Analytical biochemistry最新文献

{"title":"Colorimetric assays for the detection of C4- and C2-epimerization of NDP-glucose to NDP-galactose and NDP-mannose","authors":"Ulrike Vogel,&nbsp;Renger Dijkstra,&nbsp;Koen Beerens,&nbsp;Tom Desmet","doi":"10.1016/j.ab.2025.115947","DOIUrl":"10.1016/j.ab.2025.115947","url":null,"abstract":"<div><div>CDP-tyvelose 2-epimerase-like enzymes and UDP-glucose 4-epimerases belong to the Nucleotide Sugar active Short-Chain Dehydrogenase/Reductase (NS-SDR) superfamily and convert NDP-Glc to NDP-Man and NDP-Gal, respectively. The product detection is well established using HPLC-based methods, capillary electrophoresis and thin layer chromatography for both epimerization reactions. However, the lack of a reliable colorimetric assays and poor substrate availability slow down the screening process of these enzymes, for example during enzyme engineering projects. After testing 16 phenolic compounds with different aldohexoses in concentrated sulfuric acid, thymol was found suitable for the detection of (NDP-) mannose and 1-naphthol for the detection of (NDP-) galactose in the presence of (NDP-) glucose. As proof-of-concept, the optimized assay was used for the biochemical characterization of the CDP-tyvelose 2-epimerase from <em>Ardenticatenia bacterium</em>.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115947"},"PeriodicalIF":2.6,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144671018","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":"Decoding ulcerative colitis: Plasma protein-mediated antibody immune responses influence disease risk","authors":"Xuyong Chen,&nbsp;Haidong Wu,&nbsp;Liudan Wang,&nbsp;Yifan Guo,&nbsp;Xinpu Miao","doi":"10.1016/j.ab.2025.115946","DOIUrl":"10.1016/j.ab.2025.115946","url":null,"abstract":"<div><h3>Objective</h3><div>Previous Mendelian randomization (MR) studies have explored the role of plasma proteins in ulcerative colitis (UC), but the underlying mechanisms, particularly how plasma proteins influence UC risk via immune-mediated pathways, remain unclear. This study integrates two-sample MR and mediation analysis to investigate whether plasma proteins influence UC risk through antibody-mediated immune responses, aiming to identify novel biomarkers and therapeutic targets.</div></div><div><h3>Methods</h3><div>We analyzed 4907 plasma proteins from a genome-wide association study, 46 immune antibody responses, and UC data from the FinnGen consortium. Two-sample MR was used to assess causal relationships among plasma proteins, antibody responses, and UC. Mediation MR analysis was conducted to evaluate whether specific antibody responses mediate the effect of plasma proteins on UC risk.</div></div><div><h3>Results</h3><div>A total of 80 plasma proteins with significant causal associations with UC were identified (P &lt; 0.05). Two antibody responses, Epstein-Barr virus (EBV) EA-D antibody levels and anti-HSV-1 IgG seropositivity, were significantly and inversely associated with UC risk (EBV: OR = 0.794, 95 % CI: 0.646–0.974, P = 0.027; HSV-1 IgG: OR = 0.891, 95 % CI: 0.801–0.992, P = 0.035). Five plasma proteins showed significant causal effects on these antibody responses: UBC, TIMD4, and NEFL (linked to EBV EA-D), and TMEM70 and HIF1A (linked to HSV-1 IgG). Mediation analysis revealed that antibody responses explained 9.2 %–13.2 % of the total effects of these proteins on UC risk. For example, UBC increased UC risk partially through reduced EBV EA-D antibodies (mediated effect = 0.0588), while HIF1A contributed to UC risk via suppressed HSV-1 IgG seropositivity.</div></div><div><h3>Conclusion</h3><div>This study identifies a novel immuno-genetic pathway in UC pathogenesis, where specific plasma proteins influence disease risk through modulation of viral antibody responses. These findings suggest potential targets, such as UBC, HIF1A, and TIMD4, for biomarker development and immune-focused interventions in UC.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115946"},"PeriodicalIF":2.6,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144654331","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":"Enhancing lateral flow assay performance: Buffer additives and protein-membrane interactions","authors":"Alexander Spreinat ,&nbsp;Carola Wilczek ,&nbsp;Christin Ronsör ,&nbsp;Andrea Ernst","doi":"10.1016/j.ab.2025.115943","DOIUrl":"10.1016/j.ab.2025.115943","url":null,"abstract":"<div><div>Lateral Flow Assays (LFA) have been valuable tools for point-of-care diagnostics for decades. During periods of high testing demand, as recently in the Covid-19-pandemic, it is necessary to deepen the understanding of test and control line signal intensities. This study investigates the interaction between proteins and structurally different nitrocellulose (CN) membranes and the resulting effects on signal intensity in an LFA under the influence of various buffer additives. The experiments focused on quantitative protein adsorption, protein stability, protein line printing and changes in signal intensity in a human chorionic gonadotropin (hCG)-assay. A method was established for detecting the widths and intensities of fluorescent protein lines. We were able to show that changes in signal intensity of LFAs are driven by accessibility of the antibodies, by hydrophilicity of the membrane or assisted adsorption of antibodies onto the membrane. Additionally, the inclusion of sodium chloride, polysorbate 80 and sodium dodecylbenzenesulfonate can enhance signal intensity. The method developed for protein line analysis has proven to be effective and can help to understand protein-membrane-interactions on a macroscopic level. We demonstrate that LFA-manufacturers have a range of options to fine-tune assay performance without major modifications to assay components.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115943"},"PeriodicalIF":2.6,"publicationDate":"2025-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144631456","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":"Proton-coupled electron transfer in hallachrome, a natural 1,2 anthraquinone: Linking electrochemical properties to biological activity","authors":"Felice C. Simeone","doi":"10.1016/j.ab.2025.115945","DOIUrl":"10.1016/j.ab.2025.115945","url":null,"abstract":"<div><div>Hallachrome is a 1,2-anthraquinone secreted by marine polychaete worms whose toxicity and antimicrobial properties have been reported; the origin of this biological activity, however, remains elusive. Voltammetric studies reveal reversible redox behavior in the pH range 1–10, consistent with a two-electron, two-proton (2e⁻/2H⁺) mechanism. The electron-donating substituents in hallachrome (hydroxyl and methyl groups) stabilize the protonated hydroquinone form and result in a substantial cathodic shift of 0.4–0.5 V compared to unsubstituted 1,2-benzoquinone.</div><div>The electrochemical analysis reveals that hallachrome (redox potential −0.12 V vs. SHE at pH 7) might be capable, from a thermodynamic point of view, of oxidizing key cellular antioxidants, including NADH (−0.32 V), NADPH (−0.32 V), and glutathione (−0.24 V). These findings provide fundamental insights into the structure-activity relationships governing quinone electrochemistry and establish a foundation for understanding hallachrome's biological activity.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115945"},"PeriodicalIF":2.6,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144625290","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":"Simultaneous multi-element determination in whole blood and urine via dual-mode ICP-MS","authors":"Yan Li ,&nbsp;Jian-Yuan Zhong ,&nbsp;Ya-qi Mo ,&nbsp;Li-mei Qin ,&nbsp;Michael Aschner ,&nbsp;Yue-ming Jiang","doi":"10.1016/j.ab.2025.115941","DOIUrl":"10.1016/j.ab.2025.115941","url":null,"abstract":"<div><div>The content of elements in the body has a significant relationship with human health. However, the wide variation in the concentrations of different elements in blood or urine poses challenges for accurate detection. This study presents a dual-mode ICP-MS methodology integrating kinetic energy discrimination (KED) and dynamic reaction cell (DRC) technologies, specifically designed to address the analytical challenge of quantifying 22 physiologically critical elements (Be, V, Cr, Mn, Fe, Ca, Mg, Ba, Co, Cd, Cu, Zn, As, Se, Ti, Sr, Ni, Mo, Sn, Sb, Tl, Pb) spanning six orders of magnitude in concentration (μg/L to g/L) within complex biological matrices. Rejection parameter q (RPq) adjusts the low-mass cutoff, while rejection parameter a (RPa) controls the high-mass cutoff. Optimizing both extends the low-interference dynamic range. The method's core innovation lies in the synchronized optimization of quadrupole parameters RPa and RPq, which enables simultaneous suppression of matrix-derived polyatomic interferences while maintaining linear detector response across ultra-wide dynamic ranges. The method employs a dual-mode analysis approach: (1) In urine analysis, the KED-He mode is suitable for analyzing all elements. (2) In whole blood analysis, DRC-NH₃ mode is used for interference-prone elements (Mn, Cr, Ca), and KED-He mode is used for the remaining elements. Strategic optimization of quadrupole rejection parameters (RPa/RPq) achieved intra-day precision of 0.5–7.2 % (blood) and 1.6–5.5 % (urine), with inter-day variations ≤9.6 %, demonstrating robust performance for multi-element profiling across clinically relevant concentration ranges.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115941"},"PeriodicalIF":2.6,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144625291","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":"Spectroscopic method for measuring activity of cis-aconitate decarboxylase, an important metabolic regulator of immune responses","authors":"Kevin Knowlan,&nbsp;Cody L. Hoop,&nbsp;Nadya I. Tarasova","doi":"10.1016/j.ab.2025.115944","DOIUrl":"10.1016/j.ab.2025.115944","url":null,"abstract":"<div><div><em>Cis</em>-aconitate decarboxylase (ACOD1) is a key enzyme converting <em>cis</em>-aconitate to itaconate, which has therapeutic potential for inflammatory diseases. Existing methods to measure ACOD1 activity and itaconate are often expensive and complex. We developed a novel, high-throughput spectrophotometric assay using the Fürth-Herrmann reaction. Our method quantifies ACOD1-catalyzed itaconate production by leveraging distinct absorbance ratios of <em>cis</em>-aconitate and itaconate at 386 nm and 440 nm. We optimized parameters, characterized human ACOD1 kinetics, and determined an IC₅₀ for citraconate consistent with previous reports. This simple, fast, and reliable assay, requiring only a UV–Vis spectrophotometer, will accelerate screening for ACOD1 modulators, speeding up therapeutic development.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115944"},"PeriodicalIF":2.6,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144614768","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":"Oxford Nanopore third generation sequencing for analysis of FMR1 5′UTR CGG repeat expansions","authors":"Xu Yang ,&nbsp;Bowei Han ,&nbsp;Jie Huang ,&nbsp;Min Zhang ,&nbsp;Shi Weng ,&nbsp;Guojun Ouyang ,&nbsp;Wanqing Han ,&nbsp;Wenyu Wang ,&nbsp;Li Zhang ,&nbsp;Juanjuan Chen ,&nbsp;Juan Du ,&nbsp;Yingsong Wu ,&nbsp;Xuexi Yang","doi":"10.1016/j.ab.2025.115931","DOIUrl":"10.1016/j.ab.2025.115931","url":null,"abstract":"<div><h3>Objective</h3><div>Fragile X syndrome is mainly caused by the expansion of GC-rich cytosine-guanine-guanine (CGG) repeat in <em>FMR1</em> 5′UTR region, as well as rare gene point mutations or deletions in its open reading frame. Currently, third-generation long-read sequencing is a potential technology for simultaneously detecting CGG repeat expansions, point mutations, and deletions. However, a major challenge remains in obtaining the target long-fragment CGG repeat region with ultra-high GC content for sequencing.</div></div><div><h3>Methods</h3><div>We developed a novel approach combining long-fragment ultra-high GC polymerase chain reaction (PCR) amplification with Oxford Nanopore sequencing to detect the full spectrum of <em>FMR1</em> 5′UTR CGG repeat mutations. The method was validated using 10 standard cell line samples (males: n_normal = 1, n_intermediate = 1, n_pre-mutation = 1, and n_{full mutation} = 2; females: n_normal = 1, n_intermediate = 1, n_pre-mutation = 2, and n_{full mutation} = 1) and 53 retrospective clinical blood samples (males: n_normal = 7, n_pre-mutation = 3, n_{full mutation} = 15, and n_mosaic _mutaion = 2; females: n_normal = 9, n_pre-mutation = 13, and n_{full mutation} = 4).</div></div><div><h3>Results</h3><div>Our method demonstrated that the 100 % concordance with the triplet repeat-primed PCR and Southern blot analysis in genotyping 10 cell line samples and 53 clinical samples. Additionally, CGG repeat numbers showed strong correlation with reference mehods (male cell lines, n = 5, R² = 0.9996; female cell lines, n = 5, R² = 0.9972; clinical male samples, n = 26, R² = 1.0000; clinical female samples, n = 25, R² = 0.9854).</div></div><div><h3>Conclusion</h3><div>This study presents a simple and cost-effective strategy for preparing <em>FMR1</em> 5′UTR CGG repeat regions with long-fragment ultra-high GC content for third-generation sequencing. The approach could serve as a model for detecting other challenging disorders caused by short tandem repeat expansions, such as myotonic dystrophy and Huntington’ s disease.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115931"},"PeriodicalIF":2.6,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144625289","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":"Mass spectrometric identification of novel truncated α-synuclein species following optimized immunoprecipitation from human brain tissue","authors":"Kim-Thanh Van ,&nbsp;Fatimah Nabeebaccus ,&nbsp;Foudil Lamari ,&nbsp;Susana Boluda ,&nbsp;François Fenaille ,&nbsp;Nicolas Villain ,&nbsp;François Becher","doi":"10.1016/j.ab.2025.115942","DOIUrl":"10.1016/j.ab.2025.115942","url":null,"abstract":"<div><div>α-synuclein is a protein central to neurodegenerative diseases, and its functions are affected by multiple posttranslational modifications. Mass spectrometry is powerful for the characterization of α-synuclein forms but requires prior efficient immunoprecipitation conditions. In this study, we refined the immunoprecipitation of α-synuclein from human brain tissues by evaluating key parameters that influence recovery and specificity. We assessed the performance of tosyl-activated magnetic beads versus sheep antibody beads, identifying the optimal bead type for enhanced binding efficiency. Various elution conditions were rigorously tested to maximize protein yield. We also evaluated a range of antibodies specific to α-synuclein and delineated the effects of antibody amount and bead volume on the recovery of α-synuclein. The optimized immunoprecipitation protocol was effectively combined with high-resolution mass spectrometry for characterizing brain-derived α-synuclein from Parkinson's disease patients and controls. The assay identified a total of 38 N- or C-terminal truncated α-synuclein forms, including 22 novel sites. Our findings provide analytical tools for the reliable enrichment and characterization of α-synuclein from complex biological matrices, with potential applications in biomarker discovery and the investigation of pathogenic mechanisms underlying synucleinopathies.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115942"},"PeriodicalIF":2.6,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144615792","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":"Rapid and accurate detection of urinary iodine by single crystal silver iodide sensor","authors":"Jianmin Yang,&nbsp;Kai Tang,&nbsp;Shuyan Xiong,&nbsp;Liang Shi,&nbsp;Aidong Tang","doi":"10.1016/j.ab.2025.115939","DOIUrl":"10.1016/j.ab.2025.115939","url":null,"abstract":"<div><div>Iodine deficiency or excess impacts thyroid health, necessitating accurate urinary iodine monitoring. However, existing detection methods face challenges including complicated sample handling, poor anti-interference ability, and the detection range does not cover the range of human urinary iodine concentration. Conventional iodide ion-selective electrode is based on polycrystalline membranes, which has high detection limit and is susceptible to chloride ion interference. It is also not suitable for accurate detection of human urinary iodine. In this study, we introduced a single-crystal AgI electrode for rapid and interference-free detection of urinary iodine. The dense (220) crystal plane of the single-crystal AgI electrode provided excellent selectivity and sensitivity, with a detection limit as low as 59 μg L⁻¹ and good linearity in the range of 100–1000 μg L⁻¹. This fully covers the optimal range of urinary iodine concentrations recommended by the World Health Organization (100–500 μg L⁻¹). Importantly, the single-crystal AgI electrode has excellent resistance to chloride interference, which is a significant improvement over conventional polycrystallaline iodide ion-selective electrodes. Our results show that the electrode has excellent precision, stability and accuracy without interference from common urine ions. This work addresses the limitations of existing methods and provides a rapid, inexpensive and reliable method for urinary iodine testing.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115939"},"PeriodicalIF":2.6,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144604946","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":"Multi-protein-triggered, aptamer-tethered auto-cycling proximity recorder in formalin-fixed paraffin-embedded tissues","authors":"Xiaofang Zheng ,&nbsp;Daikun Zheng ,&nbsp;Linfeng Zheng ,&nbsp;Peng Wang ,&nbsp;Yong Liu ,&nbsp;Xixi You ,&nbsp;Xiaoling Zheng ,&nbsp;Xinyuan Cao ,&nbsp;Jiao Luo ,&nbsp;Wansong Chen ,&nbsp;Guoli Li ,&nbsp;Ruizi Peng","doi":"10.1016/j.ab.2025.115940","DOIUrl":"10.1016/j.ab.2025.115940","url":null,"abstract":"<div><div>Unraveling tumor-associated membrane proteins' spatial proximity holds a significance for clinically relevant multiple biomarker detection. Herein, based on aptamer binding to target proteins in clinical formalin-fixed paraffin-embedded (FFPE) tissue samples, we developed aptamer-tethered auto-cycling proximity recording (APR) probes as a proof-of-concept methodology for biosensing membrane proteins' spatial proximity. This APR enables continuous and repetitive recording of spatial neighboring pairs of DNA probes in FFPE breast cancer tissue samples in situ, without altering the probes themselves. Finally, this aptamer-based auto-cycling proximity recorder is able to identify spatial proximity of three membrane proteins in FFPE breast cancer tissues. Our research establishes a foundation for proximity-driven biosensing platform and provides a perspective for exploration in aptamer-based bioimaging in clinical tissue samples.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115940"},"PeriodicalIF":2.6,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144605504","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}