Pub Date : 2025-11-19DOI: 10.1016/j.ab.2025.116015
Adam Yeh , James Saliba , Volker Blank
Dextran sodium sulfate (DSS) is widely used to model colonic inflammation and colorectal cancer. However, it is a potent inhibitor of DNA polymerase, necessitating its removal prior to downstream analysis. Currently, there is no robust, widely applicable method of removing DSS from DNA samples. Here, we demonstrate that cetyltrimethylammonium bromide (CTAB) buffer enables rapid and efficient removal of DSS from mouse stool DNA. In the absence of CTAB, DSS inhibited PCR reactions at concentrations of 136 nM and above. CTAB effectively removed DSS from DNA samples processed by both phenol-chloroform extraction and column cleanup kits. 700 μL of CTAB was able to remove up to 93 nmol (4 mg) of DSS after one round of cleanup, while two rounds removed up to 465 nmol (20 mg) of DSS. Purified genomic DNA was suitable for subsequent quantitative PCR analysis.
{"title":"Rapid purification of DNA samples from dextran sodium sulfate (DSS) contaminants","authors":"Adam Yeh , James Saliba , Volker Blank","doi":"10.1016/j.ab.2025.116015","DOIUrl":"10.1016/j.ab.2025.116015","url":null,"abstract":"<div><div>Dextran sodium sulfate (DSS) is widely used to model colonic inflammation and colorectal cancer. However, it is a potent inhibitor of DNA polymerase, necessitating its removal prior to downstream analysis. Currently, there is no robust, widely applicable method of removing DSS from DNA samples. Here, we demonstrate that cetyltrimethylammonium bromide (CTAB) buffer enables rapid and efficient removal of DSS from mouse stool DNA. In the absence of CTAB, DSS inhibited PCR reactions at concentrations of 136 nM and above. CTAB effectively removed DSS from DNA samples processed by both phenol-chloroform extraction and column cleanup kits. 700 μL of CTAB was able to remove up to 93 nmol (4 mg) of DSS after one round of cleanup, while two rounds removed up to 465 nmol (20 mg) of DSS. Purified genomic DNA was suitable for subsequent quantitative PCR analysis.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116015"},"PeriodicalIF":2.5,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145562489","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 : 2025-11-17DOI: 10.1016/j.ab.2025.116012
Rania A. Hussien
{"title":"Corrigendum to“Development of a green polymer-based sensor for enhanced iron detection in diverse biological, food and environmental matrices” [Analyt. Biochem. 705 (2025) 115927]","authors":"Rania A. Hussien","doi":"10.1016/j.ab.2025.116012","DOIUrl":"10.1016/j.ab.2025.116012","url":null,"abstract":"","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116012"},"PeriodicalIF":2.5,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145538562","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 : 2025-11-13DOI: 10.1016/j.ab.2025.116011
Rithwik Pradeep , Alexander Zhdanov , Evin Allen
Tumour necrosis factor-alpha (TNF-α) plays a central role in inflammation and autoimmune pathology, making it a key therapeutic target. This study systematically evaluated the performance of monoclonal anti-human TNF-α antibodies derived from hybridoma cells—originally developed during early TNF research—using two widely adopted in vitro potency assays: a cytotoxicity-based assay in L929 fibroblasts and an NF-κB reporter assay in HEK293 Blue cells. Antibody performance was assessed in terms of neutralisation efficiency, signal reproducibility, and assay sensitivity. While both assays yielded comparable IC50 values (1.98 for L929 and 1.34 for HEK293 Blue), they differed in dynamic range, sensitivity, and biological relevance. The HEK293 Blue assay provided rapid, robust, and high-throughput-compatible detection of early TNF-α signalling events, with minimal background and superior reproducibility. In contrast, the L929 assay offered physiologically relevant insights into the later consequences of TNF-α signalling, such as apoptosis and metabolic disruption. ATP quantification using the CellTiter-Glo® assay proved to be a practical and sensitive method for viability assessment, though alternative metabolic and membrane integrity assays may complement interpretation. Overall, our findings highlight the complementary strengths of these assay platforms and support a dual-assay approach for comprehensive evaluation of anti-TNF-α antibodies.
{"title":"Comparative analysis of common potency assays for assessing human TNF-alpha neutralising antibodies","authors":"Rithwik Pradeep , Alexander Zhdanov , Evin Allen","doi":"10.1016/j.ab.2025.116011","DOIUrl":"10.1016/j.ab.2025.116011","url":null,"abstract":"<div><div>Tumour necrosis factor-alpha (TNF-α) plays a central role in inflammation and autoimmune pathology, making it a key therapeutic target. This study systematically evaluated the performance of monoclonal anti-human TNF-α antibodies derived from hybridoma cells—originally developed during early TNF research—using two widely adopted in vitro potency assays: a cytotoxicity-based assay in L929 fibroblasts and an NF-κB reporter assay in HEK293 Blue cells. Antibody performance was assessed in terms of neutralisation efficiency, signal reproducibility, and assay sensitivity. While both assays yielded comparable IC<sub>50</sub> values (1.98 for L929 and 1.34 for HEK293 Blue), they differed in dynamic range, sensitivity, and biological relevance. The HEK293 Blue assay provided rapid, robust, and high-throughput-compatible detection of early TNF-α signalling events, with minimal background and superior reproducibility. In contrast, the L929 assay offered physiologically relevant insights into the later consequences of TNF-α signalling, such as apoptosis and metabolic disruption. ATP quantification using the CellTiter-Glo® assay proved to be a practical and sensitive method for viability assessment, though alternative metabolic and membrane integrity assays may complement interpretation. Overall, our findings highlight the complementary strengths of these assay platforms and support a dual-assay approach for comprehensive evaluation of anti-TNF-α antibodies.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116011"},"PeriodicalIF":2.5,"publicationDate":"2025-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145530424","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 : 2025-11-11DOI: 10.1016/j.ab.2025.116010
Žiga Medoš , Marija Bešter-Rogač , Epameinondas Leontidis , Joel Tellinghuisen
Using experiments based on the NaCl dilution calibration method, we examine several previously unstudied instrumental effects that can lead to systematic errors in isothermal titration calorimetry (ITC). We confirm earlier results that power feedback can affect estimated heats and find further that it can also affect cell temperature and introduce temperature “noise.” To estimate effects from loss of titrant from the syringe through diffusion, and concentration changes in the cell from mixing with the overflow volume, we conduct experiments with injections separated by time intervals Δt from 8 to 60 min, giving run durations from 6 h to over 2 days. Because the effects are small, we have developed a global least-squares fitting algorithm to analyze 20 datasets simultaneously, from which three parameters governing diffusion and mixing (“leakage”) are estimated, along with calibration parameters for heat and cell volume. Expressed as an effective volume loss from the syringe, Δvdif is found to be of order ∼0.03 μL per injection for NaCl into water. Its effect is predicted to be negligible in many ITC applications. The effects from leakage are more significant but their neglect is still predicted to affect both NaCl calibration and 1:1 binding results by ∼1 %, which is marginally significant for calibration but seldom so for 1:1 binding. The calibration results for the cell volume V0 are particularly sensitive to temperature and to the literature source for the relative molar enthalpy function for NaCl(aq).
{"title":"Systematic errors in isothermal titration calorimetry: The role of feedback power and effects of mixing and diffusion on concentrations","authors":"Žiga Medoš , Marija Bešter-Rogač , Epameinondas Leontidis , Joel Tellinghuisen","doi":"10.1016/j.ab.2025.116010","DOIUrl":"10.1016/j.ab.2025.116010","url":null,"abstract":"<div><div>Using experiments based on the NaCl dilution calibration method, we examine several previously unstudied instrumental effects that can lead to systematic errors in isothermal titration calorimetry (ITC). We confirm earlier results that power feedback can affect estimated heats and find further that it can also affect cell temperature and introduce temperature “noise.” To estimate effects from loss of titrant from the syringe through diffusion, and concentration changes in the cell from mixing with the overflow volume, we conduct experiments with injections separated by time intervals Δ<em>t</em> from 8 to 60 min, giving run durations from 6 h to over 2 days. Because the effects are small, we have developed a global least-squares fitting algorithm to analyze 20 datasets simultaneously, from which three parameters governing diffusion and mixing (“leakage”) are estimated, along with calibration parameters for heat and cell volume. Expressed as an effective volume loss from the syringe, Δ<em>v</em><sub>dif</sub> is found to be of order ∼0.03 μL per injection for NaCl into water. Its effect is predicted to be negligible in many ITC applications. The effects from leakage are more significant but their neglect is still predicted to affect both NaCl calibration and 1:1 binding results by ∼1 %, which is marginally significant for calibration but seldom so for 1:1 binding. The calibration results for the cell volume <em>V</em><sub>0</sub> are particularly sensitive to temperature and to the literature source for the relative molar enthalpy function for NaCl(<em>aq</em>).</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116010"},"PeriodicalIF":2.5,"publicationDate":"2025-11-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145511445","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 : 2025-11-08DOI: 10.1016/j.ab.2025.116009
Anna Makovska , Erik Walinda , Ryo Aoyama , Minsoo Kim , Daichi Morimoto
NEDD8, a ubiquitin-like modifier essential for cullin-RING ligase regulation, is difficult to refold from Escherichia coli inclusion bodies due to aggregation during denaturant removal. Conventional refolding methods such as rapid dilution, one-step dialysis, and stepwise dialysis often produce poorly soluble proteins, limiting structural and functional analyses. To overcome this, we developed a simplified slow dialysis system using a single peristaltic pump to generate a gradual urea gradient over 96 h, enabling efficient refolding of NEDD8 under gentle conditions. Both wild-type and Q40E-mutant NEDD8, which mimics the bacteria-mediated deamidation observed during infection, refolded successfully into monomeric protein, as confirmed by size-exclusion chromatography. NMR spectra showed folded protein that matched reference assignments, demonstrating suitability for atomic level structural analysis. The method also enabled recovery of previously insoluble ISG15 mutants, highlighting its broad applicability for structural studies of diverse challenging proteins.
{"title":"A minimal slow dialysis method for refolding inclusion body proteins: Structural application to NEDD8 and its Q40E mutant","authors":"Anna Makovska , Erik Walinda , Ryo Aoyama , Minsoo Kim , Daichi Morimoto","doi":"10.1016/j.ab.2025.116009","DOIUrl":"10.1016/j.ab.2025.116009","url":null,"abstract":"<div><div>NEDD8, a ubiquitin-like modifier essential for cullin-RING ligase regulation, is difficult to refold from <em>Escherichia coli</em> inclusion bodies due to aggregation during denaturant removal. Conventional refolding methods such as rapid dilution, one-step dialysis, and stepwise dialysis often produce poorly soluble proteins, limiting structural and functional analyses. To overcome this, we developed a simplified slow dialysis system using a single peristaltic pump to generate a gradual urea gradient over 96 h, enabling efficient refolding of NEDD8 under gentle conditions. Both wild-type and Q40E-mutant NEDD8, which mimics the bacteria-mediated deamidation observed during infection, refolded successfully into monomeric protein, as confirmed by size-exclusion chromatography. NMR spectra showed folded protein that matched reference assignments, demonstrating suitability for atomic level structural analysis. The method also enabled recovery of previously insoluble ISG15 mutants, highlighting its broad applicability for structural studies of diverse challenging proteins.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116009"},"PeriodicalIF":2.5,"publicationDate":"2025-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145487379","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 : 2025-11-05DOI: 10.1016/j.ab.2025.116008
Thomas Olsen , Amany Elshorbagy , Jay E. Johnson , Sailendra N. Nichenametla , Zhen Dong , Kumar Sambamurti , John P. Richie Jr. , Kathrine J. Vinknes
Dietary restriction of the sulfur amino acids methionine and cysteine (sulfur amino acid restriction [SAAR]) is a well-established paradigm for delaying disease onset and the aging process in several experimental models. In vivo, SAAR's anti-aging effects appear to be mediated by decreased growth hormone/insulin-like growth factor-1 (GH/IGF-1) signaling, along with improvement in insulin sensitivity and overall metabolic health. SAAR-fed animals also exhibit reduced regional and total adiposity, as well as oxidative stress and inflammation. Recent experimental studies suggest that SAAR improves cognition, induces significant changes in gut microbiome composition, and that its benefits may depend on the age at which the intervention begins. In humans, observational studies have shown that higher plasma total cysteine levels are positively correlated with adiposity, insulin resistance, and an increased incidence of diabetes. Likewise, high dietary methionine and cysteine intake has been linked to increased risk of cardiovascular disease and diabetes-related mortality. Human dietary intervention studies have only been partly successful in translating the benefits of SAAR, and practical challenges for implementation remain to be addressed. This review summarizes recent advances in the SAAR field and discusses its translational potential for promoting metabolic health and reducing the risk of age-related diseases.
{"title":"Sulfur amino acids, metabolic health and beyond: Recent advances, translational implications, and future research considerations","authors":"Thomas Olsen , Amany Elshorbagy , Jay E. Johnson , Sailendra N. Nichenametla , Zhen Dong , Kumar Sambamurti , John P. Richie Jr. , Kathrine J. Vinknes","doi":"10.1016/j.ab.2025.116008","DOIUrl":"10.1016/j.ab.2025.116008","url":null,"abstract":"<div><div>Dietary restriction of the sulfur amino acids methionine and cysteine (sulfur amino acid restriction [SAAR]) is a well-established paradigm for delaying disease onset and the aging process in several experimental models. <em>In vivo</em>, SAAR's anti-aging effects appear to be mediated by decreased growth hormone/insulin-like growth factor-1 (GH/IGF-1) signaling, along with improvement in insulin sensitivity and overall metabolic health. SAAR-fed animals also exhibit reduced regional and total adiposity, as well as oxidative stress and inflammation. Recent experimental studies suggest that SAAR improves cognition, induces significant changes in gut microbiome composition, and that its benefits may depend on the age at which the intervention begins. In humans, observational studies have shown that higher plasma total cysteine levels are positively correlated with adiposity, insulin resistance, and an increased incidence of diabetes. Likewise, high dietary methionine and cysteine intake has been linked to increased risk of cardiovascular disease and diabetes-related mortality. Human dietary intervention studies have only been partly successful in translating the benefits of SAAR, and practical challenges for implementation remain to be addressed. This review summarizes recent advances in the SAAR field and discusses its translational potential for promoting metabolic health and reducing the risk of age-related diseases.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116008"},"PeriodicalIF":2.5,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145470385","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 : 2025-11-04DOI: 10.1016/j.ab.2025.116007
Wenxia Zhang , Guimei Wang , Zhenyu Wang , Guoqi Zhou , Hanliang Jiang
Background
Tyrosine metabolism (TM) plays an important role in the progression of cancer, but its role in lung adenocarcinoma (LUAD) is still unclear. This study aims to construct TM-related prognostic features for LUAD.
Methods
Transcriptomes and clinical data of LUAD were collected from public databases. A TM-related risk score (TMRS) model was constructed using 42 TM-related genes (TMRGs). The prognostic value of the model was comprehensively analyzed through survival analysis, enrichment analysis, immune assessment, and drug sensitivity prediction. The expression of key genes was also verified in LUAD cell lines and patient PBMCs.
Results
A 14-gene prognostic model (TMRS) was constructed. TMRS was an independent prognostic factor for LUAD. The low TMRS group has a more active immune microenvironment and may be more sensitive to immunotherapy. Patients with high TMRS may be more sensitive to various chemotherapy drugs. The model genes were specifically expressed in different cell types, suggesting that they may be involved in metabolic reprogramming and tumor progression.
Conclusion
This study establishes a foundation for personalized risk assessment and treatment decisions in LUAD, highlighting the prognostic significance of TM.
{"title":"The role of tyrosine metabolism-associated genes in predicting lung adenocarcinoma prognosis and their clinical relevance","authors":"Wenxia Zhang , Guimei Wang , Zhenyu Wang , Guoqi Zhou , Hanliang Jiang","doi":"10.1016/j.ab.2025.116007","DOIUrl":"10.1016/j.ab.2025.116007","url":null,"abstract":"<div><h3>Background</h3><div>Tyrosine metabolism (TM) plays an important role in the progression of cancer, but its role in lung adenocarcinoma (LUAD) is still unclear. This study aims to construct TM-related prognostic features for LUAD.</div></div><div><h3>Methods</h3><div>Transcriptomes and clinical data of LUAD were collected from public databases. A TM-related risk score (TMRS) model was constructed using 42 TM-related genes (TMRGs). The prognostic value of the model was comprehensively analyzed through survival analysis, enrichment analysis, immune assessment, and drug sensitivity prediction. The expression of key genes was also verified in LUAD cell lines and patient PBMCs.</div></div><div><h3>Results</h3><div>A 14-gene prognostic model (TMRS) was constructed. TMRS was an independent prognostic factor for LUAD. The low TMRS group has a more active immune microenvironment and may be more sensitive to immunotherapy. Patients with high TMRS may be more sensitive to various chemotherapy drugs. The model genes were specifically expressed in different cell types, suggesting that they may be involved in metabolic reprogramming and tumor progression.</div></div><div><h3>Conclusion</h3><div>This study establishes a foundation for personalized risk assessment and treatment decisions in LUAD, highlighting the prognostic significance of TM.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116007"},"PeriodicalIF":2.5,"publicationDate":"2025-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145457608","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 : 2025-11-03DOI: 10.1016/j.ab.2025.116005
Mingxian Lu , Taigang Liu
Acidophilic, alkaliphilic, and halophilic proteins function under extreme conditions and hold great industrial value. However, their experimental identification is time-consuming and costly. Here, we introduce AHAPC, a unified computational framework for multiclass classification of extremophilic proteins. First, we construct a new benchmark dataset, TriExtrem, by combining three rigorously curated datasets. Then, we extracted two types of protein features, i.e., embeddings from pretrained protein language models (PLMs) and hand-crafted embeddings which include evolutionary descriptors derived from position-specific scoring matrix (PSSM), and sequence features, followed by the feature fusion and selection. Finally, convolutional neural network (CNN), gated recurrent unit (GRU), and bidirectional long short-term memory (BiLSTM) were used for three binary classification tasks respectively, while a multi-branch BiLSTM was adopted for the multiclass setting. Comprehensive evaluation and visualized analysis demonstrate that AHAPC achieves strong performance and provides interpretable predictions, facilitating reliable discovery of extremophilic proteins.
{"title":"AHAPC: Multi-source feature fusion and ensemble learning for multiclass extremophilic protein prediction","authors":"Mingxian Lu , Taigang Liu","doi":"10.1016/j.ab.2025.116005","DOIUrl":"10.1016/j.ab.2025.116005","url":null,"abstract":"<div><div>Acidophilic, alkaliphilic, and halophilic proteins function under extreme conditions and hold great industrial value. However, their experimental identification is time-consuming and costly. Here, we introduce AHAPC, a unified computational framework for multiclass classification of extremophilic proteins. First, we construct a new benchmark dataset, TriExtrem, by combining three rigorously curated datasets. Then, we extracted two types of protein features, i.e., embeddings from pretrained protein language models (PLMs) and hand-crafted embeddings which include evolutionary descriptors derived from position-specific scoring matrix (PSSM), and sequence features, followed by the feature fusion and selection. Finally, convolutional neural network (CNN), gated recurrent unit (GRU), and bidirectional long short-term memory (BiLSTM) were used for three binary classification tasks respectively, while a multi-branch BiLSTM was adopted for the multiclass setting. Comprehensive evaluation and visualized analysis demonstrate that AHAPC achieves strong performance and provides interpretable predictions, facilitating reliable discovery of extremophilic proteins.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116005"},"PeriodicalIF":2.5,"publicationDate":"2025-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145450530","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}
The elevated expression of microRNA-21 (miRNA-21) in exosomes derived from early-stage non-small cell lung cancer (NSCLC) holds significant promise for early diagnosis. However, detecting exosomal miRNA-21 remains challenging due to its low abundance. Here, we developed an electrochemiluminescence (ECL) biosensor integrating a 3D DNA walker with rolling circle amplification (RCA) for ultrasensitive miRNA-21 detection. Gold-silver nanoclusters (AuAgNCs, Au:Ag = 4:1) served as ECL emitters, while ferrocene-modified DNA (Fc-DNA) quenched the signal ("signal-off"). Target miRNA-21 activated the 3D DNA walker, generating primers for RCA. The RCA products displaced Fc-DNA, restoring ECL ("signal-on"). The biosensor achieved a wide linear range (1 fM-1 nM) with a 0.14 fM detection limit. Critically, it distinguished NSCLC patients (n = 10) from healthy controls (n = 10) and COPD patients (n = 10) with statistical significance (P < 0.001) in clinical serum samples. Excellent stability (84 % signal retention after 25 days) and reproducibility (RSD = 3.95 %) further support its utility for early lung cancer screening.
{"title":"Bimetallic nanoclusters and dual-amplification tactic: an ultrasensitive electrochemiluminescence biosensor for miRNA-21 detection.","authors":"Tong Shen, Hongwei Yu, Xin Xu, Ze Zhang, Huanzhang Yang, Dong Chang","doi":"10.1016/j.ab.2025.115948","DOIUrl":"10.1016/j.ab.2025.115948","url":null,"abstract":"<p><p>The elevated expression of microRNA-21 (miRNA-21) in exosomes derived from early-stage non-small cell lung cancer (NSCLC) holds significant promise for early diagnosis. However, detecting exosomal miRNA-21 remains challenging due to its low abundance. Here, we developed an electrochemiluminescence (ECL) biosensor integrating a 3D DNA walker with rolling circle amplification (RCA) for ultrasensitive miRNA-21 detection. Gold-silver nanoclusters (AuAgNCs, Au:Ag = 4:1) served as ECL emitters, while ferrocene-modified DNA (Fc-DNA) quenched the signal (\"signal-off\"). Target miRNA-21 activated the 3D DNA walker, generating primers for RCA. The RCA products displaced Fc-DNA, restoring ECL (\"signal-on\"). The biosensor achieved a wide linear range (1 fM-1 nM) with a 0.14 fM detection limit. Critically, it distinguished NSCLC patients (n = 10) from healthy controls (n = 10) and COPD patients (n = 10) with statistical significance (P < 0.001) in clinical serum samples. Excellent stability (84 % signal retention after 25 days) and reproducibility (RSD = 3.95 %) further support its utility for early lung cancer screening.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"115948"},"PeriodicalIF":2.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144726511","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 : 2025-10-31DOI: 10.1016/j.ab.2025.116006
Guangxin Yuan , Meng Xia , Ya Li , Honglei Tian , Jianyu Zhu , Yanshuang Wang
Deer placenta and Placenta Hominis are valued traditional Chinese medicines often adulterated with cheaper pig or cow placenta. Current identification methods frequently lack the throughput, speed, or simplicity for effective field monitoring. This study aimed to develop a high-throughput, rapid method to simultaneously identify deer, human, pig, and cow placenta. The approach involved screening a rapid genomic DNA extraction protocol and designing specific primers for the mitochondrial cytochrome b gene of each species, with primers labeled with FAM and Biotin. Following the optimization of PCR amplification conditions, results were visually detected using immunocolloidal gold test strips. The method was rigorously evaluated for its specificity, reproducibility, sensitivity, and stability. The results showed that the extracted DNA was of satisfactory concentration, purity, and integrity. Under optimized conditions (59 °C annealing temperature, 20 cycles), authentic samples for all four placenta types produced two distinct red bands on the test strips, while adulterated and blank controls showed only one control band. Agarose gel electrophoresis confirmed specific amplification for each target with no cross-reactivity. The method demonstrated strong specificity, excellent reproducibility and stability, and a high sensitivity of 0.01 ng μL−1, surpassing conventional electrophoresis. In conclusion, this PCR-based test strip method enables the visual, simultaneous authentication of the four placenta types in a single test, making it highly suitable for on-site rapid monitoring and quality control.
{"title":"A high-throughout PCR test strip method for the rapid identification of four placentas","authors":"Guangxin Yuan , Meng Xia , Ya Li , Honglei Tian , Jianyu Zhu , Yanshuang Wang","doi":"10.1016/j.ab.2025.116006","DOIUrl":"10.1016/j.ab.2025.116006","url":null,"abstract":"<div><div>Deer placenta and Placenta Hominis are valued traditional Chinese medicines often adulterated with cheaper pig or cow placenta. Current identification methods frequently lack the throughput, speed, or simplicity for effective field monitoring. This study aimed to develop a high-throughput, rapid method to simultaneously identify deer, human, pig, and cow placenta. The approach involved screening a rapid genomic DNA extraction protocol and designing specific primers for the mitochondrial <em>cytochrome b</em> gene of each species, with primers labeled with FAM and Biotin. Following the optimization of PCR amplification conditions, results were visually detected using immunocolloidal gold test strips. The method was rigorously evaluated for its specificity, reproducibility, sensitivity, and stability. The results showed that the extracted DNA was of satisfactory concentration, purity, and integrity. Under optimized conditions (59 °C annealing temperature, 20 cycles), authentic samples for all four placenta types produced two distinct red bands on the test strips, while adulterated and blank controls showed only one control band. Agarose gel electrophoresis confirmed specific amplification for each target with no cross-reactivity. The method demonstrated strong specificity, excellent reproducibility and stability, and a high sensitivity of 0.01 ng μL<sup>−1</sup>, surpassing conventional electrophoresis. In conclusion, this PCR-based test strip method enables the visual, simultaneous authentication of the four placenta types in a single test, making it highly suitable for on-site rapid monitoring and quality control.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116006"},"PeriodicalIF":2.5,"publicationDate":"2025-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145429918","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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