Analytical biochemistry最新文献

{"title":"Rapid screening plate of cadmium-chelating activity based on a quaternary complex of Cd(II)-Chrome azurol S CAS-2,2′-dipyridyl dipy-cetylpyridinium bromide CPB","authors":"Lukman Iddrisu ,&nbsp;Haimei Meng ,&nbsp;Evodia Moses Mkulo ,&nbsp;Felix Danso ,&nbsp;Salifu Ibrahim ,&nbsp;Shumei Zhang ,&nbsp;Jiesen Su ,&nbsp;Zhijia Fang ,&nbsp;Bin Wu ,&nbsp;Qi Deng ,&nbsp;Lijun Sun ,&nbsp;Ravi Gooneratne","doi":"10.1016/j.ab.2025.115911","DOIUrl":"10.1016/j.ab.2025.115911","url":null,"abstract":"<div><div>Heavy metal ions, such as Cd, Hg, Pb, and As, tend to persist in soil without natural degradation and can be absorbed by crops, leading to the accumulation of agricultural products that pose a significant threat to human health. However, the development of a rapid and efficient technique for identifying heavy metals in agricultural products is essential to ensure health and safety. With the knowledge of the extent of damage caused by heavy metals, it becomes imperative to detect the presence of cadmium in the soil, water, and the environment. This study introduces a novel plate approach for quick and precise colorimetric detection of cadmium ions using the Cd(II)-Chrome Azurol S CAS-2,2′-dipyridyl dipy-Cetylpyridinium Bromide CPB quaternary complex. Our innovative method has shown that at a reaction solution pH of 11, the optimal concentration ratio is CAS (5 × 10⁻³ M): dipy (0.1 M): CPB (1.0 × 10⁻³ M) = 4 mL: 1 mL: 1 mL. The most significant fading alert was observed when the ethylenediaminetetraacetic acid (EDTA) chelator was added dropwise to the CAS detection plate, indicating strong chelation of Cd by EDTA. This laboratory-based study established a foundation for future applications in real environmental sample analysis.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"705 ","pages":"Article 115911"},"PeriodicalIF":2.6,"publicationDate":"2025-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144191382","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An immunoprecipitation-based assay to assess cysteine-106-dependent catalytic activity of human-protein DJ-1 in cell line lysates","authors":"Nicolas Mathas ,&nbsp;Lucie Larigot ,&nbsp;Catherine Laurent ,&nbsp;Béatrice Le-Grand ,&nbsp;Julien Dairou ,&nbsp;Erwan Galardon","doi":"10.1016/j.ab.2025.115910","DOIUrl":"10.1016/j.ab.2025.115910","url":null,"abstract":"<div><div>DJ-1 is a protein with a wide range of protective cellular functions and implicated in several pathologies, from neurodegenerative Parkinson's disease to cancer. Its physiological functions rely on its ability to form protein complexes and on the highly conserved, redox-sensitive, cysteine residue C106 located in the enzyme's active site. The later plays a key role in the protection against the modification of biomolecules by glycolytic metabolites. However, to date, only an assay based on the highly efficient enzymatic hydrolysis of the reactive intermediate cyclic 3-phosphoglyceric anhydride (cPGA) by DJ-1 can quantify its activity in biological fluids. In this work, we propose a new immunoprecipitation assay using fluorescence to assess DJ-1 catalytic activity in crude cell lysates. This assay efficiently differentiates a wild-type cell line from its DJ-1 knock-out version, and the activity recorded in five human cell line lysates were validated by the good correlation obtained with the activities observed using the cPGA assay. To conclude, this assay is a complementary expansion to the toolbox for studying DJ-1 activity and the associated C106 redox state in cell lysates, as it makes for some of the shortcomings of the previous assay.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"705 ","pages":"Article 115910"},"PeriodicalIF":2.6,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144186271","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comprehensive review and meta-analysis of magnesium chloride optimization in PCR: Investigating concentration effects on reaction efficiency and template specificity","authors":"Hadja Fatima Tbahriti ,&nbsp;Aicha Zerrouki ,&nbsp;Ali Boukadoum ,&nbsp;Mostefa Kameche ,&nbsp;Sergey Povetkin ,&nbsp;Alexander Simonov ,&nbsp;Muthu Thiruvengadam","doi":"10.1016/j.ab.2025.115909","DOIUrl":"10.1016/j.ab.2025.115909","url":null,"abstract":"<div><div>Optimizing the polymerase chain reaction (PCR) continues to be a major challenge in molecular biology, and obtaining the correct magnesium chloride (MgCl₂) concentration is key to a successful reaction. A clear understanding of how MgCl₂ affects PCR thermodynamics and kinetics is crucial for creating efficient and reliable protocols that work consistently. A systematic meta-analysis was conducted of 61 peer-reviewed studies published between 1973 and 2024. The study selection adhered to rigorous PICOS criteria, prioritizing experimental investigations that specifically examined the effects of magnesium chloride (MgCl₂) on key PCR parameters. Data extraction and subsequent analyses were performed using standardized methodologies, with particular emphasis on template characteristics, reaction conditions, and their interplay in influencing PCR efficiency and specificity. The analysis showed a strong logarithmic relationship between MgCl₂ concentration and DNA melting temperature, with an optimal ranges of 1.5 and 3.0 mM. Every 0.5 mM increase in MgCl₂ within this range was associated with a 1.2 °C increase in melting temperature. Template complexity significantly affected the optimal MgCl₂ requirements, with genomic DNA templates requiring higher concentrations than the more straightforward templates. This meta-analysis offers quantitative insights and evidence-based guidelines for optimizing magnesium chloride (MgCl₂) concentration using polymerase chain reaction (PCR). These results demonstrate that the precise modulation of MgCl₂ concentration, tailored to specific template characteristics, can significantly improve both the efficiency and specificity of PCR. These findings provide a robust theoretical framework for the development of template-specific optimization strategies and advance the design of more reliable and effective PCR protocols.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"705 ","pages":"Article 115909"},"PeriodicalIF":2.6,"publicationDate":"2025-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144172392","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A systematic investigation of TMB substrate composition for signal enhancement in ELISA","authors":"Pavel Khramtsov ,&nbsp;Anastasia Novokshonova ,&nbsp;Zarina Galaeva ,&nbsp;Maria Morozova ,&nbsp;Tatiana Bezukladnikova ,&nbsp;Mikhail Rayev","doi":"10.1016/j.ab.2025.115908","DOIUrl":"10.1016/j.ab.2025.115908","url":null,"abstract":"<div><div>3,3′,5,5′-Tetramethylbenzidine (TMB) remains one of the most widely utilized chromogenic substrates for horseradish peroxidase (HRP) in colorimetric immunoassays, including enzyme-linked immunosorbent assays (ELISA). Despite its introduction into ELISA workflows over four decades ago, limited research has been conducted to systematically optimize TMB substrate formulations. Recent advancements in the field have proposed innovative approaches to enhance HRP catalysis, such as the use of deep eutectic solvents and ionic liquids, alongside investigations into the chemical properties of TMB and its analogs to identify more efficient alternatives. However, the development of stable and high-performance TMB solutions for clinical diagnostics requires a comprehensive understanding of how formulation parameters influence signal intensity and stability. In this study, we address these gaps by conducting a systematic evaluation of key factors affecting TMB substrate performance, including buffer pH, composition and molarity, specific ion effects, incorporation of organic solvents, and the use of polymer stabilizers. Additionally, novel strategies for signal amplification, identified through an extensive review of literature and patents, were experimentally tested. Based on these findings, we developed an optimized TMB formulation comprising 0.2 mol/L sodium citrate buffer (pH 4.5), 5 % DMSO, 0.37 mmol/L CaCl₂, 0.4 mmol/L 2-hydroxy-β-cyclodextrin, 0.8 mmol/L TMB, and 1.3 mmol/L H₂O₂ as final concentrations. The performance of this optimized formulation was evaluated in comparison to previously reported formulations from the literature.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"704 ","pages":"Article 115908"},"PeriodicalIF":2.6,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144135102","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A fundamental approach to buoyant density determination by DGE-AUC","authors":"Alexander E. Yarawsky ,&nbsp;Paola Cardenas Lopez ,&nbsp;Johannes Walter ,&nbsp;Michael T. DeLion ,&nbsp;Lake N. Paul","doi":"10.1016/j.ab.2025.115907","DOIUrl":"10.1016/j.ab.2025.115907","url":null,"abstract":"<div><div>Density gradient equilibrium analytical ultracentrifugation (DGE-AUC) was first introduced in 1957. The method saw significant use over the following decade. Since then, DGE-AUC has been used by polymer and genomic DNA fields. Emerging medicine has revived interest in the technique for characterization of cell and gene therapeutics. While several <em>model</em>-<em>dependent</em> approaches exist to determine density at any point along a density gradient at equilibrium, there is ample evidence in the vast density gradient literature that indicates the presence of pressure effects, solvent compressibility, and general nonideal behavior of the gradient medium that are not easily accounted for in models describing the density gradient. These complications mandated the general use of reference materials and standard conditions. With an interest in buoyant density determination for particles of various composition, an approach that does not rely on standards is desirable. The current manuscript details a fundamental <em>model</em>-<em>independent</em> method for determination of buoyant density by DGE-AUC. An examination of this novel method is presented in the context of NISTmAb and DNA in a CsCl gradient, as well as polystyrene beads in a sucrose gradient. The method described herein is broadly applicable to determine the buoyant density of a particle in a density gradient medium.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"704 ","pages":"Article 115907"},"PeriodicalIF":2.6,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144126635","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simple azide labeling of biotin-binding proteins using microbial transglutaminase: A practical note","authors":"Takahiko Matsushita ,&nbsp;Ryo Takano ,&nbsp;Tetsuo Koyama ,&nbsp;Ken Hatano ,&nbsp;Koji Matsuoka","doi":"10.1016/j.ab.2025.115905","DOIUrl":"10.1016/j.ab.2025.115905","url":null,"abstract":"<div><div>Microbial transglutaminase (MTG) is a Ca²⁺-independent enzyme that enables site-specific protein labeling under mild conditions. However, how substrate structure and protein-specific environments influence MTG reactivity is not yet fully understood. In this study, we synthesized a novel glutamine-based azide donor (compound <strong>1</strong>) and used it alongside a commercially available amine-based azide acceptor (3-azido-1-propylamine) to evaluate MTG-mediated labeling of five biotin-binding proteins: Avidin, Streptavidin, NeutrAvidin, Tamavidin 2, and Tamavidin 2-LPI. Azide-modified proteins were visualized using strain-promoted azide–alkyne cycloaddition (SPAAC) with DBCO-Cy5, and labeling efficiency was assessed by comparing MTG+ and MTG− conditions. Tamavidin 2 showed high reactivity toward both substrates, while Tamavidin 2-LPI exhibited little to no labeling. Streptavidin was labeled only by the amine-based substrate, whereas Avidin and NeutrAvidin were mainly labeled by the glutamine-based substrate. Notably, NeutrAvidin also showed strong fluorescence in the absence of MTG, suggesting non-specific interaction. These results indicate that MTG-mediated labeling is governed by both substrate compatibility and protein-dependent factors. Although the exact modification sites remain unidentified, this study provides a practical framework for selective biotin-protein conjugation using MTG and complementary azide reagents.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"704 ","pages":"Article 115905"},"PeriodicalIF":2.6,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144126636","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ultrasensitive detection of epinephrine by a Cd@HMCNs-based electrochemiluminescent sensor","authors":"Ziqi Wang,&nbsp;Yahui Ji,&nbsp;Nana You,&nbsp;Xiaoping Hu,&nbsp;Fangxin Du,&nbsp;Gen Liu","doi":"10.1016/j.ab.2025.115906","DOIUrl":"10.1016/j.ab.2025.115906","url":null,"abstract":"<div><div>Epinephrine (EP), a vital hormone and neurotransmitter, is central to the body's fight-or-flight mechanism, boosting physical strength, mental focus, and reaction speed in high-stress scenarios. Measuring EP levels is key for analyzing stress reactions, identifying health disorders, and maintaining balanced physiological performance. In this work, we successfully synthesized cadmium-doped hollow mesoporous g-C₃N₄ spheres (Cd@HMCNs) with a high surface area and high porosity. An electrochemiluminescent (ECL) sensor for EP detection was developed since EP can effectively suppresses the ECL emission of the Cd@HMCNs/K₂S₂O₈ system. The sensor successfully detected EP within the concentration range of 5.0 × 10⁻¹⁰ mol L⁻¹ to 1.0 × 10⁻⁵ mol L⁻¹, with a detection limit as low as 1.67 × 10⁻¹⁰ mol L⁻¹. When applied to pharmaceutical sample testing, the proposed biosensor yielded reliable and satisfactory results.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"704 ","pages":"Article 115906"},"PeriodicalIF":2.6,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144105126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phylogenetic analysis of prevalent Mycobacterium species in Northeastern Iran based on hsp65 and tuf genes","authors":"Roghayeh Mohammadzadeh ,&nbsp;Nafiseh Izadi ,&nbsp;Mojtaba Sankian ,&nbsp;Mohammad Javad Najafzadeh ,&nbsp;Hadi Farsiani","doi":"10.1016/j.ab.2025.115904","DOIUrl":"10.1016/j.ab.2025.115904","url":null,"abstract":"<div><div>Phylogenetic analysis of <em>Mycobacterium</em> species can provide valuable insights into their evolutionary relationships and help to identify species and strains. In this study, two genetic markers, including the heat shock protein 65 (<em>hsp65</em>) gene and the elongation factor (EF)-Tu (<em>tuf</em>) gene were used for phylogenetic analysis of <em>Mycobacterium</em> species.</div><div>Clinical samples were collected from patients suspected of tuberculosis. Bacterial isolates were obtained from sputum samples and cultured on Löwenstein-Jensen medium. Thirty <em>Mycobacterium</em> isolates (acid-fast +, culture +), were included in our study. After DNA extraction, <em>hsp65</em> (441 bp) and <em>tuf</em> (741 bp) genes were PCR-amplified and sequenced. The Neighbor-Joining method was employed to infer the evolutionary history of the isolates and the analyses were conducted with MEGA X software. The phylogenetic trees were validated using bootstrap analysis with 1000 replicates. Bootstrap values above 70 % considered indicative of well support for the branches. The phylogenetic trees revealed the overall natural relationships among <em>Mycobacterium</em> species. Our results demonstrated that the <em>tuf</em> gene provides superior resolution for identifying distinct mycobacterial species, closely aligning its phylogenetic profile with the <em>hsp65</em> gene. However, neither of the markers was effective in distinguishing members of the <em>Mycobacterium tuberculosis</em> complex (MTBC). This study highlights the high discriminatory power of the <em>tuf</em> gene, recommending its use as a primary genomic marker for phylogenetic analysis.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"704 ","pages":"Article 115904"},"PeriodicalIF":2.6,"publicationDate":"2025-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144085691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Influence of experimental conditions on the adsorption of disease biomarker proteins to InP/ZnS quantum dots","authors":"Nathaniel A. Gomez,&nbsp;Daniel Blumel,&nbsp;Davies Dueñas,&nbsp;Bronson Young,&nbsp;Matt Hazel,&nbsp;Ming Yu","doi":"10.1016/j.ab.2025.115903","DOIUrl":"10.1016/j.ab.2025.115903","url":null,"abstract":"<div><div>The spontaneous formation of quantum dot (QD)-protein assemblies in the physiological environment exhibits challenges or benefits for nanomedicine applications. In this study, we investigated the QD-protein assemblies spontaneously formed with the greener water soluble InP/ZnS–COOH QDs and isolated disease biomarker proteins under various environmental conditions, including QDs size, solution pH, incubation time, ionic strength, different salts, as well as the lowest concentrations of the proteins that started the formation of detectable assemblies. It was shown that higher ionic strength or valence charge disrupted the assembly's formation. The basic pH 8.5 facilitated the formation to a greater extent than the pH 7.4 did. The heat shock protein 90-alpha (HSP90α) adsorbed on QDs surface more readily than cytochrome C (CytoC) and lysozyme (Lyz) in the basic environment. Among the three-sized QDs compared, the medium-sized QDs were the most effective in promoting the assemblies' formation. The detectable assemblies started at as low as 0.4 ng/mL of CytoC, 1.0 ng/mL of HSP90α, or 1.8 ng/mL of Lyz, respectively. The findings add insights into how the biomarker proteins interacted with the QDs under different environmental conditions, which promotes the understanding of QD-protein assemblies' collaborative behaviors when they facilitate bioimaging and biomedicine applications.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"704 ","pages":"Article 115903"},"PeriodicalIF":2.6,"publicationDate":"2025-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144069344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AFP-MCDF: Multi and cross-dimensional feature fusion methods for antifreeze protein prediction","authors":"Jinfeng Li,&nbsp;Fan Zhang,&nbsp;Zhenguo Wen,&nbsp;Chun Fang","doi":"10.1016/j.ab.2025.115881","DOIUrl":"10.1016/j.ab.2025.115881","url":null,"abstract":"<div><div>Antifreeze proteins can effectively inhibit the formation of ice crystals and enhance cell survival in low-temperature environments. They protect the texture prolong the shelf life of food and maintain cell and tissue integrity in medical treatments, thereby improving the success rate of surgery and transplantation. Accurate prediction of Antifreeze proteins is important to advance these fields. Traditional wet-experiment methods, while providing reliable validation results, are usually time-consuming and costly. And existing computational methods still have room for improvement in predicting performance. In this study, a novel antifreeze protein prediction method, AFP-MCDF, is proposed. The AFP-MCDF method first extracts one- and two-dimensional feature representations of Antifreeze protein sequences using the pre-trained protein language models ProtBERT and ESM-2. Subsequently, these features are fused multidimensionally via BiLSTM and TextCNN to capture long-term dependencies and local features. Finally, the method predicts the frost resistance of Antifreeze protein sequences by cross-dimensional fusion and linear mapping from N to 2 dimensions. Experimental results show that AFP-MCDF performs well in the antifreeze protein prediction task, outperforming traditional computational methods and reaching the current state-of-the-art.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"704 ","pages":"Article 115881"},"PeriodicalIF":2.6,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143935882","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}