Analytical biochemistry最新文献

{"title":"One-step purification of a bioactive PAK1-derived peptide","authors":"Djamali Muhoza ,&nbsp;Emily P. Esquivel ,&nbsp;Stacy R. Hunter ,&nbsp;Patience S. Okoto ,&nbsp;Thallapuranam K.S. Kumar ,&nbsp;Paul D. Adams","doi":"10.1016/j.ab.2025.116014","DOIUrl":"10.1016/j.ab.2025.116014","url":null,"abstract":"<div><div>The serine/threonine kinase PAK1 serves as a mediator of cytoskeletal reorganization and cancer-related signaling downstream of the small GTPases. Due to the challenges in purifying PAK1 complexes, a 46-residue peptide from PAK1, is widely used to study PAK1-Cdc42 signaling. Traditionally, this purification involved multi-step chromatography of recombinant GST-PBD46 complexes, yielding approximately 1 mg per 1.5 L culture. In this study, a 30 min heat treatment step after thrombin cleavage was used to precipitate GST while leaving pure PBD46 in solution. This step eliminated the need for further affinity and size-exclusion chromatography steps. This improved protocol produces proteins with a 6.5-fold higher yield, halves the purification processing time, and produces peptides with ≥95 % purity. Mass spectrometry, CD, fluorescence, and ¹H–¹⁵N HSQC NMR confirmed the heat-purified PBD46's identity, structure, folding, and binding to Cdc42. The method also successfully separated other small peptides (e.g., ACK1) but not larger folded proteins. This rapid and scalable approach facilitates peptide production and biochemical studies without compromising the structural or functional integrity. We believe that the method described herein is applicable to other stable GST-fused recombinant proteins.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116014"},"PeriodicalIF":2.5,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145562406","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid purification of DNA samples from dextran sodium sulfate (DSS) contaminants","authors":"Adam Yeh ,&nbsp;James Saliba ,&nbsp;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":"2026-02-01","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}

{"title":"Electrochemical strategies for decoding serotonin using advanced carbonaceous and 5th generation materials: A comprehensive review","authors":"Rimpa Mondal ,&nbsp;Sk Faruque Ahmed ,&nbsp;Nillohit Mukherjee","doi":"10.1016/j.ab.2025.115990","DOIUrl":"10.1016/j.ab.2025.115990","url":null,"abstract":"<div><div>Carbonaceous materials are placed in the category of advanced and future materials for their very unique physicochemical attributes like tuneable electronic, electrical and optical properties associated with good chemical reactivity, biocompatibility and high surface to volume ratio that make them good candidates for various device applications including biosensors. Recently, such carbonaceous materials and their composites are being widely used as the electrode materials for enzyme-free electrochemical detection of various bioanalytes. Among the bioanalytes, the neurotransmitters are known to play crucial roles in maintaining proper balance in the human nervous systems. However, there are many challenges like selectivity, sensitivity and rapidity; that are normally faced during the detection of such neurotransmitters using the conventional enzymatic way. Electrochemical biosensors made up of advanced carbonaceous materials could be a single-key answer to all such challenges. Serotonin, a catecholamine group neurotransmitter, also known as the “happy hormone”, is known to control human emotions. So, real-time monitoring of its level in human serum carries immense importance, however; it comes with various challenges. This review article covers the developments of advanced carbonaceous, as well as, the 5th generation materials like Mxenes and related composites in the enzyme-free electrochemical sensing of serotonin since the inception of such materials in electrochemical biosensing. A detailed literature review exhibited that, impressive results, like very low limit of detection (LoD) of 8.0 nM could be achieved using multiwalled carbon nanotube (MWCNT) based electrodes, whereas, nitrogen incorporated graphene quantum dot (N-GQD) modified electrodes can help to reach a notably high sensitivity of 24.0 μAμM⁻¹.cm⁻². Again, carbon spheres (CS) have been established as a good candidate to bring a balance between LoD (4.0 nM) and sensitivity (5.5 μAμM⁻¹.cm⁻²) in combination with metal oxide electrode.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 115990"},"PeriodicalIF":2.5,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145312138","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":"Advances in the detection of azodicarbonamide and the metabolic product semicarbazide","authors":"Zijuan Miao ,&nbsp;Bowen Kan ,&nbsp;Pan Liu ,&nbsp;Yongming Guo","doi":"10.1016/j.ab.2025.116018","DOIUrl":"10.1016/j.ab.2025.116018","url":null,"abstract":"<div><div>Azodicarbonamide (ADA) is widely used as a foaming and oxidizing agent in the food and plastic industries. However, long-term exposure to it could cause serious health problems. Its metabolic product, semicarbazide (SEM), has raised health concerns due to findings of potential carcinogenicity and genotoxicity in some studies, though the evidence is context-dependent and sometimes conflicting. Therefore, the development of efficient, sensitive, and rapid detection methods for ADA and SEM is of great significance for food safety assurance. This review systematically summarizes recent advances in detection techniques for ADA and SEM, including high-performance liquid chromatography, capillary electrophoresis, Raman spectroscopy, immunoassays, electrochemical methods, near-infrared spectroscopy, colorimetry, fluorescence, chemiluminescence, photoacoustic, and photothermal detection. Subsequently, the sensitivity, selectivity, applicability, and limitations of these methods are compared and analyzed. Lastly, the challenges and future research trends are discussed. This review will pave the way for the development of advanced sensing strategies for ADA and SEM.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116018"},"PeriodicalIF":2.5,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145627607","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":"Structural, optical, and electrochemical characterization of TiO2-x thin films for non-enzymatic amperometric glucose sensing","authors":"J.K. Olarte Villamizar,&nbsp;M. Zapata Torres,&nbsp;N. Cruz González,&nbsp;G. Silva Galindo","doi":"10.1016/j.ab.2025.116016","DOIUrl":"10.1016/j.ab.2025.116016","url":null,"abstract":"<div><div>This work presents a novel approach to non-enzymatic glucose sensing based on oxygen-deficient titanium dioxide (TiO_2-x) thin films deposited on metallic titanium substrates. The TiO_2-x films were fabricated via RF magnetron reactive sputtering using a titanium target, and their structural, morphological, optical, and electrochemical properties were comprehensively characterized through X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), UV–Vis spectroscopy, and electrochemical techniques. The electrochemical performance was evaluated using cyclic voltammetry (CV) and chronoamperometry. The CV results revealed that the oxidation in phosphate buffer solution occurs at an applied potential of −0.54 V, increasing with a glucose concentration. Chronoamperometric analysis demonstrated a high sensitivity of 27.55 μA cm⁻² mM⁻¹ and a low detection limit of 0.11 mM. The sensor also exhibited a linear dynamic range from 0.2 mM to 2.4 mM and excellent selectivity against common interfering species, including histidine (His), cysteine (Cys), uric acid (UA), ascorbic acid (AA), and sucrose. These findings highlight the potential of Ti/TiO_2-x electrodes as efficient, stable, and selective platforms for developing non-enzymatic glucose sensors suitable for biomedical and wearable applications, with possible use in detecting glucose concentrations in sweat (0.27 mM–1 mM) and saliva (0.23 mM–1.78 mM).</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 116016"},"PeriodicalIF":2.5,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145595681","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":"Glutaminase I and glutamine transaminase–ω-amidase pathways in colorectal cancer: Metabolic reprogramming and emerging therapeutic strategies","authors":"Sarah F. Voor,&nbsp;Arthur J.L. Cooper,&nbsp;John T. Pinto","doi":"10.1016/j.ab.2025.115989","DOIUrl":"10.1016/j.ab.2025.115989","url":null,"abstract":"<div><div>Colorectal cancer (CRC) cells exhibit a pronounced dependence on <span>l</span>-glutamine to support anabolic growth, redox balance, and mitochondrial metabolism, a phenomenon known as “glutamine addiction.” The canonical glutaminase I pathway is mediated by a liver type glutaminase isozyme (LGA; GLS1), a kidney type glutaminase isozyme (KGA; GLS2), and a shortened form (glutaminase C, GAC). GLS1 and GLS2 convert glutamine to glutamate and subsequently to α-ketoglutarate (α-KG) by glutamate dehydrogenase, fueling the TCA cycle. GLS1 inhibitors, such as CB-839 (Telaglenastat), are under clinical evaluation and shows promise in treatment of CRC, particularly in combination therapies. In addition to the canonical pathway, the glutamine transaminase–ω-amidase (GTωA) pathway, a noncanonical route involving transamination of glutamine to α-ketoglutaramate (KGM) by KYAT1/2 and subsequent hydrolysis by ω-amidase (NIT2), offers metabolic flexibility under hypoxic or nutrient-limited conditions. Preclinical studies suggest that GTωA may compensate for GLS1 inhibition, contributing to therapeutic resistance. This review explores the dual roles of glutamine metabolism in CRC, emphasizing the GTωA pathway as a potentially targetable metabolic route that may contribute to therapeutic resistance. While GLS1 inhibitors are under clinical evaluation, emerging evidence suggests that dual targeting of both pathways may enhance treatment efficacy by overcoming metabolic compensation. Understanding the regulatory mechanisms driving the “glutamine shift” between these pathways is critical for developing effective metabolic interventions in CRC.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"709 ","pages":"Article 115989"},"PeriodicalIF":2.5,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145278856","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":"Systematic errors in isothermal titration calorimetry: The role of feedback power and effects of mixing and diffusion on concentrations","authors":"Žiga Medoš ,&nbsp;Marija Bešter-Rogač ,&nbsp;Epameinondas Leontidis ,&nbsp;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>_dif 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>₀ 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":"2026-02-01","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}

{"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":"2026-01-01","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":"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":"2026-01-01","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}

{"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":"2026-01-01","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}