ELECTROPHORESIS最新文献

{"title":"A Comprehensive Evaluation of Analytical Method Parameters Critical to the Reliable Assessment of Therapeutic mRNA Integrity by Capillary Gel Electrophoresis","authors":"Jessica P. Tran,&nbsp;Jun Gao,&nbsp;Casey Lansdell,&nbsp;Barry Lorbetskie,&nbsp;Michael J. W. Johnston,&nbsp;Lisheng Wang,&nbsp;Xuguang Li,&nbsp;Huixin Lu","doi":"10.1002/elps.8123","DOIUrl":"10.1002/elps.8123","url":null,"abstract":"<p>In recent years, messenger ribonucleic acid (mRNA)-lipid nanoparticle (LNP) biotherapeutics have demonstrated significant promise in disease treatment and prevention given their rapidly modifiable production processes and considerable capacity to adapt to complex or low-yielding proteins of interest. As a result, many products are currently being developed in this space. Critically, well-characterized and appropriately designed assays are required to monitor purity and integrity in order to maintain the efficacy and consistency of these novel products. Currently, capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ion-pair reversed-phase liquid chromatography (IP-RPLC) are techniques of choice for mRNA integrity analysis. However, most methods proposed for biotherapeutic analysis have been developed using naked mRNA without LNP components or proprietary buffer formulations, which can obscure undiscovered impurities or complex interactions between mRNA and the sample matrix. In this study, we addressed these methodological challenges by using a biotherapeutically relevant commercial mRNA-LNP sample (approx. 4200 b) to refine and optimize a customizable CGE-LIF method currently under consideration for mRNA-LNP biotherapeutic analysis. We systematically characterized how critical method parameters—such as denaturant type, concentration, and usage—and LNP disruption protocols can interfere with accurate mRNA integrity analysis in CGE-LIF and IP-RPLC. We found that optimal conditions for CGE-LIF assay sensitivity, variability, and resolution included sample precipitation by isopropanol, high urea concentrations, no formamide as a sample diluent, and high concentrations of dye. Finally, the advantages and disadvantages of both CGE-LIF and IP-RPLC are highlighted, and a discussion of key considerations when using or designing methods for mRNA integrity assessment is presented.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"46 7-8","pages":"365-375"},"PeriodicalIF":3.0,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elps.8123","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143699925","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Numerical Study of Falling Droplets in a Vertical Electric Field","authors":"Hailong Zhang,&nbsp;Tingting Zhang,&nbsp;Yuxin Lu,&nbsp;Qingzhen Yang,&nbsp;Hui Xing","doi":"10.1002/elps.8136","DOIUrl":"10.1002/elps.8136","url":null,"abstract":"<div>\u0000 \u0000 <p>It is of fundamental importance to study and understand the behavior of falling droplets. External fields, such as electric and magnetic fields, are considered promising methods for controlling falling droplets, especially at small scales. Although some experimental and theoretical work on falling droplets in an electric field has been conducted, a fully numerical model for electrohydrodynamic falling droplets is still lacking. In this article, we proposed a phase field numerical model and studied the falling droplets in a vertical electric field. In particular, the influence of the electric field on the velocity and interfacial morphology of the falling droplets was investigated. It was found that the vertical electric field can elongate the droplet in the vertical direction and increase its falling velocity. With a weak or no electric field (the electrical capillary number <i>Ca_E </i>&lt; 1.0), an indentation appears on the top of the falling droplet, and the droplet evolves into a bowl-like structure. In contrast, a strong electric field (<i>Ca_E</i> ≥ 1.0) causes a protrusion on the droplet, transforming it into a thumbtack-like shape. The numerical model and the obtained results can improve our understanding of falling droplets and highlight potential ways to regulate their behaviors.</p>\u0000 </div>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"46 7-8","pages":"388-403"},"PeriodicalIF":3.0,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143699926","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detection of Ultra-Short KYCDE Peptides Using Si_xN_y Nanopores.","authors":"Chaoming Gu, Kamruzzaman Joty, Matthew O'Donohue, Navod Thyashan, Lifang Hu, Moon J Kim, Sangyoup Lee, Min Jun Kim","doi":"10.1002/elps.8122","DOIUrl":"https://doi.org/10.1002/elps.8122","url":null,"abstract":"<p><p>Detection of ultra-short peptides is one of the critical steps toward deeper understanding of proteins and the sequencing of amino acids using solid-state nanopores. The ability of solid-state nanopores to detect these ultra-short peptides can help us reveal their hydrodynamic state under different conditions like the concentrations and the external voltage, which may further guide the future development in this field for deeper investigation and possible improvement. In this study, we fabricate Si_xN_y nanopores by CDB with various pore sizes and use them to detect ultra-short peptides comprised of five different amino acids. The peptide translocation events are extracted under various external voltages. Optimal experimental conditions such as the concentration of electrolytes and analytes, and the range of external voltage are investigated and compared. The statistical results based on volume exclusion analysis indicate that a significant portion of peptides exist in aggregation form. Due to the limitations of Si_xN_y nanopores such as the thickness and the noise, most of the single peptide signals are masked under the baseline noise. In addition, the results show that peptide-pore interactions are dependent upon the diameter of the nanopore. Higher voltage may also influence the degree of peptide aggregations. This study serves to further comprehend the physical and chemical properties of peptides, find possible ways to improve the performance of solid-state nanopores in the area of protein and peptide detections, and indicate the potential improvements in solid-state nanopore-based peptide sequencing.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143656537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modified Nucleosides as Potential Biomarkers of Prostate Cancer: Targeted Metabolomics of In Vitro Cell Samples by MEKC-UV.","authors":"Isabela Rocha, Ingridi Rafaela de Brito, Hernandes F Carvalho, Aline Mara Dos Santos, Ana Valéria Colnaghi Simionato","doi":"10.1002/elps.8120","DOIUrl":"https://doi.org/10.1002/elps.8120","url":null,"abstract":"<p><p>Prostate cancer is the second most common cancer among men globally, with over 1.4 million new cases and nearly 400000 deaths reported in 2022. Despite the availability of diagnostic tools such as the Prostate Specific Antigen (PSA) test, its low sensitivity reinforces the need for the exploration of more reliable biomarkers. In this context, metabolomics offers a promising approach for identifying sensitive biomarkers to improve cancer diagnosis and treatment. Therefore, this study aimed to conduct a targeted metabolomic analysis of the extracellular environment of In Vitro non-tumoral and cancer prostate cells to compare the levels of eight nucleosides using micellar electrokinetic capillary chromatography with UV detection (MEKC-UV). The method was adapted from a previously optimized protocol for blood serum, with minor adjustments to meet the Brazilian National Health Surveillance Agency (ANVISA) standards. Nucleosides were extracted via solid-phase extraction (SPE), and cell cultures were maintained under controlled conditions at 37°C with 5% CO₂ until reaching 80% confluence. The optimized MEKC-UV method demonstrated precision and accuracy, although the Youden test indicated some lack of robustness. Statistical analysis using a two-tailed t-test revealed significantly higher adenosine levels in non-tumoral cells, whereas uridine and 5-methyluridine concentrations were elevated in cancer cells. Inosine was detected exclusively in the non-tumoral cell line. Nevertheless, the method's innovative and cost-effective nature underscores its potential as a tool for cancer biomarker identification, with distinct nucleoside patterns in cancer cells offering valuable insights for disease recognition.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143656399","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluorescence-Based Multiplex Western Blot to Simultaneously Detect the Insulin-Like Growth Factor-1 (IGF-1) Isoforms","authors":"Matteo Bocconcelli,&nbsp;Fabiana Fanelli,&nbsp;Roberta Saltarelli,&nbsp;Mauro De Santi,&nbsp;Rita Barone,&nbsp;Elena Barbieri,&nbsp;Giosuè Annibalini","doi":"10.1002/elps.8116","DOIUrl":"10.1002/elps.8116","url":null,"abstract":"<p>Insulin-like growth factor-1 (IGF-1) is critical for tissue growth and development. The <i>IGF-1</i> gene contains six exons and due to alternative splicing three different isoforms might be produced: the IGF-1Ea, Eb, and Ec prohormones (proIGF-1s). These proIGF-1s share the same IGF-1 mature sequence, which is responsible for the IGF-1 receptor binding but differ in their carboxy-terminal extensions called Ea-, Eb-, and Ec-peptides. Several lines of evidence indicate that E-peptides control the intracellular proIGF-1s localization and maturation. Here, we present a multiplex Western blotting system able to simultaneously discriminate and quantify mature IGF-1, proIGF-1s and E-peptides within the same sample. HEK293 cells were transiently transfected with plasmids containing the <i>IGF-1Ea</i>, <i>IGF-1Eb</i>, or <i>IGF-1Ec</i> isoform or an empty vector. Two different primary antibodies, which recognize the mature sequence or the common region of E-peptides, were used to detect IGF-1 isoforms, which were subsequently distinguished with secondary antibodies conjugated to different fluorophores. Our results demonstrate the feasibility of simultaneously detecting different IGF-1 isoforms using two primary antibodies directed against different epitopes of proIGF-1s, combined with fluorescence-conjugated secondary antibodies. Furthermore, this dual-epitope strategy increases the specificity of protein detection, making it a valuable tool for studying the diverse roles of IGF-1 isoforms in biological processes.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"46 7-8","pages":"462-467"},"PeriodicalIF":3.0,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elps.8116","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143656539","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparing Different Light Models for Virtual Electrodes in Optoelectronic Tweezers.","authors":"Ernesto Guzman-Saleh, Victor H Perez-Gonzalez, Rodrigo Martinez-Duarte","doi":"10.1002/elps.8131","DOIUrl":"https://doi.org/10.1002/elps.8131","url":null,"abstract":"<p><p>Optoelectronic tweezers (OET) allow for the physical manipulation of particles of interest via dielectrophoresis (DEP) in microfluidic devices. To produce the nonuniform electric field required to enable DEP, light is used to expose a photoconductive film and create a so-called virtual electrode (VE). Several attempts have been made to model the light profile used to excite the photoconductive layer and produce the VE. However, no comparison of the models has been presented in the literature. Here, we present a comparative study among the rectangular, Gaussian, and saturated-Gaussian models in mapping to light profiles obtained experimentally. These models were then used to predict the activation of a VE and the distribution of the electric field in an OET system. From this comparison, it is possible to conclude that the saturated-Gaussian model should be the preferred choice to study these systems. Moreover, VEs were also compared numerically to conventional gold electrodes used regularly in DEP applications, concluding that very relevant differences exist between the electric fields produced by these two types of electrodes.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143656536","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigation of Fatty Acids in Biological Fluid Samples and Analysis by Capillary Electrophoresis: State of the Art and Applications.","authors":"Patrícia Abranches Geraldo, Maria Patrícia do Nascimento, Bruna Marchiori Berlande, Jéssica Cordeiro Queiroz de Souza, Luiz Henrique Cantarino Adriano, Marcone Augusto Leal de Oliveira","doi":"10.1002/elps.8134","DOIUrl":"https://doi.org/10.1002/elps.8134","url":null,"abstract":"<p><p>Fatty acids are vital to various physiological processes, making their analysis crucial for understanding metabolic, nutritional, and pathological conditions. Traditional methods for its analysis in biological samples, such as gas chromatography and high-performance liquid chromatography, often require complex sample preparation, including derivatization and extraction steps. Capillary electrophoresis has emerged as a promising alternative, offering simpler sample preparation, fast analysis times, and reduced consumption of solvents and reagents, which is in line with the principles of green chemistry. Despite its potential, capillary electrophoresis remains underutilized in fatty acid analysis in biological samples. In this regard, this review discusses the state of the art in capillary electrophoresis application for fatty acid analysis in biological samples, highlighting the simplified sample preparation protocols and the technique's advantages over others.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143656376","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Forensic Age Estimation From Blood Samples by Combining DNA Methylation and MicroRNA Markers Using Droplet Digital PCR","authors":"Niu Gao,&nbsp;Junli Li,&nbsp;Fenglong Yang,&nbsp;Daijing Yu,&nbsp;Yumei Huo,&nbsp;Xiaonan Liu,&nbsp;Zhimin Ji,&nbsp;Yangfeng Xing,&nbsp;Xiaomeng Zhang,&nbsp;Piao Yuan,&nbsp;Jinding Liu,&nbsp;Jiangwei Yan","doi":"10.1002/elps.8133","DOIUrl":"10.1002/elps.8133","url":null,"abstract":"<div>\u0000 \u0000 <p>Age estimation is important in criminal investigations and forensic practice, and extensive studies have focused on age determination based on DNA methylation (DNAm) and miRNA markers. Interestingly, it has been reported that combining different types of molecular omics data helps build more accurate predictive models. However, few studies have compared the application of combined DNAm and miRNA data to predict age in the same cohort. In this study, a novel multiplex droplet digital PCR (ddPCR) system that allows for the simultaneous detection of age-associated DNAm and miRNA markers, including <i>KLF14</i>, <i>miR-106b-5p</i>, and two reference genes (<i>C-LESS-C1</i> and <i>RNU6B</i>), was developed. Next, we examined and calculated the methylation levels of <i>KLF14</i> and relative expression levels of <i>miR-106b-5p</i> in 132 blood samples. The collected data were used to establish age prediction models. Finally, the optimal models were evaluated using bloodstain samples. The results revealed that the random forest (RF) model had a minimum mean absolute deviation (MAD) value of 3.51 years and a maximum <i>R</i>² of 0.84 for the validation sets in the combined age prediction models. However, the MAD was 5.66 years and the absolute error ranged from 3.16 to 10.54 years for bloodstain samples. Larger sample sizes and validation datasets are required to confirm these results in future studies. Overall, a stable method for the detection of <i>KLF14</i>, <i>miR-106b-5p</i>, <i>C-LESS-C1</i>, and <i>RNU6B</i> by 4-plex ddPCR was successfully established, and our study suggests that combining DNAm and miRNA data can improve the accuracy of age prediction, which has potential applications in forensic science.</p>\u0000 </div>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"46 7-8","pages":"424-432"},"PeriodicalIF":3.0,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143656362","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Practical Approach to Implementing ICH Q14: Tools for Analytical Quality by Design in Capillary Electrophoresis Method Development.","authors":"Ewoud van Tricht, Cari E Sänger-van de Griend","doi":"10.1002/elps.8110","DOIUrl":"https://doi.org/10.1002/elps.8110","url":null,"abstract":"<p><p>The ICH Q14 guideline introduces a structured framework for analytical method development based on Analytical Quality by Design (AQbD) principles, aiming to ensure robust, reliable, and fit-for-purpose methods throughout the product lifecycle. However, implementing ICH Q14 remains challenging due to the lack of complete examples and training resources, making it difficult for organizations to translate theory into practice. Although previous studies have applied AQbD to capillary electrophoresis method development, many have focused only on specific aspects such as the design of experiments (DoEs) or analytical target profile (ATP), leaving a gap in providing comprehensive, practical tools for the entire analytical lifecycle. This manuscript presents a novel, user-friendly approach to implementing ICH Q14 and AQbD, offering ready-to-implement tools and methodologies that simplify the process of method design, optimization, validation, and implementation. Through a stepwise process, the approach provides practical solutions for integrating AQbD principles into everyday workflows, bridging the gap between theoretical concepts and real-world applications. The approach has been thoroughly tested in diverse industrial settings, demonstrating its reliability and effectiveness. This work aims to facilitate the adoption of AQbD in analytical method development by providing structured tools, lessons learned, and best practices that align with ICH Q14 guidelines.</p>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":" ","pages":""},"PeriodicalIF":3.0,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143656535","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pharmacokinetics, Tissue Distribution, and Excretion of 9-Methylfascaplysin, a Potential Anti-Alzheimer's Disease Agent","authors":"Manman Zhang,&nbsp;Yu Xu,&nbsp;Xingjian Sun,&nbsp;Xiaolu Shi,&nbsp;Hongze Liang,&nbsp;Xiaowei Chen,&nbsp;Wei Cui,&nbsp;Yilei Fan,&nbsp;Jianfeng Ma,&nbsp;Haixing Wang","doi":"10.1002/elps.8135","DOIUrl":"10.1002/elps.8135","url":null,"abstract":"<div>\u0000 \u0000 <p>9-Methylfascaplysin, a derivative of the marine natural product fascaplysin, has shown promising anti- Alzheimer's disease (AD) potential through its anti-β-amyloid (Aβ) neuroprotective effects. However, the pharmacokinetics (PK) of 9-methylfascaplysin, crucial for its preclinical evaluation, have not been thoroughly studied. In this study, we developed and validated a sensitive and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantification of 9-methylfascaplysin in rat plasma. The method demonstrated a lower limit of detection (LLOD) of 1 ng/mL and a linear quantification range of 5–2000 ng/mL. The PK study in rat plasma was conducted. After intragastric administration, the plasma concentration of 9-methylfascaplysin peaked at a maximum concentration (<i>C</i>_max) of 193.4 ng/m and an enterohepatic circulation (EHC) phenomenon was observed. By comparing the area under the plasma concentration-time curve (AUC) values obtained from intragastric and intravenous administrations, the absolute oral bioavailability (F) of 9-methylfascaplysin was determined as 18.3%. The tissue distribution study revealed that following a single intragastric administration, 9-methylfascaplysin was most concentrated in the stomach, followed by the small intestine, large intestine, liver, kidney, brain, lung, spleen, and heart in descending order. Furthermore, the excretion profiles of 9-methylfascaplysin in rat urine and feces were studied. The results of this study provide valuable insights into the PK behavior of 9-methylfascaplysin and serve as a foundation for its further preclinical evaluation and potential clinical application as an anti-AD agent.</p>\u0000 </div>","PeriodicalId":11596,"journal":{"name":"ELECTROPHORESIS","volume":"46 7-8","pages":"452-461"},"PeriodicalIF":3.0,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143656405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}