Pub Date : 2026-01-10DOI: 10.1016/j.ab.2026.116050
Bruna Garlet Rossato , Rafael Noal Moresco
Immunoturbidimetric assays are widely used to quantify immunoglobulins and complement proteins but are susceptible to interference. This study evaluated the effects of oxidative agents, including the Fenton reaction and hypochlorous acid (HOCl), on these assays. Serum pools were oxidized in vitro, and IgA, IgG, IgM, C3, and C4 were measured. Oxidative conditions increased measured concentrations of IgA, IgM, C3, and C4, whereas IgG was unaffected by HOCl. Increased oxidative stress index values and reduced thiol levels confirmed oxidative modification, indicating potential impairment of assay accuracy under oxidative stress.
{"title":"Oxidative interference in immunoturbidimetric assays for the quantification of immunoglobulins and complement proteins","authors":"Bruna Garlet Rossato , Rafael Noal Moresco","doi":"10.1016/j.ab.2026.116050","DOIUrl":"10.1016/j.ab.2026.116050","url":null,"abstract":"<div><div>Immunoturbidimetric assays are widely used to quantify immunoglobulins and complement proteins but are susceptible to interference. This study evaluated the effects of oxidative agents, including the Fenton reaction and hypochlorous acid (HOCl), on these assays. Serum pools were oxidized <em>in vitro</em>, and IgA, IgG, IgM, C3, and C4 were measured. Oxidative conditions increased measured concentrations of IgA, IgM, C3, and C4, whereas IgG was unaffected by HOCl. Increased oxidative stress index values and reduced thiol levels confirmed oxidative modification, indicating potential impairment of assay accuracy under oxidative stress.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116050"},"PeriodicalIF":2.5,"publicationDate":"2026-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145951142","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}
Pub Date : 2026-01-08DOI: 10.1016/j.ab.2026.116049
Md Maruf Ahmed , Qin Xu
Food safety is a paramount worldwide issue that directly affects human health and well-being, underscoring the necessity for rapid and accurate detection strategies. Conventional analytical techniques, including chromatography, mass spectrometry, and immunoassays, are highly sensitive and reliable; however, they are costly, laborious, and impractical for real-time monitoring. Conversely, electrochemical sensors have benefits for food safety monitoring, including simplicity, quick response, cost-effectiveness, and portability, making them suitable for continuous monitoring. Optimal sensing performance necessitates the design and development of functional electrode materials with superior electrocatalytic activity. Over the past decade, ZnO nanomaterials have been widely used in electrochemical sensor technology due to their multifunctional properties, including high electron mobility, biocompatibility, and tunable architecture. This review summarizes recent advancements in ZnO-based electrochemical sensors for detecting food toxins. The synthesis methodologies and material characteristics are initially examined, followed by a review of their electrochemical sensing systems. The functionalization strategies for improving ZnO's sensing capabilities have been discussed. This study highlights the existing obstacles and potential opportunities for the advancement of ZnO-based electrochemical sensors in food safety monitoring.
{"title":"Recent advances in ZnO-based electrochemical sensors for food safety monitoring","authors":"Md Maruf Ahmed , Qin Xu","doi":"10.1016/j.ab.2026.116049","DOIUrl":"10.1016/j.ab.2026.116049","url":null,"abstract":"<div><div>Food safety is a paramount worldwide issue that directly affects human health and well-being, underscoring the necessity for rapid and accurate detection strategies. Conventional analytical techniques, including chromatography, mass spectrometry, and immunoassays, are highly sensitive and reliable; however, they are costly, laborious, and impractical for real-time monitoring. Conversely, electrochemical sensors have benefits for food safety monitoring, including simplicity, quick response, cost-effectiveness, and portability, making them suitable for continuous monitoring. Optimal sensing performance necessitates the design and development of functional electrode materials with superior electrocatalytic activity. Over the past decade, ZnO nanomaterials have been widely used in electrochemical sensor technology due to their multifunctional properties, including high electron mobility, biocompatibility, and tunable architecture. This review summarizes recent advancements in ZnO-based electrochemical sensors for detecting food toxins. The synthesis methodologies and material characteristics are initially examined, followed by a review of their electrochemical sensing systems. The functionalization strategies for improving ZnO's sensing capabilities have been discussed. This study highlights the existing obstacles and potential opportunities for the advancement of ZnO-based electrochemical sensors in food safety monitoring.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116049"},"PeriodicalIF":2.5,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145948399","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}
Pub Date : 2026-01-05DOI: 10.1016/j.ab.2026.116047
Piotr Olcha , Wiesław Paja , Michał Kępski , Krzysztof Pancerz , Bartosz Klebowski , Łukasz Nowakowski , Krzysztof Gałczyński , Joanna Depciuch
Endometriosis is a chronic inflammatory disorder in which endometrial tissue grows outside the uterus, leading to pelvic pain and infertility. It remains a major challenge in women's health due to delayed diagnosis and limited therapeutic outcomes. The disease is heterogeneous and manifests in three main phenotypes: superficial peritoneal lesions, ovarian endometriomas, and deep infiltrating endometriosis, the latter still poorly understood at the molecular level. In this study, Fourier-transform infrared (FTIR) spectroscopy combined with machine learning was applied to explore biochemical differences among these phenotypes. Spectral analysis demonstrated that lipid- and carbohydrate-associated vibrations, progressively increased from ovarian to bowel and were most pronounced in peritoneal endometriosis. These findings suggest phenotype-specific alterations in lipid and carbohydrate composition. Using the Boruta feature selection algorithm, discriminative spectral intervals were identified for pairwise classification. Key signals involved protein, lipid, and carbohydrate vibrations, including CO and C–O stretching and N–H bending. Machine learning models: Deep Learning (DL), Support Vector Machine (SVM), and Extreme Gradient Boosting (XGBoost), were trained on both full spectra and Boruta-reduced datasets. Extending to three-class classification, Boruta-selected features enabled robust discrimination across all phenotypes. A decision tree revealed the 1752 cm−1 ester CO band as a critical marker for phenotype differentiation. Overall, FTIR spectroscopy combined with machine learning provides valuable molecular insights into endometriosis and represents a powerful tool for distinguishing its clinical phenotypes.
{"title":"FTIR spectroscopy combined with machine learning reveals molecular signatures distinguishing three phenotypes of endometriosis","authors":"Piotr Olcha , Wiesław Paja , Michał Kępski , Krzysztof Pancerz , Bartosz Klebowski , Łukasz Nowakowski , Krzysztof Gałczyński , Joanna Depciuch","doi":"10.1016/j.ab.2026.116047","DOIUrl":"10.1016/j.ab.2026.116047","url":null,"abstract":"<div><div>Endometriosis is a chronic inflammatory disorder in which endometrial tissue grows outside the uterus, leading to pelvic pain and infertility. It remains a major challenge in women's health due to delayed diagnosis and limited therapeutic outcomes. The disease is heterogeneous and manifests in three main phenotypes: superficial peritoneal lesions, ovarian endometriomas, and deep infiltrating endometriosis, the latter still poorly understood at the molecular level. In this study, Fourier-transform infrared (FTIR) spectroscopy combined with machine learning was applied to explore biochemical differences among these phenotypes. Spectral analysis demonstrated that lipid- and carbohydrate-associated vibrations, progressively increased from ovarian to bowel and were most pronounced in peritoneal endometriosis. These findings suggest phenotype-specific alterations in lipid and carbohydrate composition. Using the Boruta feature selection algorithm, discriminative spectral intervals were identified for pairwise classification. Key signals involved protein, lipid, and carbohydrate vibrations, including C<img>O and C–O stretching and N–H bending. Machine learning models: Deep Learning (DL), Support Vector Machine (SVM), and Extreme Gradient Boosting (XGBoost), were trained on both full spectra and Boruta-reduced datasets. Extending to three-class classification, Boruta-selected features enabled robust discrimination across all phenotypes. A decision tree revealed the 1752 cm<sup>−1</sup> ester C<img>O band as a critical marker for phenotype differentiation. Overall, FTIR spectroscopy combined with machine learning provides valuable molecular insights into endometriosis and represents a powerful tool for distinguishing its clinical phenotypes.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116047"},"PeriodicalIF":2.5,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145916549","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}
Pub Date : 2026-01-03DOI: 10.1016/j.ab.2026.116046
M. Sabbir Hossain , M. Hafizur Rahman , Md. A. Rashed , Umme Salma , Jahir Ahmed , M. Faisal , Jari S. Algethami , Farid A. Harraz
In this study, a highly sensitive electrochemical sensor with low cost is designed for determining Hydroquinone (HQ) by modifying glassy carbon (GC) electrodes with PdO@SWCNTs/NiO nanocomposite. Herein, a semiconductor metal oxide (NiO) is decorated with single-walled carbon-nanotubes (SWCNTs) and palladium oxide (PdO), using a simple ultrasonication procedure, followed by a photo-reduction approach to achieve the final PdO@SWCNTs/NiO nanocomposite. The synthesized nanocomposites' morphological and structural characteristics were effectively investigated by High-Resolution Transmission Electron Microscope (HR-TEM), Field Emission Scanning Electron Microscope (FE-SEM), X-ray Diffraction (XRD), and X-ray Photoelectron Spectroscopy (XPS) techniques. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed to evaluate the electro-catalytic performance. For the sensing studies, Differential Pulse Voltammetry (DPV) and amperometric (i-t) techniques were successfully employed, where DPV demonstrated superior sensitivity with 0.74 μAμM−1cm−2 and a low limit of detection (LOD) of 0.29 μM within the concentration range of 10 μM–495 μM of HQ. Moreover, in the amperometric technique, the designed sensor revealed superb results with a sensing concentration ranging from 60 μM to 1920 μM and a sensitivity of 0.35 μA M−1cm−2 with a detection limit of 0.62 μM. Furthermore, the developed sensor exhibits acceptable reproducibility, repeatability, and stability. Under experimental conditions, the examined sensor showed outstanding selectivity towards HQ in the presence of both inorganic and organic interference components. Moreover, in real-world analysis of samples, the developed sensors recover HQ with a reliable Relative Standard Deviation (%RSD) value.
{"title":"A high-performance PdO@SWCNTs/NiO nanocomposite-based electrochemical sensor for sensitive and selective detection of hydroquinone","authors":"M. Sabbir Hossain , M. Hafizur Rahman , Md. A. Rashed , Umme Salma , Jahir Ahmed , M. Faisal , Jari S. Algethami , Farid A. Harraz","doi":"10.1016/j.ab.2026.116046","DOIUrl":"10.1016/j.ab.2026.116046","url":null,"abstract":"<div><div>In this study, a highly sensitive electrochemical sensor with low cost is designed for determining Hydroquinone (HQ) by modifying glassy carbon (GC) electrodes with PdO@SWCNTs/NiO nanocomposite. Herein, a semiconductor metal oxide (NiO) is decorated with single-walled carbon-nanotubes (SWCNTs) and palladium oxide (PdO), using a simple ultrasonication procedure, followed by a photo-reduction approach to achieve the final PdO@SWCNTs/NiO nanocomposite. The synthesized nanocomposites' morphological and structural characteristics were effectively investigated by High-Resolution Transmission Electron Microscope (HR-TEM), Field Emission Scanning Electron Microscope (FE-SEM), X-ray Diffraction (XRD), and X-ray Photoelectron Spectroscopy (XPS) techniques. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed to evaluate the electro-catalytic performance. For the sensing studies, Differential Pulse Voltammetry (DPV) and amperometric (<em>i-t</em>) techniques were successfully employed, where DPV demonstrated superior sensitivity with 0.74 μAμM<sup>−1</sup>cm<sup>−2</sup> and a low limit of detection (LOD) of 0.29 μM within the concentration range of 10 μM–495 μM of HQ. Moreover, in the amperometric technique, the designed sensor revealed superb results with a sensing concentration ranging from 60 μM to 1920 μM and a sensitivity of 0.35 μA <span><math><mrow><mi>μ</mi></mrow></math></span> M<sup>−1</sup>cm<sup>−2</sup> with a detection limit of 0.62 μM. Furthermore, the developed sensor exhibits acceptable reproducibility, repeatability, and stability. Under experimental conditions, the examined sensor showed outstanding selectivity towards HQ in the presence of both inorganic and organic interference components. Moreover, in real-world analysis of samples, the developed sensors recover HQ with a reliable Relative Standard Deviation (%RSD) value.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116046"},"PeriodicalIF":2.5,"publicationDate":"2026-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145905294","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}
Pub Date : 2026-01-03DOI: 10.1016/j.ab.2026.116045
Rodrigo Campos-Silva , Fahimeh Rahimi , Jaya Joshi , Cătălin Voiniciuc , Juannan Zhou , Andrew D. Hanson
Enzyme biochemistry can now draw on hundreds of thousands of prokaryotic genomes and metagenomes to identify orthologous genes for research, biocatalysis, and metabolic engineering. In many applications, adaptation to O2 and mild temperatures are essential. But as organism lifestyle information can be poor or absent (especially for metagenomes), it is challenging to avoid orthologous genes from anaerobes and extremophiles. Taking bacterial sulfide-dependent THI4 thiazole synthases as test-cases, we built computational pipelines that use only DNA sequence inputs to explore (i) the average oxidation state of carbon (ZC) in orthologous enzymes and (ii) the presence of O2-metabolism genes in the corresponding (meta)genomes. ZC has been proposed to be highest (least negative) in proteins of organisms from O2-rich, mesophilic environments. We found that ZC values of 2300 THI4s ranged from −0.107 (relatively oxidized) to −0.302 (strongly reduced). As predicted, genes specifying cytochrome c or o oxidases (supporting respiration at relatively high O2 levels) and, to a lesser extent, cytochrome bd oxidases (which can function to scavenge O2) were more frequent in genomes encoding THI4s with high ZC values. Eight THI4s with ZC values in the top 5 % and from (meta)genomes having cytochrome oxidases were tested for ability to complement a THI4Δ yeast strain in aerobic conditions. Three THI4 genes from a metagenome with cytochrome c/o oxidase (but without cytochrome bd) were active. These results support the feasibility of combining ZC and cytochrome oxidase profiles to identify bacterial orthologous enzymes that work in aerobic, mild temperature conditions.
{"title":"Mining bacterial (meta)genomes for enzymes active in aerobic, mesophilic conditions","authors":"Rodrigo Campos-Silva , Fahimeh Rahimi , Jaya Joshi , Cătălin Voiniciuc , Juannan Zhou , Andrew D. Hanson","doi":"10.1016/j.ab.2026.116045","DOIUrl":"10.1016/j.ab.2026.116045","url":null,"abstract":"<div><div>Enzyme biochemistry can now draw on hundreds of thousands of prokaryotic genomes and metagenomes to identify orthologous genes for research, biocatalysis, and metabolic engineering. In many applications, adaptation to O<sub>2</sub> and mild temperatures are essential. But as organism lifestyle information can be poor or absent (especially for metagenomes), it is challenging to avoid orthologous genes from anaerobes and extremophiles. Taking bacterial sulfide-dependent THI4 thiazole synthases as test-cases, we built computational pipelines that use only DNA sequence inputs to explore (i) the average oxidation state of carbon (Z<sub>C</sub>) in orthologous enzymes and (ii) the presence of O<sub>2</sub>-metabolism genes in the corresponding (meta)genomes. Z<sub>C</sub> has been proposed to be highest (least negative) in proteins of organisms from O<sub>2</sub>-rich, mesophilic environments. We found that Z<sub>C</sub> values of 2300 THI4s ranged from −0.107 (relatively oxidized) to −0.302 (strongly reduced). As predicted, genes specifying cytochrome <em>c</em> or <em>o</em> oxidases (supporting respiration at relatively high O<sub>2</sub> levels) and, to a lesser extent, cytochrome <em>bd</em> oxidases (which can function to scavenge O<sub>2</sub>) were more frequent in genomes encoding THI4s with high Z<sub>C</sub> values. Eight THI4s with Z<sub>C</sub> values in the top 5 % and from (meta)genomes having cytochrome oxidases were tested for ability to complement a <em>THI4</em>Δ yeast strain in aerobic conditions. Three THI4 genes from a metagenome with cytochrome <em>c</em>/<em>o</em> oxidase (but without cytochrome <em>bd</em>) were active. These results support the feasibility of combining Z<sub>C</sub> and cytochrome oxidase profiles to identify bacterial orthologous enzymes that work in aerobic, mild temperature conditions.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116045"},"PeriodicalIF":2.5,"publicationDate":"2026-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145905422","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}
Neutrophil extracellular traps (NETs) could help prevent and treat thrombosis-related diseases. Detecting NETs primarily relies on plasma; however, the impact of anticoagulants and plasma centrifugation on these results remains unclear.
Objectives
This study aimed to identify the most effective anticoagulant and centrifugation methods for detecting NETs in plasma.
Methods
Plasma samples were collected from sepsis rats using sodium citrate and EDTA as anticoagulants. These samples were subjected to various centrifugal forces, durations, and temperatures to determine the optimal method for NETs analysis. Similarly, plasma samples from infected patients were collected and analyzed using different centrifugation techniques to identify the optimal method. Gene expression analysis in endothelial cells stimulated by these plasma samples was conducted to compare the endothelial responses to NETs levels.
Results
In rats, EDTA-plasma exhibited increased levels of myeloperoxidase (MPO)-DNA, while dsDNA levels remained unchanged, with both concentrations being higher at 400 g. Similarly, EDTA and a centrifugal force of 400 g increased MPO-DNA levels without affecting dsDNA levels in patients. Plasma isolated using a centrifugal force of 400 g enhanced adhesion, inflammation, and chemotaxis gene expression in endothelial cells, demonstrating that low centrifugal force is effective for NETs detection.
Conclusion
For an accurate NETs assay, it is recommended to use EDTA anticoagulant and prepare plasma using a low centrifugal force, such as 400g.
{"title":"Impact of anticoagulants and centrifugal forces on plasma neutrophil extracellular traps assay outcomes","authors":"Yang Zhang , Shengqiang Pei , Dabuxilite Bayartaikishigtai , Zixin Chen , Hongtao Zhang , Wei Chen","doi":"10.1016/j.ab.2025.116037","DOIUrl":"10.1016/j.ab.2025.116037","url":null,"abstract":"<div><h3>Background</h3><div>Neutrophil extracellular traps (NETs) could help prevent and treat thrombosis-related diseases. Detecting NETs primarily relies on plasma; however, the impact of anticoagulants and plasma centrifugation on these results remains unclear.</div></div><div><h3>Objectives</h3><div>This study aimed to identify the most effective anticoagulant and centrifugation methods for detecting NETs in plasma.</div></div><div><h3>Methods</h3><div>Plasma samples were collected from sepsis rats using sodium citrate and EDTA as anticoagulants. These samples were subjected to various centrifugal forces, durations, and temperatures to determine the optimal method for NETs analysis. Similarly, plasma samples from infected patients were collected and analyzed using different centrifugation techniques to identify the optimal method. Gene expression analysis in endothelial cells stimulated by these plasma samples was conducted to compare the endothelial responses to NETs levels.</div></div><div><h3>Results</h3><div>In rats, EDTA-plasma exhibited increased levels of myeloperoxidase (MPO)-DNA, while dsDNA levels remained unchanged, with both concentrations being higher at 400 g. Similarly, EDTA and a centrifugal force of 400 g increased MPO-DNA levels without affecting dsDNA levels in patients. Plasma isolated using a centrifugal force of 400 g enhanced adhesion, inflammation, and chemotaxis gene expression in endothelial cells, demonstrating that low centrifugal force is effective for NETs detection.</div></div><div><h3>Conclusion</h3><div>For an accurate NETs assay, it is recommended to use EDTA anticoagulant and prepare plasma using a low centrifugal force, such as 400g.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116037"},"PeriodicalIF":2.5,"publicationDate":"2025-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145848761","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}
Pub Date : 2025-12-23DOI: 10.1016/j.ab.2025.116036
Carmen Mena-Iglesias, Myriam Bustamante-Rangel, Diego García-Gómez, M. Teresa Fernández-del-Campo-García, Encarnación Rodríguez-Gonzalo, José Luis Pérez Pavón
Modified nucleosides and nucleobases have raised great scientific interest due to their possible use as diagnostic and prognostic markers for various diseases. One of the most common types of modification is methylation. Sensitive and accurate methods are essential for the quantification of these compounds in biological samples. In this work we propose, for the first time, a rapid non-separative method for the determination of methylated nucleosides in urine by direct injection on electrospray ionization triple quadrupole mass spectrometer (ESI-MS/MS). The proposed method has been applied to the simultaneous determination of five methylated nucleosides in human urine samples. A separative method based on HILIC-MS/MS has been developed in parallel with the same instrumental setup to confirm the results obtained by the non-separative method. Both methods were validated in terms of linearity, matrix effect, limits of quantification, precision and recovery. The proposed method was successfully applied to the determination of the selected analytes in the urine of healthy volunteers and was compared with the results obtained by the separative method. The proposed non-separative method considerably reduced the required analysis time and simplified the sample treatment, which is a remarkable advantage over the chromatographic methods described. Therefore, this method is suitable for rapid, simple, and reliable determination of endogenous biomarkers in urine samples for routine or high-throughput analysis. The separative method is recommended as a complementary tool for confirmation in complex situations or in cases of abnormally high biomarker levels, maintaining specificity and robustness without compromising the practical advantages of the rapid method.
{"title":"Fast quantification of methylated nucleosides in urine by triple quadrupole mass spectrometry","authors":"Carmen Mena-Iglesias, Myriam Bustamante-Rangel, Diego García-Gómez, M. Teresa Fernández-del-Campo-García, Encarnación Rodríguez-Gonzalo, José Luis Pérez Pavón","doi":"10.1016/j.ab.2025.116036","DOIUrl":"10.1016/j.ab.2025.116036","url":null,"abstract":"<div><div>Modified nucleosides and nucleobases have raised great scientific interest due to their possible use as diagnostic and prognostic markers for various diseases. One of the most common types of modification is methylation. Sensitive and accurate methods are essential for the quantification of these compounds in biological samples. In this work we propose, for the first time, a rapid non-separative method for the determination of methylated nucleosides in urine by direct injection on electrospray ionization triple quadrupole mass spectrometer (ESI-MS/MS). The proposed method has been applied to the simultaneous determination of five methylated nucleosides in human urine samples. A separative method based on HILIC-MS/MS has been developed in parallel with the same instrumental setup to confirm the results obtained by the non-separative method. Both methods were validated in terms of linearity, matrix effect, limits of quantification, precision and recovery. The proposed method was successfully applied to the determination of the selected analytes in the urine of healthy volunteers and was compared with the results obtained by the separative method. The proposed non-separative method considerably reduced the required analysis time and simplified the sample treatment, which is a remarkable advantage over the chromatographic methods described. Therefore, this method is suitable for rapid, simple, and reliable determination of endogenous biomarkers in urine samples for routine or high-throughput analysis. The separative method is recommended as a complementary tool for confirmation in complex situations or in cases of abnormally high biomarker levels, maintaining specificity and robustness without compromising the practical advantages of the rapid method.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116036"},"PeriodicalIF":2.5,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145832985","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}
Pub Date : 2025-12-19DOI: 10.1016/j.ab.2025.116035
Jessica K. Bilyj, Christina M. Gregg, Trevor D. Rapson
Accurate determination of metallation states in metalloenzymes is essential for correlating metal content with enzymatic function. Inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectroscopy (ICP-OES) are among the most sensitive and practical techniques for this purpose. However, complete digestion of protein samples is critical for accurate results, and the use of acid often leads to protein precipitation before the metals are fully released into solution.
Here, we present a case study using the nitrogenase protein NifDK, which contains both Fe and Mo, to demonstrate that trypsin digestion can prevent acid-induced precipitation. A 1:1 (w/w) ratio of protein to trypsin in Tris buffer at 37 °C overnight results in complete digestion of NifDK. Upon acid addition, a homogeneous solution is obtained without precipitation. This approach yields highly reproducible ICP-MS data.
Further improvements include performing protein quantification on the exact vial used for ICP-MS analysis, applying a drift correction to the ICP-MS data, and using nitric acid with 0.1 % (v/v) HF for accurate molybdenum quantification.
{"title":"Trypsin digestion to prevent acid induced precipitation of metalloproteins for accurate ICP-MS analysis: Nitrogenase case study","authors":"Jessica K. Bilyj, Christina M. Gregg, Trevor D. Rapson","doi":"10.1016/j.ab.2025.116035","DOIUrl":"10.1016/j.ab.2025.116035","url":null,"abstract":"<div><div>Accurate determination of metallation states in metalloenzymes is essential for correlating metal content with enzymatic function. Inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectroscopy (ICP-OES) are among the most sensitive and practical techniques for this purpose. However, complete digestion of protein samples is critical for accurate results, and the use of acid often leads to protein precipitation before the metals are fully released into solution.</div><div>Here, we present a case study using the nitrogenase protein NifDK, which contains both Fe and Mo, to demonstrate that trypsin digestion can prevent acid-induced precipitation. A 1:1 (w/w) ratio of protein to trypsin in Tris buffer at 37 °C overnight results in complete digestion of NifDK. Upon acid addition, a homogeneous solution is obtained without precipitation. This approach yields highly reproducible ICP-MS data.</div><div>Further improvements include performing protein quantification on the exact vial used for ICP-MS analysis, applying a drift correction to the ICP-MS data, and using nitric acid with 0.1 % (v/v) HF for accurate molybdenum quantification.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"710 ","pages":"Article 116035"},"PeriodicalIF":2.5,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145802967","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}
Pub Date : 2025-12-18DOI: 10.1016/j.ab.2025.116034
Moses Mayonu , Saeedeh Babaee , Julie Pollak , Lin Jiang , Bo Wang
Nuclear magnetic resonance (NMR), which is famous for its nondestructive nature and high reliability, is one of the principal analytical platforms in metabolomics. NMR metabolomics has been widely used in human and environmental health studies in the past few decades. However, NMR metabolomics data processing remains challenging due to data complexity. Although automated approaches have been explored, their reliability and accuracy are still limited. One of the limitations is the lack of cross-evaluation of the same peak in all samples during raw data processing. In this study, we developed a new approach that applies machine learning models to evaluate the peak quality for all the samples in an NMR metabolomics study. Our new approach combines the automatically selected potential peaks from all samples into a new spectrum for each peak (potential metabolite), which provides an overview of all the samples to ensure the overall data quality for the downstream statistical analysis. The results indicated that two machine learning approaches, Support Vector Machine Discriminant Analysis (SVMDA) and Extreme Gradient Boosting Discriminant Analysis (XGBDA), demonstrated high prediction rates in identifying high-quality peaks. In addition, the raw data conversion resolution was tested to optimize the performance of each machine learning approach, and XGBDA showed better tolerance to data resolution. The results indicated that machine learning approaches, such as SVMDA and XGBDA, can be used to identify high-quality peaks generated through automated peak picking, ensuring data quality for metabolomics studies. Our study paves the way for automated data processing in future NMR metabolomics research.
{"title":"The application of machine learning in nuclear magnetic resonance (NMR) peak selection for metabolomics studies","authors":"Moses Mayonu , Saeedeh Babaee , Julie Pollak , Lin Jiang , Bo Wang","doi":"10.1016/j.ab.2025.116034","DOIUrl":"10.1016/j.ab.2025.116034","url":null,"abstract":"<div><div>Nuclear magnetic resonance (NMR), which is famous for its nondestructive nature and high reliability, is one of the principal analytical platforms in metabolomics<strong>.</strong> NMR metabolomics has been widely used in human and environmental health studies in the past few decades. However, NMR metabolomics data processing remains challenging due to data complexity. Although automated approaches have been explored, their reliability and accuracy are still limited. One of the limitations is the lack of cross-evaluation of the same peak in all samples during raw data processing. In this study, we developed a new approach that applies machine learning models to evaluate the peak quality for all the samples in an NMR metabolomics study. Our new approach combines the automatically selected potential peaks from all samples into a new spectrum for each peak (potential metabolite), which provides an overview of all the samples to ensure the overall data quality for the downstream statistical analysis. The results indicated that two machine learning approaches, Support Vector Machine Discriminant Analysis (SVMDA) and Extreme Gradient Boosting Discriminant Analysis (XGBDA), demonstrated high prediction rates in identifying high-quality peaks. In addition, the raw data conversion resolution was tested to optimize the performance of each machine learning approach, and XGBDA showed better tolerance to data resolution. The results indicated that machine learning approaches, such as SVMDA and XGBDA, can be used to identify high-quality peaks generated through automated peak picking, ensuring data quality for metabolomics studies. Our study paves the way for automated data processing in future NMR metabolomics research.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"710 ","pages":"Article 116034"},"PeriodicalIF":2.5,"publicationDate":"2025-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145800380","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}
Pub Date : 2025-12-15DOI: 10.1016/j.ab.2025.116033
Jack Brydon , Radovan Krejcir , Mariana Grima , Filip Zavadil-Kokas , Zuzana Kuncova , Sousan Sousan , Robin L. Pflughaupt , Jennifer R. Thomson , Paul Davies , Ella Senior , Geoffrey A. Wood , Kathryn L. Ball , David Saliba , David J. Argyle , Borivoj Vojtesek , Ted Hupp , Maciej Parys
The TIM3 receptor acts as an immune checkpoint protein. Canine cancers exhibit higher pan-cancer penetrance of TIM3 compared to PD1, highlighting the potential of TIM3 as a compelling target in comparative immuno-oncology. We have used a highly diverse naïve canine scFv phage library to isolate antibodies to canine TIM3. Alternating rounds of biopanning were performed using either Fc or GST tagged canine TIM3 synthesized in mammalian cells. scFv sequences were identified using colony screening and next generation deep sequencing (NGS). The NGS protocol identified lower abundant frequency clones, demonstrating the enhanced depth of repertoire discovery possible using sequence-tag based tracing. Three representative scFv were expressed as mouse-canine chimeric scFv-Fc fusions or as full-length chimeric IgG. These antibodies all bound to the TIM3 receptor using either ELISA or immunoblotting. All the antibodies displayed sensitivity to reducing agents, which indicates the existence of disulfide-stabilized conformational epitopes. Epitope mapping using pepscan libraries suggested that the antibodies recognize a shared structural motif within the IgV domain of TIM3, within β-sheets fixed by disulfide bonds which would form the conformational epitope. Such conformational epitopes might be functional because they overlap with ligand-binding interfaces. Consistent with this, the antibodies attenuated TIM3 binding to Galectin-9. These data affirm that naïve canine scFv antibody libraries can yield self-antigen reactive antibodies to immune blockade receptor antigens. The data also emphasize the value in using native, folded, mammalian expressed receptor antigens to increase the probability of acquiring conformationally sensitive antibodies with potential therapeutic applications in veterinary and human medicine.
{"title":"Towards canine immunotherapy models: Monoclonal antibodies with redox regulated epitopes targeting TIM3 attenuate Galectin-9 binding","authors":"Jack Brydon , Radovan Krejcir , Mariana Grima , Filip Zavadil-Kokas , Zuzana Kuncova , Sousan Sousan , Robin L. Pflughaupt , Jennifer R. Thomson , Paul Davies , Ella Senior , Geoffrey A. Wood , Kathryn L. Ball , David Saliba , David J. Argyle , Borivoj Vojtesek , Ted Hupp , Maciej Parys","doi":"10.1016/j.ab.2025.116033","DOIUrl":"10.1016/j.ab.2025.116033","url":null,"abstract":"<div><div>The TIM3 receptor acts as an immune checkpoint protein. Canine cancers exhibit higher pan-cancer penetrance of TIM3 compared to PD1, highlighting the potential of TIM3 as a compelling target in comparative immuno-oncology. We have used a highly diverse naïve canine scFv phage library to isolate antibodies to canine TIM3. Alternating rounds of biopanning were performed using either Fc or GST tagged canine TIM3 synthesized in mammalian cells. scFv sequences were identified using colony screening and next generation deep sequencing (NGS). The NGS protocol identified lower abundant frequency clones, demonstrating the enhanced depth of repertoire discovery possible using sequence-tag based tracing. Three representative scFv were expressed as mouse-canine chimeric scFv-Fc fusions or as full-length chimeric IgG. These antibodies all bound to the TIM3 receptor using either ELISA or immunoblotting. All the antibodies displayed sensitivity to reducing agents, which indicates the existence of disulfide-stabilized conformational epitopes. Epitope mapping using pepscan libraries suggested that the antibodies recognize a shared structural motif within the IgV domain of TIM3, within β-sheets fixed by disulfide bonds which would form the conformational epitope. Such conformational epitopes might be functional because they overlap with ligand-binding interfaces. Consistent with this, the antibodies attenuated TIM3 binding to Galectin-9. These data affirm that naïve canine scFv antibody libraries can yield self-antigen reactive antibodies to immune blockade receptor antigens. The data also emphasize the value in using native, folded, mammalian expressed receptor antigens to increase the probability of acquiring conformationally sensitive antibodies with potential therapeutic applications in veterinary and human medicine.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116033"},"PeriodicalIF":2.5,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145772941","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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