How versatile is the use of ultrafiltration to study biointeractions of therapeutic metallodrugs?
Abstract For a representative number of approved or investigational anticancer metallodrugs varying in lipophilicity, unspecific adsorption onto ultracentrifugal filter units was studied. It was found that for fairly hydrophilic compounds, such as cisplatin and oxaliplatin, the binding to filters does not substantially affect their amount measured (by ICP-MS) after ultrafiltration (>95%). In the case of metal complexes with moderate lipophilicity (log P> -0.1), adsorption effects turn out to be substantial. This might impede using ultrafiltration for studying the transformations of such drugs in human serum,...
Source: Analytical Biochemistry - March 26, 2020 Category: Biochemistry Authors: Foteeva LS, Kuznetsova OV, Keppler BK Tags: Anal Biochem Source Type: research

Possible Artefacts of Antioxidant Assays Performed in the Presence of Nitroxides and Nitroxide-containing Nanoparticles.
Abstract Nitroxides and nitroxide-containing nanoparticles (RNP) are excellent antioxidants. However, they have relatively high reduction potentials, which make them behave like oxidants or show little activity in some antioxidant assays. We found that stable nitroxyl radicals (TEMPO and 4-amino-TEMPO) has low reactivity in the test of scavenging of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS•). As a result, supplementation of blood plasma with nitroxides may decrease the total antioxidant capacity of blood plasma assayed with ABTS•. Nitroxides oxidize Fe2+ and in this way interfe...
Source: Analytical Biochemistry - March 25, 2020 Category: Biochemistry Authors: Pichla M, Bartosz G, Pieńkowska N, Sadowska-Bartosz I Tags: Anal Biochem Source Type: research

Identifying transglutaminase reaction products via mass spectrometry as exemplified by the MUC2 mucin - Pitfalls and traps.
This article describes a workflow for the detection of the products of transglutaminase-catalyzed reactions. In particular, possible pitfalls and traps that can arise during the mass spectrometry-based identification of isopeptide cross-links are addressed and characterised on actual samples. PMID: 32222540 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry)
Source: Analytical Biochemistry - March 25, 2020 Category: Biochemistry Authors: Arike L, Hansson GC, Recktenwald CV Tags: Anal Biochem Source Type: research

FTIR-based L-Asparaginase activity assay enables continuous measurements in optically dense media including blood plasma.
Abstract Complex heterogeneous systems, such as micelles or blood plasma, represent a particularly challenging environment to measure the catalytic parameters of some enzymes, including L-asparaginase. Existing methods are strongly interfered by the presence of plasma proteins, amino acids, as well as other components of plasma. Here we show that FTIR spectroscopy enables continuous real-time measurement of catalytic activity of L-asparaginase, in native and in PEG-chitosan conjugated form, in aqueous solutions as well as in heterogeneous non-transparent multicomponent systems, including colloidal systems or blood...
Source: Analytical Biochemistry - March 23, 2020 Category: Biochemistry Authors: Kudryashova EV, Pokrovskaya MV, Alexandrova SS, Vinogradov AA, Sokolov NN Tags: Anal Biochem Source Type: research

A Low-cost Paper-based Aptasensor for Simultaneous Trace-level Monitoring of Mercury (II) and Silver (I) ions.
This study introduces a low-cost paper-based aptasensor to monitor Hg2+ and Ag+, simultaneously. The strategy of the sensing array is according to the conformational changes of Hg2+- and Ag+-specific aptamers and their release from the GO surface after the injection of the target sample on the sensing platform. Through monitoring the fluorescence recovery changes against the concentrations of the ions, Hg2+ and Ag+ can be determined as low as 1.33 and 1.01 pM. The paper-based aptasensor can simultaneously detect the ions within about 10 minutes. The aptasensor is applied prosperously to monitor Hg2+ and Ag+ in human serum,...
Source: Analytical Biochemistry - March 19, 2020 Category: Biochemistry Authors: Khoshbin Z, Housaindokht MR, Verdian A Tags: Anal Biochem Source Type: research

Very long-term stability of lipid biomarkers in human serum.
In this study, the stability of five lipid parameters (total cholesterol, triglycerides, HDL- and LDL-cholesterol and free fatty acids) has been tested in 16 human serum samples after storage at -80 oC up at time points 0, 1, 8 and 13 y. The majority of the lipid biomarkers were stable during storage conditions, except for cholesterol. The correlations between the samples were very good at time points. Therefore, long-term storage of human serum samples allows lipid biomarker determination, provided that the samples are stored at -80 °C. PMID: 32201135 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry)
Source: Analytical Biochemistry - March 19, 2020 Category: Biochemistry Authors: Muzakova V, Beekhof PK, Jansen EHJM Tags: Anal Biochem Source Type: research

Reliability factor for identification of amylolytic enzyme activity in the optimized starch-iodine assay.
Abstract Amylolytic enzymes are a group of proteins degrading starch to its constitutional units. For high-throughput screening, simple yet accurate methods in addition to the reducing ends assays are required. In this article, the iodine assay, a photometric assay based on the intensely colored starch-iodine complex, was adapted to enable accurate and objective differentiation between enzyme and background activity using a newly introduced mathematical factor. The method was further improved by designing a simple setup for multiple time point detection and discussing the applicability of single wavelength measure...
Source: Analytical Biochemistry - March 19, 2020 Category: Biochemistry Authors: Gaenssle ALO, van der Maarel MJEC, Jurak E Tags: Anal Biochem Source Type: research

Study of imidazole performance as pseudo-affinity ligand in the purification of IgG from bovine milk.
Abstract The spherical sepharose CL-6B beads were activated by epichlorohydrin in different epoxy contents (80, 120 and 160 μmolepoxide/mLgel) and, L-histidine and imidazole as pseudo-affinity ligands were covalently immobilized to them. Some linkers with different length, (1,2-ethanediol diglycidyl ether and 1,4-butanediol diglycidyl ether) were synthesized for activation of sepharose and the activated sepharose beads modified with imidazole and the performance of these adsorbents in the purification of immunoglobulin G from bovine milk were evaluated. Among the L-histidine bearing adsorbents, higher adsorptio...
Source: Analytical Biochemistry - March 19, 2020 Category: Biochemistry Authors: Pourrostam-Ravadaneg P, Safa KD, Abbasi H Tags: Anal Biochem Source Type: research

Introduction of a New Method for Two-Dimensional NMR Quantitative Analysis in Metabolomics Studies.
In this study, instead of using the traditional methods of calculating the 2D NMR data to search for the important features, a new procedure, which applies the high-resolution 1D NMR metabolites chemical shift range to filter the 2D NMR data, was developed. This new method was demonstrated using both a mixture of standard metabolites and a case study on plant extracts using 2D non-uniform sampling (NUS) total correlation spectroscopy (TOCSY) data. As a result, our method successfully filtered out the important features with a high success rate, and the extracted peaks showed high linearity between the calculated intensitie...
Source: Analytical Biochemistry - March 17, 2020 Category: Biochemistry Authors: Jiang L, Howlett K, Patterson K, Wang B Tags: Anal Biochem Source Type: research

Biomolecular condensates in cell biology and virology: phase-separated membraneless organelles (MLOs).
Abstract Membraneless organelles (MLOs) in the cytoplasm and nucleus in the form of 2D and 3D phase-separated biomolecular condensates are increasingly viewed as critical in regulating diverse cellular functions. These functions include cell signaling, immune synapse function, nuclear transcription, RNA splicing and processing, mRNA storage and translation, virus replication and maturation, antiviral mechanisms, DNA sensing, synaptic transmission, protein turnover and mitosis. Components comprising MLOs often associate with low affinity; thus cell integrity can be critical to the maintenance of the full complement...
Source: Analytical Biochemistry - March 16, 2020 Category: Biochemistry Authors: Sehgal PB, Westley J, Lerea KM, DiSenso-Browne S, Etlinger JD Tags: Anal Biochem Source Type: research

Simultaneous monitoring of the bioconversion from lysine to glutaric acidby ethyl chloroformate derivatization and gas chromatography-mass spectrometry.
Abstract Glutaric acid is a precursor of a plasticizer that can be used for the production of polyester amides, ester plasticizer, corrosion inhibitor, and others. Glutaric acid can be produced either via bioconversion or chemical synthesis, and some metabolites and intermediates are produced during the reaction. To ensure reaction efficiency, the substrates, intermediates, and products, especially in the bioconversion system, should be closely monitored. Until now, high performance liquid chromatography (HPLC) has generally been used to analyze the glutaric acid-related metabolites, although it demands separate t...
Source: Analytical Biochemistry - March 16, 2020 Category: Biochemistry Authors: Han YH, Choi TR, Park YL, Song HS, Choi YK, Kim HJ, Bhatia SK, Gurav R, Park K, Park SH, Kim W, Yang YH Tags: Anal Biochem Source Type: research

Re-evaluation of the 18 non-human protein standards used to create the Empirical Statistical Model for Decoy Library Searching.
Abstract The Empirical Statistical Model (ESM) for decoy library searching fused the expected amino acid sequence of 18 non-human protein standards to a human decoy library. The ESM assumed a priori the standards were pure such that only the 18 nominal proteins were true positive, all other proteins were false positive, there was no overlap in the peptides of non-human proteins versus human proteins, and that the score distribution of individual peptides would resolve true positive from false positive results or noise. The results of random and independent sampling by LC-ESI-MS/MS indicated that the fundamental as...
Source: Analytical Biochemistry - March 16, 2020 Category: Biochemistry Authors: Thavarajah T, Tucholska M, Zhu PH, Bowden P, Marshall JG Tags: Anal Biochem Source Type: research

Development of a LAMP method for detecting the N75S mutant in SDHI-resistant Corynespora cassiicola.
Abstract The replacement of asparagine with serine at codon 75 of the sdhC gene (N75S) confers succinate dehydrogenase inhibitor resistance in Corynespora cassiicola, which caused by consecutive fungicide application. To rapidly detect the mutation of N75S, a method based on loop-mediated isothermal amplification (LAMP) was developed in this study. The optimal primer set among the six primer sets designed could clearly identify N75S from the wild-type genotype. The detection threshold of the optimized LAMP mixture (10 μL) was 8.8 fg of target DNA at 63°C within 60 min. This method specifically showed a colo...
Source: Analytical Biochemistry - March 11, 2020 Category: Biochemistry Authors: Zhu J, Zhang L, Li H, Gao Y, Mu W, Liu F Tags: Anal Biochem Source Type: research

Fluorescence-based biochemical analysis of human hepatitis B virus reverse transcriptase activity.
Abstract Although the unique mechanism by which hepatitis B virus (HBV) polymerase primes reverse transcription is now well-characterized, the subsequent elongation process remains poorly understood. Reverse transcriptase (RT)-RNase H sequences from polymerase amino acid 304 (the C-terminal part of spacer domain) to 843 were expressed in Escherichia coli and purified partially. RT elongation activity was investigated using the fluorescent-tagged primer and homopolymeric RNA templates. RT elongation activity depended on both Mg2+ and Mn2+, and had low affinity for purine deoxynucleotides, which may be related with ...
Source: Analytical Biochemistry - March 11, 2020 Category: Biochemistry Authors: Toyoda T, Wang Y, Wen Y, Tanaka Y Tags: Anal Biochem Source Type: research

Improving the stability of the malachite green method for the determination of phosphate using Pluronic F68.
z Flecha FL Abstract The analytical determination of phosphate constitutes a fundamental step for the evaluation of catalytic activities of many enzymes involved in the hydrolysis of phosphate-containing biomolecules. The most sensitive colorimetric methods to quantify phosphate are based on measuring the spectral changes produced by the adsorption of malachite green to 12-molybdophosphoric acid. Malachite green methods are time-consuming because they require the preparation of color reagent on the day of use, due to its low stability. In this work we propose a modification of the malachite green method that overc...
Source: Analytical Biochemistry - March 9, 2020 Category: Biochemistry Authors: Martínez Gache SA, Recoulat Angelini AA, Sabeckis ML, González Flecha FL Tags: Anal Biochem Source Type: research

Establishment of a 1, 4, 7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) based lectin microarray for efficiently detecting serum glycans in gastric cancers.
In this study, we took use of the molecular self-assembled monolayer technique to modify a gold surface with the reagent 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) in combination with 16-amino-1-hexadecanethiol hydrochloride. Cross-linking effect of DOTA-NHS-ester is brought about via activating three -OH ends to three terminals of succinylimidines, making selective binding of the terminal amino groups in proteins possible. We immobilized ten commercial lectins on the platform and measured changes of serum lectin-matched glycans in patients with gastric cancer...
Source: Analytical Biochemistry - March 7, 2020 Category: Biochemistry Authors: Gao Y, Li SG, Liu Q, Liu SS, Ye L, Song ZJ, Du WD Tags: Anal Biochem Source Type: research

Metabolomic studies of amino acid analysis in Saccharomyces cells exposed to selenium and gamma irradiation.
al ZL Abstract Metabolomic studies are essential to identify and quantify key metabolites in biological systems. Analysis of amino acids (AA) is very important in target metabolomics studies. Chromatographic methods are used to support metabolite determinations. Therefore, this work presents analysis of 17 AA in Saccharomyces cerevisiae cells (a useful model in the study of cancer metabolism) exposed to sodium selenite and gamma radiation. An improved GC/MS method using propyl chloroformate/propanol as derivatizing reagent was applied to AA determinations. The method exhibited good linearity in the range of 0.08 t...
Source: Analytical Biochemistry - March 3, 2020 Category: Biochemistry Authors: Batista JM, Neves MJ, Pereira AG, Gonçalves LS, Menezes HC, Cardeal ZL Tags: Anal Biochem Source Type: research

Comparative metabolism of schaftoside in healthy and calcium oxalate kidney stone rats by UHPLC-Q-TOF-MS/MS method.
In this study, a six-step strategy was first developed to detect and identify the metabolites in plasma, urine, bile, feces and rat intestinal bacteria samples of healthy and model rats administrated with schaftoside using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). The number and the relative peak area of metabolites in healthy rats and model rats were compared, and it was noticed that metabolites in bio-samples of healthy and model rats both had obvious differences. A total of 28 metabolites of schaftoside in healthy rats and 30 metabolites in m...
Source: Analytical Biochemistry - March 3, 2020 Category: Biochemistry Authors: Liu R, Meng C, Zhang Z, Ma H, Lv T, Xie S, Liu Y, Wang C Tags: Anal Biochem Source Type: research

Imaging of the ex vivo transglutaminase activity in liver macrophages of sepsis mice.
Abstract Sepsis is the leading cause of death in hospitalized patients and is characterized by a dysregulated inflammatory response to infection and multiple organ failure, including the liver. Transglutaminase 2 (TG2) is--> a multifunctional enzyme that exhibits transamidase, GTPase, and integrin-binding activities and has opposing roles in the regulation of cell growth, differentiation, and apoptosis. TG2 plays both pathogenic and protective roles in liver diseases, revealing the need to examine the ex vivo activities of TG2. Here, we introduce an imaging approach to examine the ex vivo transamidase activity ...
Source: Analytical Biochemistry - March 3, 2020 Category: Biochemistry Authors: Su T, Qin XY, Furutani Y, Yu W, Kojima S Tags: Anal Biochem Source Type: research

Cross-priming isothermal amplification combined with nucleic acid test strips for detection of meat species.
Abstract Adulteration of high-quality meat with their cheaper counterparts can be minimized by rapid and reliable methods for detecting meat species. Here an isothermal cross-primer amplification (CPA) technique combined with colloidal gold nucleic acid test strips (CPA strips) was developed to differentiate cow, sheep, arctic fox, and pig meat. A simple primer design for multiplex differentiation using a universal single-labeled CPA primer system and four detection-level species-specific labeling primers were analyzed by colloidal gold-based test strip assay. Moreover, simultaneous detection of fox and pig meat o...
Source: Analytical Biochemistry - March 2, 2020 Category: Biochemistry Authors: Zheng F, Li S, Wang S, Feng T, Jiang Z, Pan J Tags: Anal Biochem Source Type: research

Quick and convenient construction of lambda-cyhalothrin antigen for the generation of specific antibody.
In this study, we developed a one-step lambda-cyhalothrin hapten synthesis that transformed the cyanide group in lambda-cyhalothrin to amide. Complete antigen was assembled by coupling the amide with succinic-anhydride-activated carrier proteins, and corresponding polyclonal antibodies were generated using Balb/c mice. Using antibody generated by the method in this paper, the competitive ELISA demonstrated the lowest detection limit of 3.772 μg/L for lambda-cyhalothrin, and no significant cross-reactivity for other pyrethroid pesticides was observed. All the results suggested we have established a more efficient techniq...
Source: Analytical Biochemistry - February 29, 2020 Category: Biochemistry Authors: Cui N, Cao L, Sui J, Lin H, Han X, Chen X, Xie H, Sun X Tags: Anal Biochem Source Type: research

Glycopeptide variable window SWATH for improved Data Independent Acquisition glycoprotein analysis.
Abstract N-glycosylation plays an essential role in regulating protein folding and function in eukaryotic cells. Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH) has proven useful as a data independent acquisition (DIA) MS method for analysis of glycoproteins and their glycan modifications. By separating the entire m/z range into consecutive isolation windows, DIA-MS allows comprehensive MS data acquisition and high-sensitivity detection of molecules of interest. Variable width DIA windows allow optimal analyte measurement, as peptide ions are not evenly distributed a...
Source: Analytical Biochemistry - February 28, 2020 Category: Biochemistry Authors: Zhou C, Schulz BL Tags: Anal Biochem Source Type: research

Fast room temperature cloud point extraction procedure for spectrophotometric determination of phosphate in water samples.
Abstract A novel fast room temperature cloud point extraction (RT-CPE) procedure for preconcentration and spectrophotometric determination of phosphate based on the heteropoly blue formation was developed. The proposed method includes the formation of yellow molybdoantymonatophosphoric heteropoly complex, its extraction into Triton X-100 micellar phase obtained at room temperature and reduction of heteropoly complex by ascorbic acid solution in ethanol and absorbance measurement of heteropoly blue at 790 nm. Under optimal conditions (1% (v/v) of Triton X-100 and 0.05 M of ammonium benzoate for initiating of RT-CPE...
Source: Analytical Biochemistry - February 28, 2020 Category: Biochemistry Authors: Snigur D, Chebotarev A, Bulat K, Duboviy V Tags: Anal Biochem Source Type: research

Analysis of testosterone-hydroxylated metabolites in human urine by Ultra High Performance Liquid Chromatography-Mass Spectrometry.
In conclusion, the UPLC-MS method developed here was validated for the analysis of testosterone-hydroxylated metabolites in human urine. PMID: 32119849 [PubMed - as supplied by publisher] (Source: Analytical Biochemistry)
Source: Analytical Biochemistry - February 28, 2020 Category: Biochemistry Authors: Escobar-Wilches DC, Ventura-Bahena A, de Lourdes López-González M, Torres-Sánchez L, Figueroa M, Sierra-Santoyo A Tags: Anal Biochem Source Type: research

Quantitative structure-chromatographic retention relationship of synthesized peptides (HGRFG, NPNPT) and their derivatives.
Abstract Carapax Trionycis extract peptides (HGRFG, NPNPT) are able to protect against CCl4-induced liver fibrosis. Therefore, this study applies to deal with chromatographic lipophilicity determination of synthesized peptides (HGRFG, NPNPT) and their derivatives using reversed-phase high performance liquid chromatography (RP-HPLC) combined with methanol-water mobile phase and two reversed-phase chromatographic columns (COSMOISL 5C18-MS-II and SHIMADZU-C18). The chromatographic lipophilicity of the analyzed compounds was expressed as logkw constant and correlated with lipophilicity descriptors. Quantitative struct...
Source: Analytical Biochemistry - February 27, 2020 Category: Biochemistry Authors: Yang X, Peng H, Han N, Zhang Z, Bai X, Zhao T, Zhao J, Liu J Tags: Anal Biochem Source Type: research

Reporter cell assay-based functional quantification of TNF- α-antagonists in serum -- a proof-of-principle study for adalimumab.
Reporter cell assay-based functional quantification of TNF-α-antagonists in serum -- a proof-of-principle study for adalimumab. Anal Biochem. 2020 Feb 26;:113646 Authors: Schuster L, Sauter M, Uhl P, Meid A, Haefeli WE, Weiss J, Theile D Abstract Quantification of therapeutic antibodies is commonly based on physico-chemical assays such as enzyme-linked immunoabsorption assays (ELISA) and lately on mass spectrometry. However, the functional integrity of evaluated immunoglobulins is yet not assessed. Consequently, a commercially available reporter cell line was used to quantify the functional conc...
Source: Analytical Biochemistry - February 26, 2020 Category: Biochemistry Authors: Schuster L, Sauter M, Uhl P, Meid A, Haefeli WE, Weiss J, Theile D Tags: Anal Biochem Source Type: research

Screening and Quantification of TNF- α Ligand from Angelicae Pubescentis Radix by Biosensor and UPLC-MS/MS.
Screening and Quantification of TNF-α Ligand from Angelicae Pubescentis Radix by Biosensor and UPLC-MS/MS. Anal Biochem. 2020 Feb 25;:113643 Authors: Yang L, Hou A, Wang S, Zhang J, Man W, Guo X, Yang B, Wang Q, Jiang H, Kuang H Abstract The aim of this study is to establish a method for rapid screening of active ingredients targeting TNF-α from Chinese herbal medicines. Take Angelicae Pubescentis Radix (APR) as an example, surface plasma resonance technique was used to establish for screening small molecule inhibitors of TNF-α from APR extract. Then UPLC-MS/MS coupled with chemometr...
Source: Analytical Biochemistry - February 25, 2020 Category: Biochemistry Authors: Yang L, Hou A, Wang S, Zhang J, Man W, Guo X, Yang B, Wang Q, Jiang H, Kuang H Tags: Anal Biochem Source Type: research

Flavonoids Interference in Common Protein Assays: Effect of Position and Degree of Hydroxyl Substitution.
Abstract Flavonoids interferes with colorimetric protein assays in a concentration- and structure-dependent manner. Degree (≥3) and position (C3) of -OH substitution was associated with intensified interference (p5μM) and is particularly evident at lower protein concentrations (25-250 μg/ml). Since, healthy human urinary protein (
Source: Analytical Biochemistry - February 24, 2020 Category: Biochemistry Authors: Singh R, Lu R, Hu M Tags: Anal Biochem Source Type: research

Some Novel Rare Earth Metal Ions complexes: Synthesis, Characterization, Luminescence and Biocidal Efficiency.
Abstract New lanthanide complexes (1-3) of the general formulae [Ln(L)(NO3)(H2O)] have been synthesized by reaction of Ln(NO3)3.xH2O {Ln = La (1), Sm (2) and Yb (3)} with 2,2'-(((1E,1'E)-thiophene-2,5-diylbis(methaneylylidene))bis(azaneylylidene))diphenol (H2L). Based on elemental analysis and spectroscopic analysis (UV-Visible, FT-IR, ESI-MS 1H and 13C NMR), molar conductance and thermogravimetric analysis. The results showed that the Schiff base ligand coordinated Ln(III) ions through the azomethine nitrogens, deprotonated hydroxyl groups, and thiophene sulphur atom. The interaction of the synthetic compounds wi...
Source: Analytical Biochemistry - February 24, 2020 Category: Biochemistry Authors: Abdel Aziz AA, Sayed MA Tags: Anal Biochem Source Type: research

Innovation in the food industry using microbial transglutaminase: Keys to success and future prospects.
Abstract Transglutaminase (TG) catalyzes cross-linking between the γ-carboxyamide groups of glutamine residues and the ε-amino groups of lysine residues in polypeptide chains, yielding ε- (γ-glutamyl) lysine (G-L) bonds. By forming a network structure in the protein via G-L bonds, it is possible to increase the viscosity of protein solutions or to cause gelation. Nearly thirty years have passed since microbial TG (MTG) appeared in the food enzyme market. Since the start of research and development, MTG has been used in fishery products such as kamaboko (boiled fish paste), meat products...
Source: Analytical Biochemistry - February 22, 2020 Category: Biochemistry Authors: Miwa N Tags: Anal Biochem Source Type: research

Determination of homocysteine thiolactone in human urine by capillary zone electrophoresis and single drop microextraction.
Abstract A simple, fast, sensitive and reproducible capillary zone electrophoresis (CZE) method with single drop microextraction (SDME) for determination of homocysteine thiolactone (HTL) in human urine has been developed and validated. The method is characterized by good precision, high accuracy, short analysis time and low consumption of reagents. The procedure consists only of few steps: urine sample centrifugation, dilution with phosphate buffer and methanol, chloroform addition onto the top of donor phase, on-line SDME in CE system, sample separation by CZE and ultraviolet detection of HTL at 240 nm. The ba...
Source: Analytical Biochemistry - February 21, 2020 Category: Biochemistry Authors: Purgat K, Olejarz P, Kośka I, Głowacki R, Kubalczyk P Tags: Anal Biochem Source Type: research

Glycoproteomic measurement of site-specific polysialylation.
Abstract Polysialylation is the enzymatic addition of a highly negatively charged sialic acid polymer to the non-reducing termini of glycans. Polysialylation plays an important role in development, and is involved in neurological diseases, neural tissue regeneration, and cancer. Polysialic acid (PSA) is also a biodegradable and non-immunogenic conjugate to therapeutic drugs to improve their pharmacokinetics. PSA chains vary in length, composition, and linkages, while the specific sites of polysialylation are important determinants of protein function. However, PSA is difficult to analyse by mass spectrometry (MS) ...
Source: Analytical Biochemistry - February 20, 2020 Category: Biochemistry Authors: Pelingon R, Pegg CL, Zacchi LF, Phung TK, Howard CB, Xu P, Hardy MP, Owczarek CM, Schulz BL Tags: Anal Biochem Source Type: research

Advances of aptamers screened by Cell-SELEX in selection procedure, cancer diagnostics and therapeutics.
Abstract Aptamers are a class of short artificial single-stranded oligo(deoxy) nucleotides that can bind to different targets, which generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Due to excellent selectivity and high affinity to targets, aptamers hold considerable potential as molecular probe in diverse applications ranging from ensuring food safety, monitoring environment, disease diagnosis to therapy. This review highlights recent development and challenges about aptamers screened by Cell-SELEX, and its application about cancer diagnostics and therapeutics. Advances about some o...
Source: Analytical Biochemistry - February 19, 2020 Category: Biochemistry Authors: Zhong Y, Zhao J, Chen F Tags: Anal Biochem Source Type: research

Bona fide gene expression analysis of samples from the bovine reproductive system by microfluidic platform.
ra MF Abstract Sample types such as those from reproductive systems often yield scarce material, which limits RT-qPCR analysis to only a few targets. Recently developed high-throughput systems can potentially change this scenario, however, the nanoliter scale of such platforms requires extra processing, e.g., preamplification, which needs to be defined through observation and experience. In order to establish best practices in high-throughput PCR approaches using samples from reproductive systems, we evaluated the Biomark™ HD performance using 11 different sample types from the bovine reproductive system: bl...
Source: Analytical Biochemistry - February 19, 2020 Category: Biochemistry Authors: Fontes PK, César de Souza Castilho A, Razza EM, Gouveia Nogueira MF Tags: Anal Biochem Source Type: research

Lateral flow assay using aptamer-based sensing for on-site detection of dopamine in urine.
Abstract A lateral flow assay using DNA aptamer-based sensing for the detection of dopamine in urine is reported. Dopamine duplex aptamers (hybridized sensor with capture probe) are conjugated to 40-nm Au nanoparticles (AuNPs) with 20T linkers. The detection method is based on the dissociation of the duplex aptamer in the presence of dopamine, with the sensor part undergoing conformational changes and being released from the capture part. Hybridization between the complementary DNA in the test line and the conjugated AuNP-capture DNA produces a red band, whose intensity is related to the dopamine concentration. Th...
Source: Analytical Biochemistry - February 19, 2020 Category: Biochemistry Authors: Dalirirad S, Steckl AJ Tags: Anal Biochem Source Type: research

Re-track: Software to analyze the retraction and protrusion velocities of neurites, filopodia and other structures.
l E Abstract We have developed new software, Re-track, that will quantify the rates of retraction and protrusion of structures emanating from the central core of a cell, such as neurites or filopodia. Re-Track, uses time-lapse images of cells in TIFF format and calculates the velocity of retraction or protrusion of a selected structure. The software uses a flexible moving boundary and has the ability to correct this boundary throughout analysis. Re-Track is fast, platform independent, and user friendly, and it can be used to follow biological events such as changes in neuronal connections, tip-growing cells such a...
Source: Analytical Biochemistry - February 17, 2020 Category: Biochemistry Authors: Mousavi SI, Peace KM, Scarlata S, Tüzel E Tags: Anal Biochem Source Type: research

A sensitive LC-MS assay using derivatization with boron trifluoride to quantify curcuminoids in biological samples.
Abstract A procedure is described to measure curcumin (C), demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), tetrahydrocurcumim (TC) and their glucuronidated metabolites (CG, DMCG, and BDMCG) in plasma, brain, liver and tumor samples. The procedure involves converting the analytes to their boron difluoride derivatives and analyzing them by combined liquid chromatography coupled to an ion trap mass spectrometer operating in the negative ion MSn scan mode. The method has superb limits of detection of 0.01 nM for all curcuminoids and 0.5 nM for TC and the glucuroniated metabolites, and several representative ...
Source: Analytical Biochemistry - February 17, 2020 Category: Biochemistry Authors: Yoon AJ, Wu H, Pan RD, Teter B, Cipolla J, Chang E, Avila LZ, Basak SK, Srivatsan E, Wang MB, Cole GM, Frautschy SA, Hampton PD, Faull KF Tags: Anal Biochem Source Type: research

Insights on the heparan sulphate-dependent externalisation of transglutaminase-2 (TG2) in glucose-stimulated proximal-like tubular epithelial cells.
Abstract The extracellular matrix crosslinking enzyme transglutaminase 2 (TG2) is highly implicated in tissue fibrosis that precedes end-stage kidney failure. TG2 is unconventionally secreted through extracellular vesicles in a way that depends on heparan sulphate (HS) proteoglycan syndecan-4 (Sdc4), the deletion of which reduces experimental kidney fibrosis as a result of lower extracellular TG2 in the tubule-interstitium. Here we establish a model of TG2 externalisation in NRK-52E tubular epithelial cells subjected to glucose stress. HS-binding TG2 mutants had reduced extracellular TG2 in transfected NRK-52E, su...
Source: Analytical Biochemistry - February 16, 2020 Category: Biochemistry Authors: Furini G, Burhan I, Huang L, Savoca MP, Atobatele A, Johnson T, Verderio E Tags: Anal Biochem Source Type: research

A high affinity modified DNA aptamer containing base-appended bases for human β-defensin.
A high affinity modified DNA aptamer containing base-appended bases for human β-defensin. Anal Biochem. 2020 Feb 15;:113627 Authors: Minagawa H, Kataoka Y, Kuwahara M, Horii K, Shiratori I, Waga I Abstract We used base-appended base modification to develop a new adenine analog, which incorporates an adenine derivative at position 7 of adenine. Using the systematic evolution of ligands by exponential enrichment method with a modified DNA library including this analog, we obtained Aad1, an aptamer that binds strongly to human β-defensin 2, a biomarker of physical stress found in saliva. The di...
Source: Analytical Biochemistry - February 15, 2020 Category: Biochemistry Authors: Minagawa H, Kataoka Y, Kuwahara M, Horii K, Shiratori I, Waga I Tags: Anal Biochem Source Type: research

Automated determination of early eluting oligonucleotide impurities using ion-pair reversed-phase liquid chromatography high resolution-mass spectrometry.
Abstract A new method is presented for the automated determination of early eluting (E.E.) oligonucleotide impurities analyzed by IP-RP HPLC HRMS. E.E. are impurities shorter than the main drug component and are primarily formed by the mechanisms of coupling failure, and depurination. The method is based on the detection of the theoretically derived most abundant mass of an impurity in the experimental data. An exhaustive list of candidate impurities and their formulas is automatically generated using the parent sequence and the known mechanisms of impurity formation. The approach accounts for possible modificatio...
Source: Analytical Biochemistry - February 14, 2020 Category: Biochemistry Authors: Roussis SG, Cedillo I, Rentel C Tags: Anal Biochem Source Type: research

2-Phenoxyethanol: A novel reagent for improved sensitivity of carbohydrate quantification.
Abstract Anthrone is a routinely used reagent for estimating carbohydrates (Polysaccharides) in research, development and pharmaceutical applications. In presence of sulphuric acid, the polysaccharide gets hydrolyzed to monosaccharides in the form of hydroxymethyl furfural or furfural. The furfural then reacts with anthrone to form a green color complex with a maximum absorbance at 625 nm. Though anthrone reacts well with polysaccharides containing hexoses (such as glucose and galactose) and rhamnose, it is less reactive with uronic acids (such as glucuronic acid and galacturonic acid) and hexosamines (such as f...
Source: Analytical Biochemistry - February 14, 2020 Category: Biochemistry Authors: Rajendar B, Mulagalapati R, Reddy MVNJ, Patri S, Karthik YK, Matur RV Tags: Anal Biochem Source Type: research

Identification and characterization of substrates crosslinked by transglutaminases in liver and kidney fibrosis.
Abstract The transglutaminase (TGase) family consists of eight isozymes that catalyze the Ca2+-dependent crosslink formation between the glutamine and lysine residues of proteins. In the pathogenesis of various chronic diseases, among the TGase isozymes, TG2 in particular was upregulated and contributed to a critical role in fibrosis development and progression via the stabilization of extracellular matrix proteins and activation of TGF-β. Although TG2 has been considered a key enzyme in fibrosis, the causative role of TG2 and involvement of other isozymes remain unclear. We have recently developed a comprehe...
Source: Analytical Biochemistry - February 13, 2020 Category: Biochemistry Authors: Tatsukawa H, Takeuchi T, Shinoda Y, Hitomi K Tags: Anal Biochem Source Type: research

A four-channel paper microfluidic device with gold nanoparticles and aptamers for seized drugs.
Abstract In this note we describe a four-channel paper microfluidic device (μPAD) that utilizes gold nanoparticles (AuNPs) as colorimetric sensors and specifically tailored aptamers to capture a set of targeted drugs, including cocaine, codeine and methamphetamine. The design utilizes salt-induced aggregation of AuNPs to produce a color change that indicates the presence of target analytes. In the absence of the targets, the aptamers bind to AuNPs, preventing aggregation. This μPAD was developed by optimizing the concentrations of aggregating agents, AuNPs and aptamers. The resultant device is sensitive and ...
Source: Analytical Biochemistry - February 11, 2020 Category: Biochemistry Authors: Wang L, McCord B Tags: Anal Biochem Source Type: research

Bovine serum albumin determination based on methylene blue detection by photothermal lens spectroscopy.
We report on a new sensitive method, for bovine serum albumin quantification, which is based on the use of methylene blue as a labelling agent combined with photothermal lens detection. In the presence of sodium dodecyl sulfate, methylene blue forms a dimer which can react with bovine serum albumin producing dedimerization. We found that, the photothermal signal decreases proportional to the concentration of bovine serum albumin added to the system composed by dodecyl sulfate and methylene blue. Therefore, the change of the signal intensity is linearly proportional to the concentration of bovine serum albumin. Under the op...
Source: Analytical Biochemistry - February 11, 2020 Category: Biochemistry Authors: Nejad Asbaghi BA, Shokoufi N, Hajibaba SN Tags: Anal Biochem Source Type: research

Fluorescent probe displacement assays reveal unique nucleic acid binding properties of human nudix enzymes.
In this study we designed new fluorescence-based assays to examine the interaction of purified recombinant E. coli NudC and human Nudt1 (aka MTH1) Nudt3, Nudt12, Nudt16, and Nudt20 (aka Dcp2). All Nudix proteins except Nudt1 and Nudt12 bound both RNA and DNA stoichiometrically with high affinity (dissociation constants in the nanomolar range) and no clear sequence specificity. In stark contrast, Nudt12 binds RNA but not similar DNA oligonucleotides. Nudt12 also bound RNAs with 5' NAD+ caps more tightly than those with NADH or m7G cap. NudC was similarly selective against m7G caps but did not differentiate between NAD+ and ...
Source: Analytical Biochemistry - February 11, 2020 Category: Biochemistry Authors: Ray A, Frick DN Tags: Anal Biochem Source Type: research

Adaptation of a continuous, calorimetric kinetic assay to study the agmatinase-catalyzed hydrolytic reaction.
Abstract Ureohydrolases are members of the metallohydrolase family of enzymes. Here, a simple continuous assay for agmatinase (AGM) activity was established by following the degradation of agmatine to urea and putrescine using isothermal titration calorimetry (ITC). ITC is particularly useful for kinetic assays when substrates of interest do not possess suitable chromophores that facilitate the continuous spectrophotometric detection of substrate depletion and/or product formation. In order to assess the accuracy of the ITC-based assay, catalytic parameters were also determined using a discontinuous, colorimetric ...
Source: Analytical Biochemistry - February 10, 2020 Category: Biochemistry Authors: Wilson LA, Garcia D, Pedroso MM, Schulz BL, Guddat LW, Schenk G Tags: Anal Biochem Source Type: research

Dye binding assay reveals doxorubicin preference for DNA versus cardiolipin.
Abstract Doxorubicin (DOX) is a potent anticancer agent that binds both DNA and cardiolipin (CL). To investigate DOX binding to CL versus DNA, aqueous soluble, CL-enriched nanoparticles, termed nanodisks (ND), were employed. Upon incubation with CL-ND, but not with phosphatidylcholine ND, DOX binding was detected. DOX binding to CL-ND was sensitive to buffer pH and ionic strength. To investigate if a DOX binding preference for DNA versus CL-ND exists, an agarose gel-based dye binding assay was developed. Under conditions wherein the commercial fluorescent dye, GelRed, detects a 636 bp DNA template following electr...
Source: Analytical Biochemistry - February 8, 2020 Category: Biochemistry Authors: Fox CA, Ryan RO Tags: Anal Biochem Source Type: research

Solution-phase synthesis of the fluorogenic TGase 2 acyl donor Z-Glu(HMC)-Gly-OH and its use for inhibitor and amine substrate characterisation.
r R Abstract A reliable solution-phase synthesis of the water-soluble dipeptidic fluorogenic transglutaminase substrate Z-Glu(HMC)-Gly-OH is presented. The route started from Z-Glu-OH, which was converted into the corresponding cyclic anhydride. This building block was transformed into the regioisomeric α- and γ-dipeptides. The key step was the esterification of Z-Glu-Gly-OtBu with 4-methylumbelliferone. The final substrate compound was obtained in an acceptable yield and excellent purity without the need of purification by RP-HPLC. The advantage of this acyl donor substrate for the kinetic characteris...
Source: Analytical Biochemistry - February 8, 2020 Category: Biochemistry Authors: Wodtke R, Pietsch M, Löser R Tags: Anal Biochem Source Type: research

Advances in fluorescent probes for detection and imaging of endogenous tyrosinase activity.
Abstract Enzymes are a class of important substances and their abnormal levels are associated with many diseases. Great progress has been achieved in the past decade in the detection and imaging of enzymes in live cells and tissues. Small-molecule fluorescent probes have the ability to directly detect and image abnormal levels of enzymes in live cells, providing a powerful means for diagnostics and treatment. Combined with confocal microscopy, fluorescent probes have attracted much attention because of their high sensitivity and spatiotemporal resolution. Tyrosinase is a copper-containing oxidase that plays a key ...
Source: Analytical Biochemistry - February 6, 2020 Category: Biochemistry Authors: Chen Y Tags: Anal Biochem Source Type: research

An automated workflow approach for the analysis of flavin adenine dinucleotide by HPLC with internal standard.
In this study we have developed a fully automated method for the analysis of FAD by fluorescence detection. FAD is extracted from whole blood samples by trichloroacetic acid, with diethyl ribityl isoalloxazine used as an internal standard. Linearity for FAD was above 0.99 (r2) up to a concentration range of 1000 nmol/L. Precision of the method (intra-day and inter-day) compared against commercial quality control material was below 8% (coefficient of variation) with recovery of FAD exceeding 90%. Accuracy of the protocol was compared against a previous cycle from an external quality assurance program (RCPAQAP) (n = 12...
Source: Analytical Biochemistry - February 6, 2020 Category: Biochemistry Authors: Nguyen VL, Ah-Loo A, Fitzpatrick M Tags: Anal Biochem Source Type: research