Analytical biochemistry最新文献

{"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}

{"title":"High-Performance Sn/AuNP-Based Spectroscopic Ellipsometric Sensor for Patulin.","authors":"Zafer Üstündağ, Sinem Tunçer Çağlayan, Mustafa Oguzhan Caglayan","doi":"10.1016/j.ab.2025.115987","DOIUrl":"https://doi.org/10.1016/j.ab.2025.115987","url":null,"abstract":"<p><p>A label-free spectroscopic ellipsometric (SE) aptasensor was developed for ultra-sensitive patulin (PAT) detection on a tin (Sn) substrate modified via electrochemical grafting of a 4-mercaptobenzene diazonium-derived self-assembled monolayer. Cyclic voltammetry and high-resolution X-ray photoelectron spectroscopy confirmed uniform nanofilm formation, followed by citrate-reduced gold nanoparticle decoration and thiolated anti-PAT aptamer immobilization. Monitoring the ellipsometric phase shift (Δ) at 507 nm and 508 nm yielded linear responses over 0.01-1000 ng/mL PAT (R² > 0.98) with limits of detection of 10.7 pg/mL and 9.5 pg/mL (3.3 σ), respectively. Isotherm fitting indicated pseudo-second-order binding (R² = 1.00), consistent with surface-constrained, mass-transfer-limited interactions. Precision (%RSD < 1.1 %) and accuracy (%RE within ±5 %) were validated in both buffer and spiked apple-juice samples (recoveries 98.9-103.2 %). This label-free SE approach combines a wide dynamic range and sub-10 pg/mL sensitivity with minimal sample preparation and no external labels.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"115987"},"PeriodicalIF":2.5,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145231324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An ultrasensitive electrochemical immunosensor constructed via Ag-GO-Nf nanocomposites for the detection of pefloxacin.","authors":"Jiaojiao Zhang, Yankai Liu, Enping Liu, Yajiao Li, Jingming Zhou, Yumei Chen, Hongliang Liu, Xifang Zhu, Aiping Wang","doi":"10.1016/j.ab.2025.115985","DOIUrl":"https://doi.org/10.1016/j.ab.2025.115985","url":null,"abstract":"<p><p>Pefloxacin (PEF), a third-generation quinolone antimicrobial drug, is widely used in fisheries and animal husbandry. However, overuse poses food safety hazards and serious threats to human health. In this study, a label-free electrochemical immunosensor based on Ag-GO-Nf nanocomposites was developed for the rapid detection of PEF. Although electrochemical immunoassays are well-established for quinolone detection, no specific method has been reported for PEF. We synthesized silver nanoparticle/graphene oxide/nafion nanocomposites (Ag-GO-Nf) via in situ reduction, where the synergistic effect of GO (high conductivity) and AgNPs (redox-active) significantly enhanced electron transfer efficiency. The nanocomposites were characterized by UV-Vis, XRD, SEM, and TEM, while electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to monitor the stepwise immunosensor assembly. Key experimental parameters were optimized systematically. The developed sensor exhibited a wide linear range (0.50-100 ng/mL), an ultra-low limit of detection (0.27 ng/mL, S/N = 3), and high specificity toward structural analogues. Practical assays in spiked pork and milk samples yielded recoveries of 97.0-100.4%, while the current signal retained 90.77% of its initial value after 28 days, demonstrating excellent long-term stability. This work provides a robust technical strategy for rapid, sensitive, and efficient PEF detection.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"115985"},"PeriodicalIF":2.5,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145231327","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":"Highly sensitive luciferase-based assay with red fluorescent protein expression for accurate quantitative monitoring and real-time visualization of cell invasion","authors":"Michael Lyngbæk Christensen ,&nbsp;Tove Kirkegaard ,&nbsp;Ismail Gögenur ,&nbsp;Frederik Diness ,&nbsp;Jesper Thorvald Troelsen ,&nbsp;Stine Bull Jessen","doi":"10.1016/j.ab.2025.115986","DOIUrl":"10.1016/j.ab.2025.115986","url":null,"abstract":"<div><h3>Background</h3><div>Traditional migration and invasion assays like scratch, Transwell, and Boyden chamber are widely used but have disadvantages such as being time-consuming, lacking real-time monitoring, and relying on endpoint measurements. We addressed these limitations by developing a novel fluorescent and luciferase-based invasion assay.</div></div><div><h3>Materials and methods</h3><div>Three stable cell lines co-expressing the red fluorescent protein dTomato, and secreting luciferase were generated based on Caco-2, MDA-MB-231 and HEK293T cells. Transwell chamber membranes were coated with Matrigel for invasion assay, onto which the modified cells were seeded. To simulate non-invasive and invasive conditions, chambers were incubated for 48 h in FBS-free or FBS-supplemented medium. Following incubation, the Matrigel along with non-invasive cells were removed, and the chambers washed before being transferred into fresh media for 24 h allowing the cells to secrete luciferase. Luciferase activity was measured and compared to traditional cell counting invasion assay, with further confirmations through Z-stacking and microscopic fluorescent imaging.</div></div><div><h3>Results</h3><div>Our results demonstrated that luciferase activity accurately correlates with cell count. Applying luciferase efficiently quantifies variation in cell invasion with higher sensitivity, hence improving detection of low-level invasion as compared to cell counting techniques based on nuclear staining. The expression of the fluorescent dTomato protein proved ideal for real-time visualization of invading cells.</div></div><div><h3>Conclusion</h3><div>Overall, using luciferase and dTomato co-expressing cells for invasion assay showed reliable and accurate measurements of variations in cell invasion patterns. Introducing these cells reduced time-consuming steps, improved sensitivity, and endpoints measurements, while being capable of real-time visualization, providing advantages over traditional methods.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"708 ","pages":"Article 115986"},"PeriodicalIF":2.5,"publicationDate":"2025-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145190684","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":"Heterodimeric aptamer improves the limit of detection of sandwich ELASA and dot blot assay","authors":"Tzi Shien Yeoh,&nbsp;Marimuthu Citartan","doi":"10.1016/j.ab.2025.115984","DOIUrl":"10.1016/j.ab.2025.115984","url":null,"abstract":"<div><div>Heterodimerization, which is an amalgamation of discrete aptamers into a single functional module is a viable strategy to augment the binding strength of aptamers. In this study, we sought to derive several heterodimeric aptamers using the discrete aptamers, LepDapt-2a and LepDapt-5a prior to their binding evaluation against the target LipL32. We further integrated heterodimeric aptamer LepDapt-H1b as a detection aptamer into aptamer-based sandwich ELASA and dot blot assay for the detection of <em>Leptospira</em>, attaining a LOD of 4 and 10 CFU/mL for both assays, respectively, which are better compared to our previous study. Additionally, the LOQ acquired for sandwich ELASA assay was 11 CFU/mL. We have proven that heterodimerization has improved the performance of sandwich ELASA and dot blot assay, suggesting them to be applicable for the diagnostics of leptospirosis.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"708 ","pages":"Article 115984"},"PeriodicalIF":2.5,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145181840","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":"H-ferritin nanocages: refined strategy for optimized protein production and purification","authors":"Arianna Bonizzi,&nbsp;Marta Sevieri,&nbsp;Leopoldo Sitia,&nbsp;Raffaele Allevi,&nbsp;Francesca Gorgoglione,&nbsp;Ilaria Tagliolini,&nbsp;Beatrice Bignami,&nbsp;Serena Mazzucchelli","doi":"10.1016/j.ab.2025.115983","DOIUrl":"10.1016/j.ab.2025.115983","url":null,"abstract":"<div><div>H-ferritin (HFn) nanocages are promising tools in biomedical applications, yet producing sufficient injectable quantities remains challenging. To optimize production, we compared two lysis methods and purification protocols, focusing on the ClearColi BL21(DE3) strain, which features a modified lipopolysaccharide that avoids triggering endotoxin responses in humans. Our findings show that omitting protease inhibitors during lysis and using 20 mM Tris-HCl pH 8.0 buffer with Q-Sepharose resin significantly enhances protein yield. Importantly, these adjustments do not compromise encapsulation efficiency or cytotoxicity, offering a more efficient and cost-effective strategy for obtaining pure, stable HFn suitable for therapeutic use.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"708 ","pages":"Article 115983"},"PeriodicalIF":2.5,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145181796","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":"StackAPP: Advancing autophagy protein identification with ensemble learning","authors":"Munem Shahriar Shoyshob ,&nbsp;Kusay Faisal Al-Tabatabaie ,&nbsp;Lway Faisal Abdulrazak ,&nbsp;Md. Ashikur Rahman ,&nbsp;Md. Mamun Ali ,&nbsp;Sobhy M. Ibrahim ,&nbsp;Kawsar Ahmed ,&nbsp;Francis M. Bui ,&nbsp;Mohammad Ali Moni","doi":"10.1016/j.ab.2025.115981","DOIUrl":"10.1016/j.ab.2025.115981","url":null,"abstract":"<div><div>Autophagy is an important cell process that may be critical for various physiological activities as well as maintenance of the cellular bioenergetic and metabolic homeostasis. Identifying the proteins involved in autophagy is essential for understanding autophagy pathways and developing treatments for autophagy-related disorders. This work introduces an innovative approach to the prediction of autophagy proteins that involves the integration of stacking classifiers with the feature fusion of Amphiphilic Pseudo Amino Acid Composition and Amino Acid Composition. Initially, protein sequences are used to extract Amphiphilic Pseudo Amino Acid Composition and Amino Acid Composition features. The complementary data collected by Amphiphilic Pseudo Amino Acid Composition and Amino Acid Composition are then integrated using a feature fusion technique. Stacking classifiers combines multiple base classifiers to improve predictive performance, using the fused features as input. The proposed method proves its efficacy in the identification of autophagy proteins by achieving an impressive accuracy of 0.9606 and the Matthews correlation coefficient (MCC) of 0.9241 on the independent test. Further, our methodology is better than the standard methods in terms of predictive accuracy, as evidenced through comparative analysis. Overall, the current study provides a realistic model for the prediction of autophagy proteins with prospects for use in the protein prediction field as well as the field of bioinformatics and biomedical to enhance future research directions.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"708 ","pages":"Article 115981"},"PeriodicalIF":2.5,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145102569","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":"High-throughput protein A chromatography platform for accurate antibody quantification in IV admixtures","authors":"Tse-Hong Chen,&nbsp;Xiaoyang Liu,&nbsp;Shao-Chun Wang,&nbsp;Mohammed Shameem,&nbsp;Kenneth S. Graham","doi":"10.1016/j.ab.2025.115982","DOIUrl":"10.1016/j.ab.2025.115982","url":null,"abstract":"<div><div>Antibodies hold significant therapeutic potential, but accurate quantification of them in complex intravenous (IV) admixtures is critical for ensuring therapeutic efficacy and patient safety. Traditional reversed-phase liquid chromatography (RPLC) often faces challenges in resolving antibodies from non-product related impurities within IV matrices. This study first evaluates the specificity of Protein A (ProA) affinity chromatography for quantifying bispecific antibodies (BsAbs) in IV matrices. The method leverages the selective binding of ProA to the antibody Fc region to achieve clear separation of antibodies from potential interference, including human serum albumin (HSA), and extractables and leachables (E&amp;L) from contact materials, enabling accurate quantification down to 0.1 μg/mL. The fit-for-purpose method qualification secondly demonstrates linearity, accuracy, and precision across the tested concentration ranges. The ProA method achieves rapid and complete separation in 5 min without sample preparation, significantly enhancing throughput. This platform capability is further demonstrated by its successful application to five different BsAbs in this study. These findings highlight the ProA method as a reliable, efficient, and specific approach for accurately quantifying antibodies in the presence of challenging IV admixture matrices, supporting the development and administration of low-dose antibody therapies.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"708 ","pages":"Article 115982"},"PeriodicalIF":2.5,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145102566","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":"Real-time electrochemical detection of Ascorbic acid in mouse brain homogenate using SCN-wrapped Co@NiO Nanosensor","authors":"Shahid Habib Ansari ,&nbsp;Sana Amjad ,&nbsp;Tehreem Ul Wara ,&nbsp;Sehrish Hanif ,&nbsp;Ali Raza ,&nbsp;Hafiza Khushbakht Hussain ,&nbsp;Naeem Akhtar ,&nbsp;Mohibullah Shah ,&nbsp;Imran Imran ,&nbsp;Sonam Javaid Khan ,&nbsp;Bushra Yaqub ,&nbsp;Samra Rasheed","doi":"10.1016/j.ab.2025.115980","DOIUrl":"10.1016/j.ab.2025.115980","url":null,"abstract":"<div><div>Oxidative stress, a major key factor to neurological disorders such as Parkinson's, Alzheimer's, and Huntington's disease. Ascorbic acid (AA), a vital brain antioxidant, protects neurons by scavenging reactive oxygen species, and its fluctuations can damage neuronal function. Therefore, precise monitoring of AA in humans is critical for early diagnosis and disease management. However, despite significant advancements in noble metal-free nanocomposite based electrochemical sensors, precise detection remains challenging due to low physiological concentrations and interference from coexisting biomolecules, which limit sensor sensitivity, selectivity, and real-time applicability. To address these limitations herein, we synthesized cobalt-doped nickel oxide (Co@NiO), wrapped with thiourea, and dopamine (SCN- wrapped Co@NiO) for selective and sensitive detection of AA. The SCN groups provide abundant binding sites through hydrogen-bonding interactions with AA, thereby enhancing selectivity, while the Co@NiO nanostructure catalytically facilitates AA oxidation, significantly improving sensitivity and enabling efficient sensing efficacy against interfering species. The findings demonstrated that the fabricated material exhibits excellent sensitivity (0.1837 μA/nM/cm²), a wide linear range (5nM–20μM), and low detection limit (12.72 nM). Furthermore, the designed electrode has shown excellent selectivity towards AA even in presence of various interfering species, highlighting its potential in real-time analysis for practical applications.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"708 ","pages":"Article 115980"},"PeriodicalIF":2.5,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145090912","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":"qPCR-based method to measure plasmid internalization efficiency of non-viral nanoparticle carriers in adherent cell model","authors":"Priscila Tonon Baschirotto,&nbsp;Samara Valeria López Ramírez,&nbsp;Randall Loaiza-Montoya","doi":"10.1016/j.ab.2025.115979","DOIUrl":"10.1016/j.ab.2025.115979","url":null,"abstract":"<div><div>Non-viral nanoparticle-mediated transfection systems have gained wide acceptance in modern pharmaceutical and biotechnological applications, including RNA delivery for COVID-19 vaccines. However, these systems may show low transfection and transcription efficiencies, potentially limiting the use of reporter molecules like GFP to estimate efficacy in early development stages. Moreover, the expression of reporter molecules may not reflect internalization efficiency, which can be pertinent information for the development of these systems. Therefore, as their popularity continues to rise, more sensitive and accurate tools will be necessary.</div><div>We validated a DNA extraction and qPCR assay to quantify the internalized plasmid copy number in NIH/3T3 cells transfected using LGA-PEI (lactic-co-glycolic acid-modified polyethylenimine) nanoparticles. Optimized qPCR and DNA extraction assays exhibited high linearity, sensitivity, and robustness. Additionally, we developed an appropriate extraction and extracellular plasmid digestion protocol that eliminated potential polymer interference with the qPCR reaction and increased specificity for internalized plasmid quantification.</div><div>Using these optimized protocols, we estimated that transfected NIH/3T3 cells incorporated approximately 9 500 to 150 000 plasmids/cell for transfection reactions that yielded as few as 4.1 %–18.2 % GFP-positive cells. Overall, these results indicate that our optimized method is reliable for quantifying transfected plasmids in context of low-efficiency nanoparticle carrier system.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"708 ","pages":"Article 115979"},"PeriodicalIF":2.5,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145079502","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}