Analytical biochemistry最新文献_第8页

Isolation and functional properties of highly-purified N-terminal domain of human NaPi2b by scalable “resin overload” technique 用可扩展的“树脂过载”技术分离纯化人类NaPi2b蛋白n端结构域及其功能特性

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-18 DOI: 10.1016/j.ab.2025.115875

Daria Savenkova , Irina Makarenko , Daria Nedorezova , Ramziya Kiyamova , Mikhail Bogdanov

{"title":"Isolation and functional properties of highly-purified N-terminal domain of human NaPi2b by scalable “resin overload” technique","authors":"Daria Savenkova , Irina Makarenko , Daria Nedorezova , Ramziya Kiyamova , Mikhail Bogdanov","doi":"10.1016/j.ab.2025.115875","DOIUrl":"10.1016/j.ab.2025.115875","url":null,"abstract":"<div><div>Despite the enthusiasm and advances in the purification of native and engineered full-length membrane proteins, little attention has been paid to their fragments which could serve as attractive inspiration for function, regulation, or targeting of full-length membrane protein with therapeutic antibodies (Abs). Production of recombinant fragments of “therapeutic” membrane proteins for early-stage discovery research requires their purification to near homogeneity. It is important not only for the production of biotherapeutic antibodies but also for structural and functional studies of competitive protein-Abs, protein-protein, and lipid-protein interactions which heavily rely on the purity and quality of the isolated protein fragment of interest. The development of novel strategies for simple but still highly efficient protein purification remains a one of main research focus in the biotechnology and biomedicine because conventional purification approaches require complex manipulation steps and are timely and costly. Here, we would like to introduce a simple and rapid protein purification strategy for the human NaPi2b N-terminal (NT) sequence recombinantly expressed in a bacterial host at a laboratory scale. We demonstrate that “resin overload” e.g. the conditions when loading exceeds dynamic binding capacity can be counterintuitively but intelligently utilized to isolate highly purified protein fragments and prevent non-specific low-affinity binding of contaminant endogenous host proteins. The results showed that this method allowed us to achieve the highest purity while maintaining both immunogenic (recognition by Abs) and functional (phosphorylation) properties of the NaPi2b NT sequence. Although adaptations are required on a case-to-case basis, we believe this work can inspire other researchers working with the purification of protein and protein fragments to apply this proof-of-principle in a scalable manner.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115875"},"PeriodicalIF":2.6,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143850821","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}

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Recent advances and challenges in graphene-based electrochemical biosensors for food safety 食品安全石墨烯电化学生物传感器研究进展与挑战

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-17 DOI: 10.1016/j.ab.2025.115866

Sarita Yadav , Neetu Sehrawat , Shikha Sharma , Minakshi Sharma , Sandeep Yadav

{"title":"Recent advances and challenges in graphene-based electrochemical biosensors for food safety","authors":"Sarita Yadav , Neetu Sehrawat , Shikha Sharma , Minakshi Sharma , Sandeep Yadav","doi":"10.1016/j.ab.2025.115866","DOIUrl":"10.1016/j.ab.2025.115866","url":null,"abstract":"<div><div>Ensuring food safety is a critical global concern, particularly in light of recent pandemics and rising contamination risks from pesticides, antibiotics, toxins, and allergens. These contaminants pose significant health hazards, including neurological disorders, endocrine disruption, antibiotic resistance, and carcinogenic effects. Regulatory agencies such as the Food and Agriculture Organization (FAO), the World Health Organization (WHO), and the United States Food and Drug Administration (FDA) have established strict maximum residue limits (MRLs) to mitigate these risks. However, enforcement remains challenging due to limitations in current detection methods. The increasing global population and limited food resources have exacerbated food security challenges, while contaminants can infiltrate food at various stages, including production, processing, and packaging. Despite consumer awareness, significant amounts of food are discarded due to quality concerns. To address these issues, researchers are actively developing low-cost, reliable sensing technologies for real-time food quality assessment and contamination detection. Among these, graphene-based electrochemical biosensors have emerged as a promising solution due to their high sensitivity, selectivity, and cost-effectiveness. This review provides an in-depth analysis of recent advancements in graphene-based electrochemical biosensors, focusing on their role in detecting foodborne hazards and improving food quality monitoring. By integrating selective layers, these sensors enhance detection efficiency and provide an innovative solution for safeguarding public health. The findings underscore the transformative potential of graphene-derived biosensors in food safety diagnostics, paving the way for more reliable and sustainable food monitoring systems.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115866"},"PeriodicalIF":2.6,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143859113","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}

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Dual-screen-printed electrode platform for simultaneous detection of IgG and IgM antibodies using magnetic beads nanocomplexes 利用磁珠纳米复合物同时检测IgG和IgM抗体的双丝网印刷电极平台

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-16 DOI: 10.1016/j.ab.2025.115864

Ezgi Ayni , Ehsan Sanattalab , Nimet Yildirim-Tirgil

{"title":"Dual-screen-printed electrode platform for simultaneous detection of IgG and IgM antibodies using magnetic beads nanocomplexes","authors":"Ezgi Ayni , Ehsan Sanattalab , Nimet Yildirim-Tirgil","doi":"10.1016/j.ab.2025.115864","DOIUrl":"10.1016/j.ab.2025.115864","url":null,"abstract":"<div><div>This study uses a dual-screen-printed electrode platform to investigate the electrochemical behavior of magnetic platforms with varying sizes for sensitive detection of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies. Magnetic beads of 500 nm and 1 μm in diameter were functionalized with amino ferrocene and the receptor-binding domain of the severe acute SARS-CoV-2 spike protein. The sensor utilized a dual screen-printed electrode (SPE) system with spatially separated working electrodes, each immobilized with either anti-IgG or anti-IgM capture antibodies. A bipotentiostat was employed to independently monitor the electrochemical signals from these two working electrodes. Following incubation with target antibodies within a concentration range of 0.1 μg/mL to 200 μg/mL, the magnetic bead complexes were captured on their respective electrodes, and simultaneous electrochemical measurements of both IgG and IgM were conducted. The sensor's performance was evaluated in both buffer solutions and a complex serum-like matrix. Results showed distinct and quantifiable electrochemical responses for IgG and IgM, with minimal interference between the analytes. While matrix effects were observed, the sensor demonstrated its potential for simultaneous detection of SARS-CoV-2 antibodies in complex biological samples.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115864"},"PeriodicalIF":2.6,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143850822","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}

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Urease immobilization on alginate-based composite micro-beads 海藻酸盐基复合微球脲酶的固定化

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-14 DOI: 10.1016/j.ab.2025.115867

Demet Baybaş

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Simultaneous voltammetric determination of 3-methyladenine and adenine at disposable screen-printed carbon electrode 一次性丝网印刷碳电极上3-甲基腺嘌呤和3-甲基腺嘌呤的同时伏安测定

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-10 DOI: 10.1016/j.ab.2025.115863

José Gouveia S. Neto , Wellington Eneias Rodrigues , Éverton Marcelo P. Diniz , Vagner B. dos Santos , Severino Carlos B. Oliveira

{"title":"Simultaneous voltammetric determination of 3-methyladenine and adenine at disposable screen-printed carbon electrode","authors":"José Gouveia S. Neto , Wellington Eneias Rodrigues , Éverton Marcelo P. Diniz , Vagner B. dos Santos , Severino Carlos B. Oliveira","doi":"10.1016/j.ab.2025.115863","DOIUrl":"10.1016/j.ab.2025.115863","url":null,"abstract":"<div><div>The anodic behavior of 3-methyladenine (3-mAde) and adenine (Ade) were investigated in aqueous media on commercial screen-printed carbon electrodes (SPCEs), using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The electrochemical performance of the SPCEs was first evaluated by CV and using the [Fe(CN)<sub>6</sub>]<sup>3-</sup> redox probe. The electroactive areas of some SPCEs were also determined and discussed. Overall, the results were very satisfactory and indicated good repeatability and reproducibility of the SPCEs. Regarding 3-mAde its oxidation occurred in a single irreversible step pH dependent controlled by a diffusion layer and at more positive potential, ∼ +200.00 mV, in relation to Ade, demonstrating the possibility of simultaneous detection of these two biomarkers. By DPV experimental conditions were explored to increase the sensitivity and selectivity of the 3-mAde electrooxidation signal, such as influence of the composition and pH of the medium, effect of 3-mAde concentration and presence of possible interferents. A simple, sensitive, selective and fast electroanalytical method, using DPV and commercial SPCE, was developed for determination of 3-mAde in hydrolyzed DNA samples; with a linear range from 2.00 to 10.00 μmol L<sup>−1</sup>, limit of detection (LOD) of 0.35 μmol L<sup>−1</sup> and recoveries ranged from 96.0 % to 98.3 % in acetate buffer (pH = 4.10). For the simultaneous quantification of Ade and 3-mAde the analytical data were: concentration range of 0.50–10.00 μmol L<sup>−1</sup>, LOD of 0.34 μmol L<sup>−1</sup> and the mean recovery was 95.2 % for Ade and from 2.00 to 10.00 μmol L<sup>−1</sup>, LOD of 0.64 μmol L<sup>−1</sup> and the mean recovery was 101.3 % for 3-mAde.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115863"},"PeriodicalIF":2.6,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143844827","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}

引用次数: 0

A comprehensive review of computational methods for Protein-DNA binding site prediction 蛋白质- dna结合位点预测的计算方法综述

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-08 DOI: 10.1016/j.ab.2025.115862

Zi Liu , Wang-Ren Qiu , Yan Liu , He Yan , Wenyi Pei , Yi-Heng Zhu , Jing Qiu

引用次数: 0

Integrative biophysical characterization of molecular interactions: A case study with the sfGFP–nanobody complex 分子相互作用的综合生物物理表征：sfgfp -纳米体复合物的案例研究

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-07 DOI: 10.1016/j.ab.2025.115859

Aysha K. Demeler , James Bosco , Matthew J. Sydor , Michelle Nemetchek , Saeed Mortezazadeh , Liam Kerr , Sophia Bird , Roza Gabdullina , Cindee Yates-Hansen , Levi J. McClelland , Ekaterina Voronina , Trushar R. Patel , Borries Demeler

{"title":"Integrative biophysical characterization of molecular interactions: A case study with the sfGFP–nanobody complex","authors":"Aysha K. Demeler , James Bosco , Matthew J. Sydor , Michelle Nemetchek , Saeed Mortezazadeh , Liam Kerr , Sophia Bird , Roza Gabdullina , Cindee Yates-Hansen , Levi J. McClelland , Ekaterina Voronina , Trushar R. Patel , Borries Demeler","doi":"10.1016/j.ab.2025.115859","DOIUrl":"10.1016/j.ab.2025.115859","url":null,"abstract":"<div><div>This study compares several analytical biophysical methods for investigating protein-protein interactions (PPIs) in solution, using the interaction between superfolder green fluorescent protein (sfGFP) and its anti-sfGFP nanobody enhancer as a model system. Techniques evaluated include microscale thermophoresis, fluorescence correlation spectroscopy, analytical ultracentrifugation with multi-wavelength and fluorescence detection, isothermal titration calorimetry, and analytical size exclusion chromatography coupled to multi-angle static light scattering and dynamic light scattering. Each method was assessed for information content, dynamic range, precision, and complementarity. The results consistently indicate a single-digit nanomolar dissociation constant and 1:1 stoichiometry for the interaction. While each technique offers unique insights into binding affinity, thermodynamics, and stoichiometry of the interaction, the multi-method approach provides a more complete and reliable characterization of PPIs. The study demonstrates how combining multiple complementary techniques enhances the robustness of PPI analysis in solution-phase conditions.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"703 ","pages":"Article 115859"},"PeriodicalIF":2.6,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143855566","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}

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In-gel refolding allows fluorescence detection of fully denatured GFPs after SDS-PAGE 凝胶内再折叠允许在SDS-PAGE后对完全变性的gfp进行荧光检测

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-05 DOI: 10.1016/j.ab.2025.115861

Misa Shiratori , Rio Tsuyuki , Miwako Asanuma , Saki Kawabata , Hiromasa Yoshioka , Kenji Ohgane

{"title":"In-gel refolding allows fluorescence detection of fully denatured GFPs after SDS-PAGE","authors":"Misa Shiratori , Rio Tsuyuki , Miwako Asanuma , Saki Kawabata , Hiromasa Yoshioka , Kenji Ohgane","doi":"10.1016/j.ab.2025.115861","DOIUrl":"10.1016/j.ab.2025.115861","url":null,"abstract":"<div><div>Green fluorescent proteins (GFPs) have been widely used as fusion tags, especially to visualize subcellular localization and dynamics of the fused partner proteins. Also, GFPs serve as fluorescent tags in size-exclusion chromatography and native-PAGE, facilitating the evaluation of expression levels and quality of the expressed fusion proteins. However, the fluorescent detection of GFPs is generally incompatible with denaturing SDS-polyacrylamide gel electrophoresis (PAGE), where the samples are heat-denatured before loading. Accordingly, detecting GFP-fused proteins after SDS-PAGE usually relies on western blotting with anti-GFP antibodies. To enable in-gel fluorescence detection of SDS-PAGE-separated GFPs, some protocols employ mild denaturing conditions to keep the GFPs intact. However, such mild denaturation sometimes results in partial denaturation of the proteins and irregular electrophoretic mobility that is not proportional to their molecular weights. Here, we demonstrate that the fully denatured GFPs can be refolded within the gel by cyclodextrin-mediated removal of SDS in the presence of 20 % methanol, enabling the in-gel fluorescence detection of the GFP-fused proteins. The protocol is compatible with subsequent total protein staining and western blotting. Although future studies are needed to clarify the scope and generality, the technique developed here would provide a simple, time- and cost-effective alternative to the immunodetection of GFPs.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"702 ","pages":"Article 115861"},"PeriodicalIF":2.6,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143800049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

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Development of radioligand binding assays for REV-ERBα and REV-ERBβ rev - erba和rev - erbb β放射配体结合试验的建立。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-04 DOI: 10.1016/j.ab.2025.115858

Thomas Koelblen, Elise P. Burris, Thomas P. Burris

{"title":"Development of radioligand binding assays for REV-ERBα and REV-ERBβ","authors":"Thomas Koelblen, Elise P. Burris, Thomas P. Burris","doi":"10.1016/j.ab.2025.115858","DOIUrl":"10.1016/j.ab.2025.115858","url":null,"abstract":"<div><div>REV-ERBα and REV-ERBβ are atypical nuclear receptors that function as ligand-dependent repressors of transcription. They play critical roles in the regulation of the circadian rhythm, inflammation, and metabolism. The natural ligand for the REV-ERBs is heme, and synthetic ligands (both agonists and antagonists) have been designed and utilized to probe the potential clinical utility of targeting REV-ERBs for drug development. Although biochemical assays that can detect REV-ERB ligands have been developed based on protein-protein interactions, no classical fluorescent- or radioligand-binding assay has yet been developed that directly detects ligand binding. Here, we describe the development of the first radioligand binding assay (RLBA) using scintillation proximity assay (SPA) technology for both REV-ERBα and REV-ERBβ using labeled STL1267, a potent REV-ERBα/β agonist we recently described. <sup>3</sup>H-STL1267 displayed saturable binding to the ligand binding domains of both REV-ERBα and REV-ERBβ with equilibrium dissociation constants (K<sub>d</sub>s) of 392 nM and 202 nM, respectively. In competition radioligand binding assays, we used unlabeled STL1267 or the well-characterized first-generation REV-ERB agonist SR9009 as competitors to <sup>3</sup>H-STL1267 binding. STL1267 displayed K<sub>i</sub>s for REV-ERBα and REV-ERBβ of 253 ± 30 nM and 98 ± 14 nM, respectively. As expected, SR9009 displayed considerably lower potency than STL1267, with a K<sub>i</sub> of 692 ± 209 nM for REV-ERBα and 2546 ± 127 nM for REV-ERBβ. Although developing an RLBA has been challenging due to the lack of high-affinity ligands that can be used as probes, our results demonstrate the feasibility of such an assay for both receptors, providing a robust assay with utility for ligand/drug discovery.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"702 ","pages":"Article 115858"},"PeriodicalIF":2.6,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143794462","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}

引用次数: 0

Comparative assessment of four virus neutralization assays for detection of SARS-CoV-2 neutralizing antibodies 四种病毒中和法检测SARS-CoV-2中和抗体的比较评价。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2025-04-03 DOI: 10.1016/j.ab.2025.115860

Faezeh Maghsood , Navid Dashti , Tannaz Bahadori , Forough Golsaz-Shirazi , Christiane Moog , Mohammad Mehdi Amiri , Fazel Shokri

{"title":"Comparative assessment of four virus neutralization assays for detection of SARS-CoV-2 neutralizing antibodies","authors":"Faezeh Maghsood , Navid Dashti , Tannaz Bahadori , Forough Golsaz-Shirazi , Christiane Moog , Mohammad Mehdi Amiri , Fazel Shokri","doi":"10.1016/j.ab.2025.115860","DOIUrl":"10.1016/j.ab.2025.115860","url":null,"abstract":"<div><div>Neutralizing antibodies (NAbs) targeting receptor-binding domain (RBD) or spike of SARS-CoV-2 play an important role in blocking virus entry to the host cells and detecting their levels is critical for the assessment of humoral protective immune response following vaccination or recovery from SARS-CoV-2 infection. Here, we compared the performance of four virus neutralization tests to measure neutralizing antibodies in various sample types. We analyzed 25 serum samples obtained from mice or rabbits immunized with different vaccine platforms, and also 11 mouse anti-RBD monoclonal antibodies (MAbs) using surrogate virus neutralization test (SVNT), pseudovirus neutralization test (PVNT), conventional virus neutralization test (CVNT), and one-step or two-step inhibition flowcytometry virus neutralization test (IFVNT). All four VNTs showed significant correlations with each other, though PVNT and CVNT displayed significantly lower limit of detection (LoD) compared to the other two assays. In conclusion, our findings indicate that all four VNT assays give valid and accurate results and could be employed to determine the level of SARS-CoV-2 neutralizing monoclonal and polyclonal antibodies.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"702 ","pages":"Article 115860"},"PeriodicalIF":2.6,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143787545","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}

引用次数: 0