: Publication date: Available online 3 August 2018Source: Analytical BiochemistryAuthor(s): Sathya Srinivasan, Ranganathan Velu, Maria C. DeRosa, Bhaskar Mohan MurariWe report two label-free fluorescent aptasensor methods for the detection of S. typhimurium. In the first method, we have used a ‘‘turn off’’ approach in which the aptamer is first intercalated with SYBR Green I (SG), leading to a greatly enhanced fluorescence signal. The addition of S. typhimurium (approximately 1530–96900 CFU/mL), which specifically binds with its aptamer and releases SG, leads to a linear decrease in fluorescence intensity. The lowest detection limit achieved with this approach was in the range of 733 CFU/mL. In the second method, a ‘‘turn on’’ approach was designed for S. typhimurium through the Förster resonance energy transfer (FRET) between Rhodamine B (RB) and gold nanoparticles (AuNPs). When the aptamer and AuNPs were mixed with RB, the fluorescence of RB was significantly quenched via FRET. The aptamer adsorbs to the AuNP surface to protect them from salt-induced aggregation, which leads to the fluorescence quenching of RB in presence of AuNPs. Upon the addition of S. typhimurium, S. typhimurium specifically binds with its aptamer and loses the capability to stabilize AuNPs. Thus, the salt easily induces the aggregation of AuNPs, resulting in the fluorescence recovery of the quenched RB. S. typhimurium concentrations ranging from 1530 to 96900 CFU/mL with the detection limit of 464 CFU/mL was achieved with this methodology. Given these data, some insights into the molecular interactions between the aptamer and the bacterial target are provided. These aptasensor methods also may be adapted for the detection of a wide variety of targets.Graphical abstractLabel-free fluorescent aptasensor methods are investigated in turn-off and turn-on modes to effectively detect Salmonlla typhimurium at low concentrations.Image 1
  • Immunochromatographic strip biosensor for the rapid detection of
           N-glycolylneuraminic acid based on aptamer-conjugated nanoparticle
    • Abstract: Publication date: Available online 20 July 2018Source: Analytical BiochemistryAuthor(s): Sheng Gong, Honglin Ren, Chao Lin, Pan Hu, Ruiyun Tian, Zengshan Liu, Yansong Li, Yu Zhou, Shiying LuAbstractN-glycolylneuraminic acid (Neu5Gc) is a type of sialic acid that is not typically produced in healthy humans but detective in some visceral cancer cells. As a new carcinoma biomarker, the level change in the serum and urine from the patient could potentially have the relation to the disease progression. So the measurement of the Neu5Gc will help to take a better response to therapeutic schedule for the sufferers. A sensitive and rapid aptamer-nanoparticle immunochromatographic strip for the visual detection of Neu5Gc was developed. The assay is based on the competitive reaction of binding the DNA aptamer targeting the candidate molecule selected by SELEX between Neu5Gc and complementary DNA. The sensing results indicated that the aptamer-based strip was sufficiently sensitive to detect Neu5Gc. The visual limit of detection (LOD) for semi-quantitative detection was 30 ng/mL under the optimal conditions and a quantitative detection limit of 5.38 ng/mL could be obtained using a scanning strip reader. The average recovery of the spiked cancer cell samples was 88.86%, with a coefficient of variation (CV) of 5.27%. The detection could be performed in less than 15 min using a simple procedure without any complicated equipment, demonstrating that this aptamer-nanoparticle biosensor strip has great potential for use to Neu5Gc-related cancer diagnosis.
  • 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 BeldAbstractThe 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.
  • 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. KellyToll-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. WheelerAbstractMeasurement 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.
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